Single-cell profiling coupled with lineage analysis reveals vagal and sacral neural crest contributions to the developing enteric nervous system

  1. Jessica Jacobs-Li
  2. Weiyi Tang
  3. Can Li
  4. Marianne E Bronner  Is a corresponding author
  1. Division of Biology and Biological Engineering, California Institute of Technology, United States

Abstract

During development, much of the enteric nervous system (ENS) arises from the vagal neural crest that emerges from the caudal hindbrain and colonizes the entire gastrointestinal tract. However, a second ENS contribution comes from the sacral neural crest that arises in the caudal neural tube and populates the post-umbilical gut. By coupling single-cell transcriptomics with axial-level-specific lineage tracing in avian embryos, we compared the contributions of embryonic vagal and sacral neural crest cells to the chick ENS and the associated peripheral ganglia (Nerve of Remak and pelvic plexuses). At embryonic day (E) 10, the two neural crest populations form overlapping subsets of neuronal and glia cell types. Surprisingly, the post-umbilical vagal neural crest much more closely resembles the sacral neural crest than the pre-umbilical vagal neural crest. However, some differences in cluster types were noted between vagal and sacral derived cells. Notably, RNA trajectory analysis suggests that the vagal neural crest maintains a neuronal/glial progenitor pool, whereas this cluster is depleted in the E10 sacral neural crest which instead has numerous enteric glia. The present findings reveal sacral neural crest contributions to the hindgut and associated peripheral ganglia and highlight the potential influence of the local environment and/or developmental timing in differentiation of neural crest-derived cells in the developing ENS.

Editor's evaluation

This paper is useful for researchers in the field of enteric neuroscience and peripheral nervous system development. The single cell RNA-sequencing based analysis of the developing chicken ENS, demonstrates differential cell identity contribution from the sacral and vagal neural crest and influence of the local distal embryonic environment for final differentiation. A basic classification scheme of neuronal cell types in the chicken combined with analysis of a more mature embryonic stages and functional data will however be needed in the future to determine the role of differential stem cell origin for final neuronal composition in the distal gut of chicken.

https://doi.org/10.7554/eLife.79156.sa0

Introduction

The enteric nervous system (ENS) is the largest component of the peripheral nervous system and plays a critical role in regulating gut motility, homeostasis, and interactions with the immune system and gut microbiota (Nagy and Goldstein, 2017). In amniotes, the ENS consists of millions of neurons with motor, sensory, secretory, and signal transduction functions, as well as a larger number of supportive enteric glia. Interestingly, enteric glia recently have been shown to retain neurogenic potential via reentrance into a progenitor-like state (Laddach et al., 2023). Together these diverse neurons and glia form a highly orchestrated network of physically and chemically connected cells embedded between the muscle and mucosal layers of the gastrointestinal system (Fleming et al., 2020). Due to the vast number of cells and its capacity for autonomic regulation, the ENS is often referred to as ‘a second brain’ (Gershon, 1999).

The neurons and glia of the ENS arise from the neural crest, a migratory stem cell population, emigrating from the closing neural tube. This transient population consists of four subpopulations designated from rostral to caudal along the body axis as cranial, vagal, trunk, and sacral. Best studied in mouse and chick embryos, much of the ENS is derived from ‘vagal’ neural crest cells that arise in the caudal hindbrain (adjacent to somites 1–7) at chick Hamburger Hamilton (HH) stage 10, approximately embryonic (E) day 1.5. These cells enter the foregut and migrate caudally to populate the entire length of the gut by E8 in the chick embryo (Le Douarin and Teillet, 1973), as well as giving rise to nerve-associated Schwann cell precursors (SCPs) that later invade the gut (Uesaka et al., 2015; Espinosa-Medina et al., 2017). However, there is an additional neural crest contribution to the ENS from the sacral neural crest population (Le Douarin and Teillet, 1973). First observed by Le Douarin and Teillet in quail-chick chimeric grafts, the sacral neural crest arises caudal to somite 28 at HH 17–18 (E2.5), migrates to the dorsal side of the developing gut, and forms the paired pelvic plexuses and Nerve of Remak at E3.5 (Anderson et al., 2006; Burns and Douarin, 1998; Yntema and Hammond, 1955; Figure 1A). The Nerve of Remak, which is specific to bird, is closely associated with the hindgut and has been described as a staging ground for many neural crest-derived cells which migrate to the gut along extrinsic axons to colonize the post-umbilical gut by E8 and rapidly expand in number by E10 (Burns and Douarin, 1998; Pomeranz et al., 1991).

Figure 1 with 2 supplements see all
Bulk RNA-seq of vagal and sacral neural crest derived cells in the post-umbilical enteric nervous system (ENS).

(A) Schematic diagram describing experimental procedure for viral labeling. Vagal and sacral neural crest cells were labeled by H2B-YFP (green) in separate embryos. The post-umbilical gastrointestinal tracts, including accompanying ganglia, were dissected at E10 for dissociation. (B) YFP+ cells from the post-umbilical region derived from vagal or sacral neural crest (NC) were sorted via FACS. (C) Volcano plot describing differentially expressed genes of sacral (sacral-post, blue) and vagal neural crest cells in the post-umbilical gut (vagal-post, red). Genes with fold change greater than 2 and p-value<0.05 are colored. (D) Heatmap highlighting selected genes related to neuronal functions from differential gene expression analysis in sacral and vagal-post ENS populations (with two replicates per condition). Genes are ordered based on significance level and fold change.

Dysregulation of ENS development is responsible for enteric neuropathies such as Hirschsprung’s disease which affects 1 in 5000 live births (Amiel et al., 2008), and is characterized by a paucity or absence of neurons in the distal colon resulting in potentially lethal obstruction and increased risk of infection (Lake and Heuckeroth, 2013; Ji et al., 2021; Lourenção et al., 2016). While the etiology of Hirschsprung’s disease is not completely understood, it is thought that insufficient migration or proliferation of vagal neural crest precursors results in neuronal deficits, particularly in the hindgut due to the long distance needed for precursor cells to reach their destination. Grafting sacral neural crest cells in place of ablated vagal neural crest results in isolated ganglia in myenteric and submucosal plexuses. However, the grafted sacral neural crest’s contribution to the ENS was insufficient to compensate for the lack of vagal-derived cells, suggesting that there may be intrinsic differences between these two populations (Burns et al., 2000). Molecular cues such as GDNF (Young et al., 2001) and ET3 (Nagy and Goldstein, 2006) are essential for migration and differentiation of vagal neural crest during early development and mutations in these genes are common in patients with Hirschsprung’s disease (Kenny et al., 2010). However, it is unknown if these genes are involved in the development of the sacral neural crest.

Recent studies have proposed that lack of rostrocaudal migration of the vagal neural crest may not be the only cause of enteric neuropathies. A distinction between the ENS of the foregut/midgut versus hindgut is that the former arises solely from vagal neural crest-derived cells, whereas the latter is populated by both vagal and sacral neural crest-derived cells (Burns and Douarin, 1998). Thus, a complete understanding of ENS ontogeny requires more thorough characterization of possible differences between vagal neural crest contributions to the pre-umbilical versus post-umbilical gut and characterization of sacral neural crest-derived contributions to the hindgut and associated peripheral ganglia. Open questions include: What cell types are derived from the sacral neural crest? Is the sacral neural crest population distinct from the vagal, or do they have shared derivatives? Does the post-umbilical gut possess special cell types absent in the pre-umbilical region? Defining the transcriptional landscape of sacral and vagal neural crest-derived cells along the entire length of the gut holds the promise of revealing similarities and differences between these populations.

To tackle these questions, we combined single-cell transcriptomics with a recently developed lineage tracing method in which replication-incompetent avian (RIA) retroviruses can be used to infect specific axial levels of the neural tube of the developing chick embryos to permanently express an inherited fluorophore (Tang et al., 2019). By infecting either vagal or sacral neural crest populations, RIA retroviral infection permits region-specific lineage tracing without the need for transplantation or Cre-mediated recombination that can result in ectopic expression. This enables transcriptional profiling of vagal- or sacral-derived RIA-labeled ENS cells in the pre- and post-umbilical gut at single-cell resolution. To characterize these cell populations, we chose E10 (similar to E16 mouse and 8 wk post-conception in human) as a starting time point. By E10, the vagal neural crest is undergoing the process of differentiation along the entirety of the gut while in the post-umbilical gut the sacral neural crest has formed the Nerve of Remak and has begun neurogenesis. Thus, this time point reflects a stage in which cells from each population contain both precursors and some differentiated neuronal subtypes.

Our results reveal both interesting similarities and differences between pre-umbilical vagal, post-umbilical vagal, and sacral neural crest-derived cells. While the vagal neural crest is the only contributor to CALB2/TAC1/PBX3+ neurons in both the pre- and post-umbilical gut at E10, the sacral neural crest contributes over 50% of cells to neuronal subtypes that are unique to the post-umbilical gut. Our in vivo analysis at E10 reveals sacral-derived neurons in the submucosal and myenteric plexuses as well as a major population residing within the Nerve of Remak. Interestingly, the vagal neural crest population in the post-umbilical gut shares more clusters with the sacral neural crest than the pre-umbilical vagal neural crest. The exception is that the sacral-derived population at this time point appears to be depleted in a neuronal/glial precursor present in the vagal-derived population. Trajectory analysis suggests that many sacral neural crest-derived cells are predicted to be enteric glia/SCPs, which have been shown to maintain a neurogenic potential in vitro and in an injury model (Laddach et al., 2023). Collectively, the data provide a transcriptomic reference for the developing chick ENS and associated peripheral nerves, expanding our understanding of the role of the sacral neural crest, a largely understudied stem cell population. Our results further suggest that the hindgut environment and/or developmental timing may influence cell fate decisions in the ENS.

Results

Sacral and vagal neural crest exhibit distinct transcriptional profiles at the population level

As a first step in assessing contributions of vagal and sacral neural crest in the post-umbilical region, we selectively labeled either the vagal or sacral neural crest population using a novel RIA retrovirus lineage-tracing technique (Tang et al., 2019; Tang et al., 2021) for bulk RNA-seq analysis. To this end, the neural tube at the level of the caudal hindbrain was injected with RIA retrovirus carrying a YFP expression cassette at HH10 (~E1.5) to label vagal neural crest cells, or below the level of somite 28 at HH17 (E2.5) to label the sacral neural crest (Figure 1A).

Injection of lineage tracer into the neural tube of chick embryos is a frequently used and accurate method to label the premigratory neural crest (Nakamura and Funahashi, 2001; Serbedzija et al., 1991). RIA virus has been shown to specifically label the vagal neural crest (Tang et al., 2019; Tang et al., 2021), but has not been previously applied to the sacral level. As it takes ~2 d for YFP expression mediated by RIA infection to become detectable, we turned to an alternative lineage label, the lipophilic dye DiI, to demonstrate the specificity of labeling the sacral neural tube/neural crest since it is immediately visible upon injection. DiI was injected in the same manner as RIA into the sacral neural tube of HH17 (E2.5) chick embryos. Shortly after injection, we observed specific labeling confined to the lumen of the neural tube with no labeling of adjacent tissue; subsequently, DiI was observed in the neural crest migratory streams 48 hr after injection (~HH24-25, E5) (Figure 1—figure supplement 1).

Next, we used the RIA virus encoding YFP to obtain pure populations of either vagal or sacral neural crest cells from the E10 post-umbilical gut, including the closely associated Nerve of Remak and pelvic plexuses (Figure 1A, Figure 1—figure supplement 2). The E10 time point reflects a stage at which both vagal and sacral neural crest cells have populated the post-umbilical gut for 2 d, enabling capturing of both precursors and some differentiated neuronal subtypes. After dissociation, YFP+ cells were sorted using FACS (Figure 1B). Similar regions from three guts were pooled as a replicate, with each library containing 2000 cells.

Differential gene expression analysis revealed intriguing distinctions between vagal and sacral neural crest cells in the post-umbilical gut at the population level. Genes enriched in the sacral population include SST1/SSTR, DBH, TH, DDC, PNMT, and SLC18A2. GRM3 expression is more abundant in the sacral population, which may reflect a transient state of differentiation. In addition, we observed upregulation of GFRA3, the receptor for artemin and CXCL12 which is related to signaling during cell migration (Figure 1C and D). Conversely, the vagal post-umbilical population expresses the adrenergic receptor ADRA1B, enzyme GAD1, CALB2, and NTS. Additionally, the population expresses genes related to neural crest and neuronal migration such as SEMA3D and TNC. HES5 and DLL1 expression indicate functions for Notch/Delta signaling in the vagal-post-umbilical population (Figure 1C and D).

Single-cell transcriptome profiling of the chick ENS

Whereas portions of the mammalian including the human ENS have been transcriptionally profiled at the single-cell level (Nakamura and Funahashi, 2001; Serbedzija et al., 1991), there is much less information about the developing chick ENS, and particularly the sacral-derived subpopulation. To understand the transcriptional profile of vagal and sacral neural crest-derived cells at single-cell resolution, we performed viral labeling as described above (Figure 1A) and collected three distinct neural crest populations at E10: vagal neural crest from the pre-umbilical gut (vagal-pre; three guts pooled per replicate), vagal neural crest from the post-umbilical gut and associated peripheral ganglia (including the Nerve of Remak) (vagal-post; six guts pooled per replicate), and sacral neural crest from the post-umbilical region plus associated peripheral ganglia (sacral; six guts pooled per replicate). After FACS isolation of YFP+ cells, 4.6k–5k cells were sequenced for each replicate producing transcriptomic profiles for cells. In total, 26,993 of these cells (3.2k–5.4k cells per replicate) possessed RIA viral genome transcripts (two or more), indicating true infection, and were used to generate a single-cell profile that organized into 15 clusters (Figure 2A; Zheng et al., 2017; Bray et al., 2016; Butler et al., 2018). To ascribe cluster identity, we first performed gene expression heatmap analysis for the top 10 gene markers in clusters 0–14 (C0–C14), displaying the most upregulated genes with focus on genes relevant to neuronal, progenitor, glial, and non-neural identity (Figure 2B). UMAPs were generated for pre-umbilical vagal, post-umbilical vagal, and post-umbilical sacral (Figure 2C). We also calculated the proportion of cells that each population contributes to the clusters (Figure 2—figure supplement 1).

Figure 2 with 1 supplement see all
Single-cell RNA-seq of vagal and sacral-contributions to the enteric nervous system (ENS) and associated peripheral ganglia.

(A) Uniform manifold approximation and projection (UMAP, resolution 0.3) representation of all RIA+ cells (>2 RIA transcripts; 26,993 cells) collected from the vagal and sacral-labeled embryos in both post-umbilical (including the Nerve of Remak and pelvic plexus) and pre-umbilical gastrointestinal tracts. (B) Expression heatmap for top 10 gene markers in clusters 0–14 (subsampled) with arrows pointing to marker genes for neural crest progenitor and glial (SOX8, HES5, CDH19, SFRP1), neuronal (ELAVL4, CHRN3A, VIP, SST, NFEM), fibroblast (COL1A1, LUM), melanocyte (MLANA), epithelial (EPCAM), vascular muscle (MEIS2A.2, VCAN), and endothelial (PECAM1). (C) UMAP representation (resolution 0.3) of each population: vagal-derived cells in the pre-umbilical gut, vagal-derived cells in the post-umbilical gut, and sacral-derived cells in the post-umbilical gut. (D) Key for putative cluster identities. See Figure 3A for greater detail.

Gene markers were analyzed for each cluster (Supplementary file 1) and violin plots were generated to observe differences in gene expression profiles between clusters (Figure 3B). Based on known cell-type markers, clusters were preliminarily classified as neuronal, glial, progenitor, or non-neural (Figure 3A). Neuronal clusters (C2, C4) had high expression of ELAVL4 (Figure 3A and B), a marker of early post-mitotic neurons (Akamatsu et al., 2005). Both C2 and C4 express the neuropeptide GAL, cholinergic receptor CHRNA3, and tyrosine signaling kinase RET. C2 expresses the neuropeptide VIP and tachykinin (TAC1) and is predominantly vagal-derived (post-umbilical vagal – 81%; pre-umbilical vagal – 16%) (Figure 2C, Figure 2—figure supplement 1). In contrast, C4 expresses DDC, PNMT, CHGB and is specific to the post-umbilical gut (vagal –35%; sacral – 65%).

Gene expression analysis of markers associated different enteric nervous system (ENS) cell types.

(A) Schematic diagram demonstrating genes associated with particular clusters of neuronal (ELAVL4+), glial (PLP1+, SOX10+), progenitor (SOX10+), and non-neural cell identities. (B) Violin plot of key genes reflecting neuronal/glial/progenitor cell fates. (C) Violin plot of key genes for non-neural cell identities (fibroblast, melanocyte, epithelial, vascular muscle, mesenchymal, endothelial, and myeloid progenitor).

We identified enteric glial clusters (C1, C6, C8) based on expression of the neural crest marker SOX10 (Kim et al., 2003) and enteric glial markers PLP1 (Rao et al., 2015), PMP22 (Hagedorn et al., 1999), S100A10 (Ferri et al., 1982), FRZB, and ZEB2 (Hegarty et al., 2015; Figure 3A and B). Within this enteric glial group, there is heterogeneity in expression of glial genes, including SCP genes. Both C1 and C8 express the gene CDH19, while only C8 expresses the canonical SCP marker MPZ (Jessen and Mirsky, 2019). C6’s expresses HEYL, FRZB, and ZEB2. These clusters are mainly comprised of post-umbilical gut populations. C1 and C8 have high proportions of sacral cells (60%, 59%) versus post-umbilical vagal (35%, 39%) and minimal contribution from the pre-umbilical vagal (5%, 2%). Likewise, C6 has low contribution from the pre-umbilical vagal but has larger contribution of vagal cells compared to sacral in the post-umbilical gut (61% vs 39%).

To highlight enriched signaling pathways and transcription factors, we generated tables of genes implicated in signaling (signal transduction [GO:0007165], potential cell–cell signaling [GO:0007267], transcription regulation [DNA-binding transcription factors; GO:0003700], or transcription factor binding [GO:0008134]) (Supplementary file 2). All three clusters expressed genes associated with signaling pathways such as Wnt (FRZB, SFRP2/5, NKD1), Vegf (ETS1), Tgfb (JUN, TGFB1), and Notch (JAG), GABA (GNG5).

C0, C3, and C7 were classified as progenitors due to expression of SOX10 and low expression of the neuronal and glial fate markers ELAVL4 and PLP1 (Figure 3A and B). C0 has high expression of EDNRB (Liu et al., 2019) and SOX8 (Cheung and Briscoe, 2003), and low ASCL1 (Castro et al., 2011). This putative neuroblast cluster is vagal-derived with the largest population present in the pre-umbilical gut (92%) versus post-umbilical (7%) gut (Figure 2C, Figure 2—figure supplement 1). C7 is similar but coupled with higher expression of ASCL1. Interestingly, both C7 and C0 are depleted in the sacral neural crest (Figure 2C, Figure 2—figure supplement 1). C3 has no clear gene profile (Figure 2B) and lacks differentiation markers (Figure 3B), potentially indicating a stem cell progenitor identity, supported by expression of the neural crest gene LMO4 (Ochoa et al., 2012) and stem cell genes LAMA4 and LAMB1 (Ulloa-Montoya et al., 2007; Figure 3B). Sacral neural crest contributes 52% of cells in C3 compared with pre-umbilical (15%) and post-umbilical vagal (33%) (Figure 2C, Figure 2—figure supplement 1).

Several small clusters C5/C9–14 contain profiles not typically associated with the ENS (Figure 3A). While these may be contaminating cells captured due to autofluorescence, we cannot exclude the possibility that some may be neural crest-derived since these clusters all express RIA transcript. C5/C9–C14 are likely non-neural cells due to the absence of clear neuronal or glial markers (Figure 3B). C5 (60% sacral, 8% post-umbilical vagal, 32% pre-umbilical vagal) (Figure 2C, Figure 2—figure supplement 1) expresses the fibroblast genes LUM, COL1A1 (Muhl et al., 2020), and TWIST1 (Vincentz et al., 2013; García-Palmero et al., 2016; Figure 3C). C9, present only in the post-umbilical gut (Figure 2C), has a large sacral contribution (80%) with high expression of the melanocyte genes MITF (Levy et al., 2006) (alternative name CMI9; Mochii et al., 1998), MLANA (Chen et al., 2021), and KIT (Wehrle-Haller, 2003). C10 (40% sacral, 40% post-umbilical vagal, 20% pre-umbilical vagal) expresses the epithelial genes EPCAM (Trzpis et al., 2007) and the keratin genes KRT7/1947 (Athwal et al., 2019 ) cells (Figure 2C, Figure 2—figure supplement 1). C11 expresses vascular muscle genes PDGFRA, MYL9, and VCAN (Sorokin et al., 2020). C12 has high expression of the mesenchymal genes CDH15, MYOD (Kasprzycka et al., 2019), and PITX2 (Gage et al., 2014). C13 expresses PECAM1 (Watt et al., 1995), CDH5 (Sauteur et al., 2014), and ID3 (Das et al., 2015; Gadomski et al., 2020), potentially indicating an endothelial cell type. C11, C12, and C13 are almost exclusively derived from the sacral neural crest (99, 97, and 95%, respectively) (Figure 2C, Figure 2—figure supplement 1). C14 expresses the macrophage genes CX3CR1, GPR34, MERTK (Verheijden et al., 2015) and is found in both sections of the gut, with greatest contribution from the post-umbilical vagal (42%) and sacral (56%) neural crest (Figure 2—figure supplement 1).

Validation of marker expression by vagal versus sacral neural crest using dual retroviral lineage tracing

We next sought to validate the gene expression differences identified by single-cell RNA-seq between sacral and vagal neural crest contributions to the gut in differentially labeled cell populations. To this end, we utilized axial-level-specific retroviral labeling to sequentially mark vagal or sacral neural crest cells with different fluorophores in the same embryo. For identifying the vagal neural crest, RIA retrovirus expressing nuclear H2B-RFP was injected into the neural tube adjacent to somite 1–7 at HH10 (~E1.5). Embryos were then allowed to develop until HH17 (E2.5), at which time RIA retrovirus carrying H2B-YFP was injected into the neural tube posterior to somite 28 to label the sacral neural crest. The entire length of the gut and associated Nerve of Remak was dissected and removed for immunohistochemistry at E10 and stained with antibodies to gene products identified as differentially expressed in our scRNA-seq dataset (Figure 3A).

The results show that the pre-umbilical gut contained only H2B-RFP+ (yellow in figure), suggesting that only vagal but not sacral neural crest cells contributed to this region (Figure 4—figure supplement 1). This is consistent with previous studies using quail-chick chimerae demonstrating the absence of sacral-derived cells in the pre-umbilical gut (Le Douarin and Teillet, 1973). Immunohistochemistry revealed vagal RIA retrovirus-labeled cells (yellow) that co-expressed acetylcholine receptor (ACHR) (Figure 4A–A’, Figure 4—figure supplement 1A–A’’’’) and HUC/D (ELAV) in the pre-umbilical (Figure 4B–B’, Figure 4—figure supplement 1B–B’’’’), consistent with differentiated neurons. Additionally, there were sparsely distributed neurons marked by TH (Figure 4C–C’, Figure 4—figure supplement 1C–C’’’’) and DBH (Figure 4D–D’, Figure 4—figure supplement 1D–D’’’’). H2B-RFP (yellow) and P0 double-positive Schwann cells were present along the pre-umbilical region (Figure 4E–E’, Figure 4—figure supplement 1E–E’’’’), as well as enteric progenitors or glial cells as determined by SOX10 expression (Figure 4F–F’, Figure 4—figure supplement 1F–F’’’’).

Figure 4 with 2 supplements see all
In vivo validation of vagal neural crest contributions to neuronal, glial, and progenitor cells in the pre-umbilical gut.

Transverse sections through the E10 preumbilical gut reveal (A) acetylcholine receptor expression in some vagal neural crest-derived cells along the pre-umbilical small intestine. (B) Vagal neural crest also gave rise to HUC/D+ neurons in this region. (C) A small number of neurons expressing TH were observed sparsely distributed in the myenteric plexus of the pre-umbilical intestine. (D) DBH expressing vagally derived cells were also observed in both the myenteric and submucosal plexus of the pre-umbilical intestine. (E) Vagal-derived glial cells (P0+) were present in the pre-umbilical intestine. (F) Enteric progenitor or glial cells expressing nuclear SOX10 were present in the pre-umbilical gastrointestinal tract. Insets (A’–F’) show magnified regions in the corresponding dashed box. Sacral neural crest cells were absent from the pre-umbilical gut. White arrows indicate double-positive cells. Scale bars for main figure (A–F): 80 μm. Scale bars for insets (A’–F’): 10 μm.

In contrast to the pre-umbilical gut, the post-umbilical ENS contained both H2B-RFP+ cells (yellow) and H2B-YFP+ (cyan) cells, indicating a collective contribution from sacral neural crest (cyan) and vagal neural crest (yellow) (Figure 1A, Figure 5). Consistent with our scRNA-seq data, neural crest cells from different axial origins appeared to express different markers. ACHR+ cells were more abundant in the vagal-derived population throughout the post-umbilical region (Figure 5A and A’, Figure 5—figure supplement 1A–A’’’’’), whereas ACHR+ cells from sacral neural crest cells were observed primarily in the myenteric plexus of the post-umbilical gut (Figure 5A and A”, Figure 5—figure supplement 2A–A’’’’’). Both vagal (Figure 5B and B’, Figure 5—figure supplement 1B–B’’’’’) and sacral (Figure 5B and B”, Figure 5—figure supplement 2B–B’’’’’) neural crest populations differentiated into HUC/D+neurons, with the vagal-derived HUC/D+ cells residing within the myenteric and submucosal plexus. While the majority of sacral-derived cells were in the Nerve of Remak, others were observed in the submucosal and myenteric plexuses (Figure 5E), some of which at are ACHR+ cells (Figure 5A”). Similarly, sacral-derived cells that expressed TH+ (Figure 5C and C”, Figure 5—figure supplement 2C–C’’’’’) and DBH+ (Figure 5D and D”, Figure 5—figure supplement 1D–D’’’’’) cells mostly resided in the Nerve of Remak while the vagal-derived cells were located in the myenteric or submucosal plexuses (Figure 5—figure supplement 1C–C’’’’’, TH; Figure 5—figure supplement 1D–D’’’’’, DBH). Both vagal and sacral neural crest contributed to Schwann cells and progenitors/glia expressing SOX10 (Figure 5E–E”, Figure 5—figure supplement 1E–E’’’’’, vagal; Figure 5—figure supplement 2E–E’’’’’, sacral) and P0 (Figure 5F–F”, Figure 5—figure supplement 1F–F’’’’’, vagal; Figure 5—figure supplement 2F–F’’’’’, sacral). These results confirm that the sacral neural crest contributes to both a large portion of the Nerve of Remak as well as a subset of neurons in the post-umbilical gut at E10.

Figure 5 with 2 supplements see all
Relative contributions of vagal and sacral neural crest cells to the post-umbilical gut.

(A) Acetylcholine receptor was broadly expressed by vagal neural crest along the post-umbilical gut (inset A’); ACHR was also present in sacral neural crest-derived cells (inset A”). (B, C) Differentiated neuronal markers HUC/D and TH were expressed by vagal (insets B’, C’) neural crest cells in the hindgut, while sacral neural crest cells were most present in the Nerve of Remak (insets B”, C”). (D) DBH expressing vagally derived cells were observed primarily in the myenteric plexus of the hindgut while sacral-derived DBH+ cells were predominantly in the Nerve of Remak (D”). (E) Both populations contributed P0+ glial cells within the plexuses of the hindgut (insets E’–E”). (F) SOX10+ progenitors were derived from both populations with vagal cells residing within the hindgut (inset F’) and sacral cells predominantly located within the Nerve of Remak (inset F”). Insets (A’–F’, A”–F”) show magnified regions in the corresponding dashed box. White arrows indicate double-positive cells. Scale bars for main figure (A–F): 80 μm. Scale bars for insets: 10 μm.

Subclassification of vagal and sacral neural crest-derived neuronal cell types

To clarify contributions to neuronal subtypes, we extracted all cells from neuronal (ELAVL4+) clusters C2 and C4 (Figure 4A’) and re-clustered them into 11 subclusters (sC0-10) to identify genes characteristic of each cluster (Table S3; Figure 6A). We plotted expression of receptors, neurotransmitters, and neuropeptides (Figure 6B and C) and generated a table of transcription factors associated with each cluster (Table S4). sC0, sC1, and sC9 were identified as neuroblast-like due to high expression of the neural crest genes SOX10 (Kim et al., 2003) and ZEB2, plus ASCL1 (Castro et al., 2011), PENK, NEFM, GAL, CHGA, DDC, and CHRNA7. sC5 has similar gene expression to sC0 and sC1 but was considered to be immature neurons undergoing neurogenesis due expression of Ascl1 and transcription factors ETV5 (Liu and Zhang, 2019) and ETV1 (Wright et al., 2021).

Figure 6 with 1 supplement see all
Subclassification of neuronal clusters.

(A) Subclustering of neuronal clusters (resolution of 0.4) (c4, c2 inset A’) resulted in 11 distinct populations (sc0-10). (B) Schematic diagram demonstrating marker genes of each cluster. (C) Violin plots of neurotransmitters, neuropeptides, receptors, and key genes representing neuroblasts and specific neuronal functions such as catecholaminergic, serotonergic, cholinergic, and nitrergic. (D) UMAP representation (resolution 0.4) of each population’s subclustered neuronal cells.

sC2 represents neurons that are CHRNA7/GAL/DLX6 (Vohra et al., 2006)/HMX3 (Heanue and Pachnis, 2006) positive; however, the expression of ASCL1 points to an immature population. sC10 expresses CHAT, TAC1, PENK, CALB2, and MEIS1, associated with neurogenesis and neural crest invasion in zebrafish ENS development (Uribe and Bronner, 2015). sC3 is similar to sC10, with expression of CHAT/TAC1/CHRNA7/MEIS1, but also expresses GAL, VIP, NPY, MYTL, ZFHX3. Only low expression of the nitrergic gene NOS1 was observed. Interestingly, both sC10 and sC3 express PBX3, a transcription factor that has been previously linked to postmitotic interneruons in mice (Morarach et al., 2021), but whose role in chick remains unknown. sC6 and sC7 express CHAT, NEFM, CALB1, ISL1. sC7 is marked by expression of SST, DDC, NEFM, and GAL, whereas sC4 is similar to sC7 except for low expression of SST and absence of catecholaminergic genes (DBH/TH). sC8 expresses the catecholaminergic genes DBH and TH, and the serotonergic genes SLC18A2 and DDC.

We next separated each population into separate UMAPs of vagal pre-umbilical gut, vagal post-umbilical gut, and sacral post-umbilical gut (Figure 6D). Additionally, we calculated the proportion of cells in each cluster contributed by each population (Figure 6—figure supplement 1). As predicted by each population’s UMAP of all RIA+ cells (Figure 2C), there is a distinct distribution pattern across the subclusters based on population of origin and gut location. There are no subclusters that are exclusively sacral-derived, while sC10 (CALB2/TAC1/PBX3) is comprised of only vagal cells (50% post- and pre- umbilical).

Comparing pre-umbilical and post-umbilical populations, we find no clusters that are unique to the pre-umbilical environment. However, the pre-umbilical vagal does heavily contribute to the putative progenitor subclusters 0 and 5 (88 and 83%, respectively) compared to the post-umbilical vagal (9%, 15%). This pattern is also seen in sC3 (GAL/VIP/NPY+) and sC2 (CHRNA7/GAL+), in which the post-umbilical gut cells form only 23 and 15%, respectively. There is minimal sacral contribution to these clusters. Conversely, sC4 (CHRN7A/NEFM+), sC6 (CALB1/CHAT+), and sC7(SST/DDC+) are unique to the post-umbilical environment and interestingly are comprised of over 50% sacral neural crest. Although not exclusive to the post-umbilical environment, SOX10+ putative neuronal progenitor sC1/sC9 and sC8 (TH/SLC18A2/DDC/SST+) have higher percentages of sacral cells (72%, 83%, 66%) and limited contribution from the post-umbilical vagal (27%, 10%, 33%).

RNA velocity analysis reveals developmental trajectories leading to ENS differentiation

In order to better identify potential differences in vagal and sacral contributions to neuronal and glial lineages within the E10 pre- and post-umbilical gut, we isolated cells from neuronal (C2, C4), glial (C1, C6, C8), and progenitor clusters (C0, C7, C3) (defined by clustering of RIA+ cells presented in Figure 2) for RNA velocity analysis (La Manno et al., 2018). This method utilizes the ratio of spliced and unspliced mRNA to infer information regarding terminal cell state and the cell’s experience of latent time. Using a dynamical model in scVelo (Bergen et al., 2020), we determined the predicted trajectory for each subpopulation (Figure 7A–C). Both pre- and post-umbilical vagal-derived populations have C0 as a progenitor pool with developmental trajectories reaching into neuronal and enteric glial clusters (Figure 7A and B). Interestingly, sacral-derived cells lack this progenitor cluster at E10 (Figure 7C). Instead, the sacral neural crest cells have differentiation trajectories within enteric glial/SCP or neuronal clusters. The enteric glial clusters within the vagal post-umbilical also have intertwined trajectories including into the putative stem cell cluster C3 indicating potential plasticity between the clusters (Figure 7B). This finding is supported by a recent study that demonstrates the ability of enteric glia to reenter a progenitor-like state and undergo neurogenesis in vitro and in an injured gut (Laddach et al., 2023). We also noted that in both the vagal- and sacral-derived post-umbilical cell populations, there are RNA velocity trajectories from neuronal clusters C2 into C4 which may reflect previously reported post-mitotic neuronal differentiation (Morarach et al., 2021).

RNA velocity and terminal fate analysis of vagal and sacral neural crest cells in the enteric nervous system (ENS).

(A) Streamlines of RNA velocity projected on UMAP for pre-umbilical vagal-derived cells. (B) Streamlines of RNA velocity projected on UMAP for post-umbilical vagal-derived cells. (C) Streamlines of RNA velocity projected on UMAP for post-umbilical sacral-derived cells. (D) Absorption probabilities for the terminal fates determined in each population: pre-umbilical vagal-derived, post-umbilical vagal-derived, and post-umbilical sacral-derived.

Further analysis was performed using CellRank (Lange et al., 2022), a method that builds upon the scVelo analysis to reconstruct single-cell dynamics in populations without a known developmental trajectory and predicts terminal states. We calculated the probability for each cell to give rise to each respective terminal state (‘absorption probability’ of the Markov Model) (Figure 7D). Based on this analysis, we observed that the vagal-derived cells in the pre-umbilical gut have a terminal state in the neuronal cluster C2 with high probability of contribution from a subsection of the progenitor pool (C0). C0 was also identified as its own terminal state in the pre-umbilical vagal cells with the highest probability in a subsection of the cluster itself, pointing to self-renewal of the progenitor population. In contrast, vagal cells in the post-umbilical state have the neuronal cluster C4 as terminal state with high probability of contribution from progenitor clusters (C0, C3, C7). Interestingly, this arises from neuronal cluster C2. Like the pre-umbilical vagal cells, a self-contributing progenitor pool (C3) was also identified as a terminal state. Sacral-derived cells have terminal states of neuronal cluster C4 and enteric glial cluster (C8). The terminal fate C4 has the highest contribution from itself while the enteric glial terminal state has high probability of contribution from other enteric glial clusters (C1, 6) and a putative stem cell cluster (C3). Taken together, this analysis reveals potential differences in the precursor pool of sacral versus vagal neural crest derived cells, with the sacral neural crest giving rise to ‘enteric glia’ (Laddach et al., 2023) that potentially reflect a transitional state that retains both glial and neuronal potential. This highlights differences in developmental potential and timing of differentiation between these two populations.

Validation of sacral contribution to enteric glial/Schwann cell precursor-like fates

Given that, in addition to neurons, the sacral neural crest has predicted terminal fates of enteric glial, we performed additional validation of glial/SCP-like contributions sections containing H2B-YFP (cyan in figure) labeled sacral neural crest. The results show that the sacral neural crest gives rise to enteric glial fates with numerous cells residing in the Nerve of Remak, proximal to the hindgut. Immunostaining detected co-expression of P0 and YFP (cyan) cells, indicating sacral neural crest contribution to an SCP population (Jessen and Mirsky, 2005; Figure 8A and A’, Figure 8—figure supplement 1A–A’’’’), as well as PLP1 (Figure 8B and B’, Figure 8—figure supplement 1B–B’’’’) a marker of glial cells that has been shown to be widely expressed by enteric glia in mice (Rao et al., 2015). Indeed, conditional removal of Plp1 expressing enteric glia disrupts gastrointestinal motility in female mice (Rao et al., 2017), indicating the importance of such cells in ENS health. Additionally, we observed a small population of sacral neural crest cells expressing the canonical glial marker GFAP (Jessen and Mirsky, 1980; Figure 8C and C’, Figure 8—figure supplement 1C–C’’’’). Together these results confirm our predicted fate at E10 of the sacral neural crest cells to enteric glia/SCP-like cells.

Figure 8 with 1 supplement see all
Sacral-derived glial fate in the Nerve of Remak.

Sacral-derived cells contributed enteric glia within the Nerve of Remak labeled with P0 (A, A’), PLP1 (B, B’), and GFAP (C, C’). Insets (A’–E’) show magnified regions in the corresponding dashed box. White arrows indicate double-positive cells. Scale bars for main figure (A–E): 80 μm. Scale bars for insets (A’–E’): 20 μm.

Discussion

The enteric nervous system regulates critical gastrointestinal functions including digestion, fluid secretion, and immune interactions. Abnormal ENS development can lead to enteric neuropathies including Hirschsprung’s disease, characterized by lack of motility and obstruction. Studies of ENS development have primarily focused on the role of the vagal neural crest in the ontogeny of ENS disorders and did not parse the respective contributions of sacral versus vagal neural crest (Amiel et al., 2008; Kenny et al., 2010). Thus, the role of the sacral neural crest, which colonizes the hindgut in close coordination with the vagal neural crest, has been largely understudied. To better understand the derivatives of the sacral neural crest and their coordination with the vagal neural crest during ENS development, here we examine the diversity of cell types arising from vagal versus sacral axial levels at single-cell resolution in the E10 embryonic chick gut.

Ablation and heterotopic grafting experiments previously have been used to study the interplay between the vagal and sacral populations but have led to contradictory interpretations. While some studies concluded that vagal and sacral neural crest exhibit autonomous migration properties independent of the environment, others suggested a role for environmental influences. In an aganglionic hindgut model created by surgically removing the caudal part of vagal neural crest, transplanted quail sacral neural crest cells migrated into the hindgut and produced a small number of neurons but failed to compensate for the absence of vagal-derived neurons. This suggested that sacral neural crest cells do not require the vagal population to migrate but lack intrinsic ability to populate the gut fully (Burns et al., 2000). Reciprocally, when vagal neural crest cells are grafted to the sacral region, they migrate earlier and produce a larger neuronal population than the endogenous sacral neural crest cells (Burns and Le Douarin, 2001). However, other studies suggested a more prominent environmental effect, such that interchanged vagal and sacral neural crest cells migrated according to the local environment (Erickson and Goins, 2000). Consistent with this, combining chick gut before neural crest colonization with chick or quail neural crest revealed that sacral neural crest cells can colonize the colorectum independent of the vagal neural crest, but require the hindgut environment to differentiate (Hearn and Newgreen, 2000). Additionally, a recent study has demonstrated that human pluripotent stem cell (hPSC)-derived sacral neural crest are required together with hPSC-derived vagal neural crest to repopulate an aganglionic murine colon (Fan et al., 2023).

Our study using axial-level-specific labeling and transcriptomic analysis helps to resolve some of these apparent discrepancies. By utilizing RIA retroviruses within the chick embryo, we provide a complementary approach to address questions of developmental potential of vagal versus sacral neural crest population. RIA enables selective labeling of each population, facilitating comparison of the relative contributions of the vagal and sacral neural crest in the pre- and post-umbilical gut as well as the role of environmental factors therein. Coupling this neural crest axial-level-specific lineage labeling technique with transcriptomic analysis further provides granular detail of cell types within the developing ENS and differences in each neural crest population’s derivatives.

Using bulk RNA-sequencing, we find some differences between sacral and vagal-derived ENS cell types at the population level at E10. For example, we find TNC expression to be specific to the vagal neural crest (Figure 1C and D), consistent with its requirement for migration into the hindgut by changing the extracellular microenvironment (Akbareian et al., 2013). In addition, the sacral neural crest expresses high levels of SOX10-mediated Cdh19 (Huang et al., 2022), as well as Pax3 and 5-HT3 for innervation and neuronal maturation in the pelvic ganglion (Deal et al., 2021; Ritter et al., 2021). Ret is also known to be upregulated in vagal neural crest cells to mediate more invasive behavior than in sacral neural crest (Delalande et al., 2008).

Our single-cell RNA-sequencing results demonstrate that the pre-umbilical gut is solely populated by the vagal neural crest, giving rise predominantly to motor neurons and fibroblasts. In the post-umbilical gut, vagal neural crest cells form enteric glia, several neuronal subtypes, and some non-neural fates. Interestingly, the sacral and vagal neural crest contributions to the post-umbilical gut overlap, indicating the importance of the environment in post-umbilical ENS development. Comparison of the two gut axial levels demonstrates that only the post-umbilical gut has the unique neuronal subclusters sC4 (CHRN7A/NEFM+), sC7 (SST/DDC+), and sC6 (CALB1/CHAT+). Interestingly, all of these unique post-umbilical clusters have large contribution from the sacral neural crest (>50%) (Figure 6—figure supplement 1).

Importantly, our data reveal large differences in cell-type contribution between vagal neural crest-derived cells that localize in the pre-umbilical versus post-umbilical gut. Indeed, the complement of post-umbilical vagal-derived clusters is more similar to the sacral’s than the profile of pre-umbilical vagal-derived clusters. Within the post-umbilical gut, there is significant overlap between vagal and sacral neural crest fates. This suggests that there may be a relatively uniform developmental potential between vagal and sacral neural crest cells and that the local environment of the hindgut may play an important role in guiding their differentiation.

Indeed, the data suggest that there is no population in the E10 chick post-umbilical ENS that is exclusively derived from the sacral neural crest; however, the sacral does contribute over 50% of cells to the glial clusters C1/8 and non-neural clusters C11/12/13 (Supplementary file 3). We identify multiple clusters derived predominantly from the vagal populations, including two progenitor clusters (C0/C7) and the GAL/VIP/NPY+ (sC3) and CALB2/TAC1/PBX3+ (sC10) neuronal subclusters. The absence of sacral contribution to these neuronal subtypes may be the result of differences in timing of differentiation, but may also partially explain the inability of the sacral neural crest to compensate for loss of the vagal (Burns et al., 2000).

RNA velocity analysis demonstrates that the vagal neural crest maintains a glial/neuronal progenitor pool while the sacral neural crest does not at E10. While the presence of a sacral-derived neuron cluster and the absence of a putative neuroblast pool could be interpreted as early termination of sacral neurogenesis, we posit there may be further sacral contributions to ENS neurons at later time points from enteric glia/SCPs that may later migrate into the gut wall from the Nerve of Remak. This is consistent with a recent study that shows the potential for enteric glia to regain progenitor-like state and undergo neurogenesis in vitro and in an injury model (Laddach et al., 2023).

Previous studies have suggested that the maintenance of proliferative capacity in the ‘wavefront’ of invading vagal neural crest is critical for successful colonization of the elongating gut (Nagy and Goldstein, 2006; Landman et al., 2007; Simpson et al., 2007). Consistent with this possibility, endothelin-3, a gene implicated in Hirschsprung’s disease (Bidaud et al., 1997; Kenny et al., 2000), is important for maintaining a pro-proliferative environment for the vagal neural crest in the avian hindgut (Nagy and Goldstein, 2006) and is required for sacral neural crest colonization in the murine hindgut (Baynash et al., 1994).

RNA velocity further suggests distinct developmental trajectories for each neural crest population. Vagal-derived cells have a terminal state of VIP/TAC1+ neurons in the pre-umbilical gut and DDC/PNMT+ neurons in the post-umbilical gut similar to the predicted terminal neuronal state of sacral-derived cells (Figure 7C). This highlights the possible importance of the local environment in ENS development. The high probability of contributions of VIP/TAC1+ neurons (C2) and DDC/PNMT+ neurons (C4) (Figure 7C) is similar to the murine data demonstrating post-mitotic differentiation mediated by the transcription factor Pbx3 (Morarach et al., 2021). We also see high expression of PBX3 in the putative motor neuron subcluster sC10 (Figure 6C), thus indicating a homologous role for this transcription factor in chick ENS development.

In addition to DDC/PNMT+ neurons, the sacral neural crest is predicted to give rise to enteric glia or SCPs (Figure 7C), confirmed by our immunohistochemical analysis (Figure 8). We noted a large number of sacral-derived enteric glial cells residing in the Nerve of Remak, a structure unique to avian embryos (Goldstein and Nagy, 2008). Original studies by Burns and Le Douarin found that the sacral neural crest cells form the Nerve of Remak at E3 and continue to reside there until entering the gut between E8 and E10 (Burns and Douarin, 1998), possibly due to the presence of the chemorepellent SEMA3A in the hindgut (Shepherd and Raper, 1999). Quail-chick chimera indicated that Nerve of Remak alone may not be sufficient to populate the hindgut; in mice, the pelvic plexus represents the staging area for sacral neural crest cells to populate the gut (Nagy et al., 2007).

Our single-cell RNA-sequencing identified an enteric glial cluster (C8) that also expresses the canonical SCP marker MPZ (P0). Both vagal and sacral neural crest contribute to this cluster in the post-umbilical gut, as confirmed by the expression of P0+ (Figures 7C and 8A). However, there is a paucity of putative SCPs in the pre-umbilical gut. Studies in chicken and mice have shown that the vagal neural crest consists of a subpopulation of cells that take on a SCP fate prior to enter the gut, migrating along extrinsic nerves, contributing a stem cell pool that undergoes neurogenesis. Vagal-derived SCPs (emerging from the neural tube adjacent to somites 1–2) in chick migrate into the pre-umbilical gut via the vagus nerve and innervate the esophagus (Espinosa-Medina et al., 2017). In mice, sacral-derived SCPs have been shown to migrate into the hindgut via the pelvic nerve, forming ~20% of neurons in the colon (Uesaka et al., 2015). Additionally, a subset of SCPs derived from somite level 3–7 migrate ventrally and invade the esophageal mesenchyme (Espinosa-Medina et al., 2017). As the labeling technique used in this study would be expected to label both early migrating neural crest cells as well as neural crest-derived SCPs, we cannot distinguish whether the putative SCPs observed at E10 are derived from a neural crest population that initially migrates into and invades the gut or a later migrating SCP population.

Previous studies in mouse, human, and zebrafish, have provided valuable information regarding gene expression, cell fate divergence, and neuronal subtypes within the ENS (Morarach et al., 2021; Lasrado et al., 2017; Memic et al., 2018; Drokhlyansky et al., 2020). By focusing transcriptome analysis on the myenteric plexus of the small intestine at postnatal day 21, a study in mice identified 12 distinct neuronal classes categorized by a combination of neurotransmitters. Our results have identified most neurotransmitter genes found in the murine system with the exception of NOS1+ nitrergic neurons, GAD2+ GABAergic neurons, or SLC17A6+ Glutamatergic neurons (Morarach et al., 2021), which may develop at a later time point than studied here (Figure 7B). Another study, utilizing RAISIN RNA-seq, identified 21 neurons and 3 glial clusters in the mouse small intestine and colon and 14 neuronal clusters in the human colon. The neuronal subsets we have identified generally agree with this study, showing an overlap with murine Tac1/Chat+ neurons (sC10) and Penk+ neurons (sC6) (Figure 6C; Drokhlyansky et al., 2020). Rather than a Nos1+ inhibitory motor neurons, we identified a Gal/Vip +expressing neuronal cluster (sC3). We do observe similarities between the murine and chick glial clusters, both of which have Pmp22/Frzb/Cdh19/Plp1+ glial clusters (C1/6/8) (Drokhlyansky et al., 2020). Similarly to studies done in zebrafish embryos, which observed enteric neural crest progenitors labeled with sox10, phox2bb, and zeb2a. Similar to our study, vip+ and pbx3+ neuronal subtypes were identified in later zebrafish stages alongside neural crest-derived melanocytes and mesenchyme in the posterior section of the larvae (Howard et al., 2021).

While previous studies did not separate vagal from sacral contributions, the small intestine and colon contain both sacral and vagal populations, which are distinct from anterior vagal derivatives (Figure 2C). Our analysis reveals that gene expression patterns are markedly different between the vagal-derived cells in the pre- and post-umbilical gut (Figure 2C), confirming the results from previous studies within proximal versus distal colon (Drokhlyansky et al., 2020). This important conclusion suggests that the ENS is not uniformly distributed throughout the gut but varies from proximal to distal. Work in chick has identified a gene regulatory network (GRN) of Tfap/Sox/Hbox/bHLH transcription factors that determines vagal neural crest fate (neural, mesenchymal, or neuronal) prior to delamination and subsequent contribution to the ENS in the pre-umbilical region (Ling and Sauka-Spengler, 2019). Whether the same GRN regulates the vagal or sacral neural crest contribution to the post-umbilical gut remains to be determined.

Taken together, the present results suggest that there are different developmental programs for vagal versus sacral neural crest population. Our results may help explain why the sacral neural crest cannot completely compensate for the loss of vagal neural crest. The cell composition of the post-umbilical ENS is distinct from that of pre-umbilical ENS, with major differences resulting from the differential contributions of the sacral neural crest. In addition, the differentiation program of vagal-derived neural crest in the post-umbilical gut is different from that of the pre-umbilical vagal population, suggesting that environmental factors have a large influence on cell fate.

Methods

Retroviral labeling and chick embryology

H2B-YFP (#96893) and H2B-RFP (#92398) obtained from Addgene were cloned into the RIA plasmid between Not1 and Cla1 sites. RIA-H2B-YFP/RFP was transfected into DF1 cells (ATCC, Manassas, VA; #CRL-12203, Lot number 62712171, Certificate of Analysis with negative mycoplasma testing available at ATCC website) using PEI standard transfection protocol. DF1 cells were maintained in Gibco Dulbecco’s Modified Eagle Medium (DMEM) supplied with 10% FBS for 4 d, with 12 ml of supernatant collected per day. The supernatant was concentrated using ultracentrifuge for at 76,000 × g for 1.5 hr to get a viral stock tittered about 107 pfu/ml, aliquoted, and stored at –80°C until use. Viral solution was supplemented with 0.3 μl of 2% food dye (Spectral Colors, Food Blue 002, C.A.S# 3844-45-9) as an indicator, injected to fill the neural tube between somite 1–7 at HH10 to label vagal neural crest and/or posterior to somite 28 at HH17 to label sacral neural crest in ovo. Embryos were supplied with Ringer’s Solution (0.9% NaCl, 0.042% KCl, 0.016% CaCl2• 2H2O wt/vol, pH 7.0), sealed with surgical tape, and incubated at 37°C until embryonic day 10.

Vital dye labeling of the sacral neural crest, sectioning, and imaging

Vital dye DiI (Invitrogen #V22885) was diluted 1:5 with 10% sucrose and injected into HH17 embryos in ovo posterior to somite 28. Embryos were supplied with Ringer’s Solution (0.9% NaCl, 0.042% KCl, 0.016% CaCl2• 2H2O wt/vol, pH 7.0), sealed with surgical tape, and incubated at 37°C until subsequent collection. Timepoint (T) 0 embryos were collected after 1 hr of incubation. T24 embryos were collected 24 hr after injection. Embryos were fixed in 4% PFA in PBS (pH 7.5) for 30 min at room temperature and washed with PBS for three times. Fixed samples were incubated in a gradient of sucrose (5% for 2 hr at room temperature, 15% at 4°C overnight) and in gelatin at 37°C overnight. Embryos were embedded in gelatin solution, flash-frozen with liquid nitrogen, and mounted with Tissue-Tek O.C.T compound (Sakura #4583) for sectioning (Microm HM550 cryostat). Embryo sections were incubated in 1× PBS at 42°C until the gelatin was dissolved and stained with DAPI (CAT #) before being mounted with Fluoromount-G Mounting Medium (Invitrogen # 00-4958-02).

All sections were imaged with Zeiss AxioImager.M2 with Apotome.2. Images were cropped and magnified for representation.

Gut cell dissociation and fluorescence-activated cell sorting (FACS)

At embryonic day 10, the gastrointestinal tract, including the associated Nerve of Remak and pelvic plexuses (Supplementary file 2), was dissected from chick embryos and washed with Ringer’s solution. Pre- and post-umbilical regions were separated, broke into pieces in chilled DPBS, and loose-fit homogenized in Accumax solution (EMD Millipore). 400 μl of Accumax-tissue mixture was aliquoted into 1.7 ml Eppendorf tubes and shaken at 37°C for 12 min. After dissociation, chilled Hanks Buffered Saline Solution (HBSS) supplemented by BSA (125 mg in 50 ml, Sigma; 0.2% w/v) and 1 M HEPES (500 μl in 50 ml, pH 7.5, Thermo Fisher) was added to quench the reaction. The dissociated cells were passed through a 70 μm cell strainer (Corning) and collected by centrifuging at 500 × g for 11 min at 4°C. The cells were resuspended in HBSS-BSA, supplemented 7-AAD Viability Staining Solution (BioLegend # 420404, 500 TESTS), and sorted for YFP+, viable single cells using Sony SY3200 cell sorter at the Caltech Flow Cytometry Facility.

Bulk transcriptome analysis

For vagal neural crest in the post-umbilical regions, sacral neural crest at post-umbilical regions, two biological replicates were processed, with each replicate containing YFP+ cells from three embryos. A total of 2000 cells per replicate were lysed to generate cDNA library using SMART-Seq v4 Ultra Low Input RNA Kit (Takara Bio). The library was sequenced with 50 million single-end reads with 50 bp length using HiSeq 2500 at the Millard and Muriel Jacobs Genetics and Genomics Laboratory Caltech. Sequencing reads were trimmed using cutadapt (Martin, 2011) and mapped to Galgal6 genome using Bowtie2 (Langmead and Salzberg, 2012). DESeq2 (Love et al., 2014) analysis was performed to find differential expressed genes between vagal and sacral neural crest at post-umbilical regions generated by HTseq-count (Anders et al., 2015). Differential gene expression was presented using Volcano Plot (coloring genes with Fold Change >2 and p-value <0.05) and Heatmap2 provided by the Galaxy platform. Because there were more upregulated genes in the sacral than vagal-post populations, we annotated genes related to neuronal function for sacral population, genes related to neuronal function, as well as genes with top fold-change and top significance in vagal-post population.

Single-cell transcriptome analysis and data processing

For vagal neural crest in pre- and post-umbilical gut, sacral neural crest in post-umbilical gut, two biological replicates were obtained. Each replicate of pre-umbilical gut was pulled from three embryos; each replicate containing post-umbilical gut was pulled from six embryos. After FACS for viable YFP+ cells, 4600–5000 cells per replicate were used for library preparation by the SPEC at Caltech. The library was sequenced on NovaSeq S4 lane with 2 × 150 bp reads by Fulgent Therapeutics. To process fastq raw data, standard ENSEMBL galgal6 reference database was used. Single-cell level gene quantification was then performed using Cellranger v3.1.0 (Zheng et al., 2017) and kallisto 0.46.2 and bustools 0.40.0 pipelines (Bray et al., 2016) with default parameters. Gene count matrices from all the samples were combined and only cells with more than 200 genes detected were kept for the downstream analysis. To further remove potential doublet cells, DoubletFinder 2.0.3 package was used. Of these, cells with greater than 2 transcripts of the RIA retrovirus promotor mRNA were then selected for further analysis. Average number of RIA retrovirus promoter mRNA transcripts per cluster are presented in Supplementary file 5. This resulted in 26,993 cells total with an average of 10,261 transcripts/cell (median of 9701) and 2734 genes/cell (median of 2854). Gene counts were normalized and scaled using Seurat v3.2.0 (Butler et al., 2018). From the pre-umbilical vagal samples, 10362 RIA+ cells were analyzed with an average of 10,395 transcripts/cell (median of 10,569 transcripts) and an average of 2794 genes/cell (median of 2945). From the post-umbilical vagal, 6454 RIA+ cells were recovered with an average of 10,307 transcripts/cell (median of 8779) and 2712 genes/cell (median of 2746). The sacral consisted of 10,177 RIA+ cells, with an average of 10,095 transcripts/cell (median of 8923) and 2688 genes/cell (median of 2760). The first 30 principal components from principal component analysis (PCA) were used to find neighbors with Findneighbors function before cell clustering with FindClusters function (resolution = 0.3). UMAP dimensionality reduction was performed using RunUMAP function with uwot-learn selected for the parameter umap.method.

Subclustering was performed on cells in C2 and C4. The first 30 principal components from the PCA were used to find neighbors with Findneighbors function before cell clustering with FindClusters function (resolution = 0.4). UMAP dimensionality reduction was performed using RunUMAP function with uwot-learn selected for the parameter umap.method.

RNA velocity analysis

Loom files containing spliced/unspliced transcript expression matrices for all cells were generated using the velocyto.py pipeline (La Manno et al., 2018). Loom files were then refined to cell IDs that remained after Seurat filtering and cutoff of >2 RIA transcripts (RIA+ cells) for trajectory analysis and concatenated with corresponding Seurat UMAP coordinates, colors, and cluster identity. scVelo (Bergen et al., 2020) dynamical modeling was then performed on all RIA+ cells and each individual population using default settings. Estimated velocities were then used for terminal state analysis in CellRank (Lange et al., 2022). Terminal states, initial states, and absorption probabilities were calculated using default settings.

Immunohistochemistry and imaging

Gastrointestinal tracts were dissected and fixed in 4% PFA in PBS (pH 7.5) for 25 min at 4°C and washed with PBS for three times. Pre- and post-umbilical regions were separately incubated in 15% sucrose at 4°C overnight and in gelatin at 37°C for 2 hr. Gut segments were embedded in gelatin solution, flash-frozen with liquid nitrogen, and mounted with Tissue-Tek O.C.T compound (Sakura #4583) for sectioning (Microm HM550 cryostat). Gut sections were incubated in 1× PBS at 42°C until the gelatin was dissolved, soaked in 0.3% vol/vol Triton-X100 in 1× PBS for permeabilization. Blocking buffer was prepared in 1× PBS with 5% vol/vol normal donkey serum and 0.3% vol/vol Triton-X100. Sections were incubated with primary antibody at 4°C overnight. Sections were washed with 1× PBS for 10 min and three times. After the washes, sections were incubated with secondary antibody for 45 min at room temperature. List of primary antibodies used: 1:20 chicken anti-AchR ratIgG2a, mAB270 (DSHB Antibody Registry ID: AB_531809); 1:500 mouse anti-HuC/D IgG2b (Invitrogen, Cat# A21271); 1:20 chicken anti-mouse P0 IgG1, IE8 (DSHB Antibody Registry ID: AB_2078498); 1:500 rabbit anti-Sox10 (Millipore, Cat# AB5727); 1:500 rabbit anti-Tyrosine Hydroxylase (Millipore, Cat# AB152); 1:500 rabbit anti-DBH (ImmunoStar, Cat# 22806); and 1:250 rabbit anti-PLP1 E9V1N (Cell Signaling Technology #28702). List of secondary antibodies used: 1:1000 donkey anti-mouse IgG2b 647 (Invitrogen A31571), 1:1000 goat anti-mouse IgG1 647 (Invitrogen A21240), 1:1000 donkey anti-rat IgG 647 (Abcam ab150155), and 1:1000 goat anti-rabbit IgG 647 (Invitrogen A21245).

All sections were imaged with Zeiss AxioImager.M2 with Apotome.2. Images were cropped and magnified for representation.

Data availability

Sequencing data has been deposited to GEO, accession number GSE242228.

The following data sets were generated
    1. Jacobs-Li J
    2. Tang W
    3. Li C
    4. Bronner ME
    (2023) NCBI Gene Expression Omnibus
    ID GSE242228. Single-cell profiling coupled with lineage analysis reveals vagal and sacral neural crest contributions to the developing enteric nervous system.

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Decision letter

  1. Kathryn Song Eng Cheah
    Senior and Reviewing Editor; University of Hong Kong, Hong Kong
  2. Ulrika Marklund
    Reviewer; Karolinska Institutet, Sweden

Our editorial process produces two outputs: (i) public reviews designed to be posted alongside the preprint for the benefit of readers; (ii) feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.

Decision letter after peer review:

Thank you for submitting your article "Single-cell profiling coupled with lineage analysis reveals distinct sacral neural crest contributions to the developing enteric nervous" for consideration by eLife. Your article has been reviewed by 2 peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Kathryn Cheah as the Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Ulrika Marklund (Reviewer #2).

The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.

Essential revisions:

1) Extend the strategy of the scRNA study to stages when ENS neurons from both vagal and sacral origin are fully differentiated, compared to the current data set in which the expression profiles indicate that the differentiation states of both populations do not match, including relatively immature cell states. This will allow solid conclusions about cell types contributed by either source of neural crest, as well statements of relative proportions of the different populations.

2) Validate the strategy for neural crest lineage tracing using the RIA retroviruses, given the substantial contribution of non-ENS cell types, such as hematopoietic cells, muscle, endothelial cells, etc.. This would be necessary to support the conclusion that the neural crest contributes to various gut cell types, which is potentially interesting given that multiple corresponding neural crest tracing studies in mouse label only limited or none of these cell populations.

Related to this point, it is important to show how far the injected virus spreads, given the viral labelling technique is new for avians.

3) Extending the bio-informatic analyses will be necessary to clarify cell type identities as well as the contribution of specific cell populations from the sacral and vagal neural crest, and thereby essential for supporting the conclusions of the study. Specifically, the following points should be addressed:

a) In addition of the merged datasets, provide separate analyses of the (pre- and postumbilical), and sacral (postumbilical) populations to elucidate the differentiation paths and relations of individual clusters with relevant methodologies (e.g. Velocity).

b) Comparison to existing ENS data sets from other species, in particular mammals, will likely help to define the various ENS populations in the chicken and place the results and conclusions of the study into the larger context of the ENS field. (recommended).

c) Related, this is the first scRNAseq study of the chicken ENS at this stage and will therefore represent a valuable resource to the ENS field across all species. Including for instance tables listing the DEG would be a highly useful supplement to the paper.

4) Validate the scRNAseq data and resulting conclusion by providing more extensive analyses of the different ENS populations employing immunohistochemistry similar to Figures 5 and 6, to i) define neuronal subpopulations and ii) quantify the size of the respective populations, which will support the comparison of the contributions from vagal and sacral neural neural crest and the conclusion that cells originating from different axial origin contribute different fates.

Central to this validation is to consider naming clusters systematically after prominent marker genes rather than possible function, since the physiology of the chicken ENS is currently not well characterised. This will allow for better comparison with other species.

[for detailed suggestions see comments by reviewer2]

5) Depending on the outcome of the above points, it will be important for the authors to choose an appropriate hypothesis to address functionally to corroborate the implication(s) of the promising scRNAseq results.

Additional points to be addressed:

i) Clarify whether there are true melanocytes in the GI-tract preparations or cell populations with a largely similar expression profile.

ii) Discuss the present results from chicken in the context of the work, with respect to neural crest-derived cell attaching to nerve bundles and obtaining a schwann cell precursor-like identity before migrating into the gut, from Uesaka et al. 2015 and Espinosa-Medina et al. 2017.

iii) use distinguishable colours when representing more than one antibody staining in data panels showing immunohistochemistry. This is important when validating the scRNAseq results.

iv) Clarify information and name of the antibody denoted as AchR is nicotinic receptor β subunit. Does this correspond to Chrnb2? In this case, please state the full name of the antigen. AchR is a blunt denotation. Moreover, it is a rat antibody not a chicken antibody.

v) ensure that information in the figures is accessible, in particular the size of text and labels.

vi) assess whether neural-crest derived fibroblasts are present in your data sets, given that you find Col1a1, Acta, etc.).

Smaller corrections to the text and figures:

1) First sentence in the abstract – (describe also the origin from nerve-associated SCP-cells, and state gastrointestinal tract not "intestinal tract".

2) Second section introduction: "During development, these cells migrate from the neural tube (not "central nervous system" as neural crest by definition is part of neural tube but not central nervous system – it's a denotion of later structures).

3) p 3 para 2 line 4-5: Much of the ENS is derived…… , enter the foregut and migrate (remove from) caudally to populate…

4) p 3 para 2 line 12 – probably better to stick with caudally rather than switching to posteriorly.

5) For clarity in the last sentence of this section, please indicate what Embryonic day stage 17-18 correspond to.

6) Citation 19 should probably be 14 in last section on subtypes markers describing Figure 7.

7) Figures5 and 6 – In these figures the authors use pre.int and post.int rather than the pre- and post-umbilical used in the text and legend. Please clarify?

8) It is stated that neurons and secretomotor neurons are merged to generate Figure 7. As secretomotor neurons are neurons, it is not clear what is meant here?

9) p 5 para 1 line 1-2 – functionally distinct units have distinct functions by definition.

10) p 4 para 1 – The idea of differences between vagal and sacral crest contributions to ENS is inserted in the middle of this paragraph on enteric neurophathies, but whether the genes mentioned are differentially expressed in the two populations is not stated. This should be clarified.

11) p 11 para 2 line 7 – What is meant by intermediate levels of cells?

12) p 13 para 2 line 2 – These are neural clusters, but some of them are not neuronal, eg those that are glial or are precursors.

13) Figure 1 – Are both the vagal and sacral populations post-umbilical? In C it indicates that sacral is post, but that is not indicated for vagal.

Reviewer #1 (Recommendations for the authors):

This manuscript is generally well constructed, although I have a number of minor suggestions below. My major concern is that no functional data are included. The gene expression differences among specific enteric neural subtypes from vagal and sacral neural crest provides the authors with a multitude of hypotheses they could test to validate the many inferences they make throughout the manuscript. Testing at least one of these, for example showing whether a gene knockout has more of an effect on a sacral population or a vagal population would significantly enhance the importance of this manuscript for the field. This is especially the case since with the exception of vascular muscle and melanocytes, it seems like vagal and sacral crest can generate similar derivatives, although in very different proportions. So it is important to understand whether or not there is any compensation or regulation when a subset of one population is diminished. In addition, the authors suggest in a number of places that environmental cues influence cell fate decisions. However this remains untested in the context of these new lineage tracing studies.

Reviewer #2 (Recommendations for the authors):

Suggestions to improve the manuscript:

Relating to issue 1: Using regular immunohistochemistry – can you validate EGFP expression in the populations that may be arising from labelled cells? Another alternative explanation to the capture of non-ENS cells in the data could be (as you also mention) contamination during FACS, but also potential leakage of the virus when injecting into the neural tube. It is possible that small amounts of virus escape and infects the gut primordia (which is also easily accessible at this stage in the chicken). These alternative explanations should be tested and discussed.

Relating to issue 4: Please provide separate analysis of vagal (pre and postumbilical), and sacral (postumbilical) and analyse the differentiation paths with relevant methodologies (Velocity?) if possible. The UMAP in its current state is hard to interpret. As reviewer I cannot address the claims made on cellular identities without access to lists of differentially expressed genes.

Relating to issue 6: Clusters could be named after the most prominent marker, or only by numbers in figures. It is fine to speculate on which functional groups they may correspond to, but leave those out from the figures, or make sure it is clear that the functional names are presumed/potential/putative. Nomenclature is an alarming complication in the ENS field at the moment, where highly speculative names are given to scRNA-seq clusters – these tend to damage and confuse the field (seen in recent papers including Drokhylansky et al., 2020 and Fawkner-Corbett et al., 2021). For example, these publications include the errorness use of CGRP as sensory marker (it is expressed in at least 4 other functionally distinct ENS classes) and the made-up "neuroendocrine enteric neurons". It would be valuable to keep an open mind in the chicken system and allow the classification system to grow in a reasonable pace based on functional validations. I hope you agree!

Related to issue 7: It would be advisable to use other colour combinations, and larger pictures displaying the cells in question. The zoomed-out gut with DAPI can be shown ones, as they don't add much information.

Related to issue 8: Marker analysis – please provide a more full-bodies analysis to support the claims that neural crest from different axial regions have different fate potencies. As an example, in Figure 6 it would be important to define the proportion of total neurons that expresses the marker genes. Please also define myenteric versus submucosal analysis. Make sure to align your validating IHC to the markers found in the scRNA-seq. In the case of AchR – why is this used? (it correspond to Chrnb2 which is not part of the scRNA-seq analysis). Other relevant markers such as Sst, Npy and more are however not shown. If adrenergic/dopaminergic/serotonergic neurons are claimed, please show IHC for these neurotransmitters (related to earlier comment most of these phenotypes aren't well validated in the mouse, and especially not in the chicken). An important extension of your work would be to analyse the markers in older chickens, this could partially resolve the issue you have in analysing the ENS with scRNA-seq at such an early stage of neuronal development.

Related to issue 9: Corrections on gene-cell-function correlations: Grm3 is the receptor for glutamate, not a sign of glutamate production. It is stated that excitatory motor neurons express Gad1, Calb2 and Nts, while Calretinin is expressed in excitatory motor neurons, there is no evidence that they would be GABAergic in other species. How Nts relates to this phenotype is also not clear. Gfra3 is the receptor for Artemin, not GDNF. Expression of neuroblast marker Hes5 could indicate differential maturity between vagal and sacral neural crest. Please state the paper showing connection between Myo9d and P75. Expression of Gal is not a sign of excitatory MN, rather inhibitory MN. Ntg1 is not a marker of IPAN, Noggin is correlated to IPANs in the mouse, but not a bona fide marker. How Calbindin relates to IPANs in the chicken is not clear, and in mouse it is also not a good marker, it is rather mostly not correlated (Morarach et al., 2021). Fut9 is in Morarach et al., a plausible marker of excitatory MN, not inhibitory MN, although not validated. Discussion: It is mentioned that Cck, Vip, Sst, Nog and Nmu are sensory markers. While Nmu was shown in Morarach et al., to specifically mark murine IPANs, Cck was shown to mark Dogiel Type 1 cells, and not classical IPANs. VIP and SST are excluded from IPANs in the mouse, while SST is expressed in human IPANs.

Additional issues/questions:

1) Some markers found (Col1a1, Acta2 and more) are also associated with neural crest-derived fibroblasts (potential mesothelial cells according to Zeisel et al., 2018 or mesenchymal cells; Ling and Sauka-Spengler, Nat Cell Biol. 2019). Please compare gene expression patterns to explore if you can find the equivalent cells in your dataset.

2) Which is your definition of schwann cells? By definition, most ENS researchers tend to define enteric glia as those part of the ganglionic plexi or scattered within muscles/villi, while schwann cell precursors giving rise to ENS are nerve-attached. Mpz is found in developing ENS marking presumed schwann-cell precursors (Morarach et al., 2021). Perhaps calling them schwann cell precursors would be ok, although if Mpz expression is found within ganglia and not attached to nerves?

3) It is highly advisable to perform scRNA-sequencing at a stage when the ENS is mature, or if not possible, at the very least at a stage when the full plethora of ENS neuron classes have differentiated from both sacral and vagal origins. Only then would you be able to evaluate the difference in fate between the two axial regions of the neural crest.

4) A recommendation would also be to scrutinize the scRNA-seq dataset from the human ENS (Elmentaite et al., Nature 2021). Markers conserved between the mouse (Morarach et al., 2021) and human may be the most relevant to assess in your scRNA-seq chicken dataset.

5) Figures need more attention. Figure 2: A – instead of displaying all genenames (which are unreadable), consider to name examples of genes instead. B – Again, the text is unreadable, please make bigger C- The C0-12 index needs to be larger and please put in the numbering in the UMAP. Comments on Figure 5-6 are already provided above. Figure 7: Images are stretched in general. Provide larger numbering in A, and bigger index.

Smaller corrections:

1) First sentence in the abstract – (describe also the origin from nerve-associated SCP-cells, and state gastrointestinal tract not "intestinal tract".

2) Second section introduction: "During development, these cells migrate from the neural tube (not "central nervous system" as neural crest by definition is part of neural tube but not central nervous system – it's a denotion of later structures).

3) Much of the ENS is derived…… , enter the foregut and migrate (remove from) caudally to populate…

4) For clarity in the last sentence of this section, please indicate what Embryonic day stage 17-18 correspond to.

5) Citation 19 should probably be 14 in last section on subtypes markers describing Figure 7.

6) Preumbilical ENS express TH in Figure 6X – however in the legend it is stated that small intestine does not express TH. What do you mean?

7) It is stated that neurons and secretomotor neurons are merged to generate Figure 7. As secretomotor neurons are neurons, it is not clear what is meant here?

Comment on data, code, reagents:

1) The chicken ENS has not been analysed by scRNA-seq at this stages before. As these datasete would be very helpful for researchers investigating the chicken ENS as well as to compare with ENS in other species, ideally, much more information on gene expression data are needed. For instance, tables listing the DEG would be a great supplement to the paper.

2) The antibody denoted as AchR is nicotinic receptor β subunit 2. Does this correspond to Chrnb2? In this case, please state the full name of the antigen. AchR is a blunt denotation. Moreover, it is a rat antibody not a chicken antibody.

[Editors' note: further revisions were suggested prior to acceptance, as described below.]

Thank you for resubmitting your work entitled "Single-cell profiling coupled with lineage analysis reveals distinct sacral neural crest contributions to the developing enteric nervous system" for further consideration by eLife. Your revised article has been evaluated by Kathryn Cheah (Senior Editor) and a Reviewing Editor.

The manuscript has been improved but there are some remaining issues that need to be addressed, as outlined below:

Reviewer #2 (Recommendations for the authors):

Several concerns have been dealt with. Outlined below are the remaining or new issues:

1) In light of Figures 4 and 5, I wonder which part of the peripheral nervous system that actually has been included in the single cell RNA-sequencing experiment. The validating figures indicate that most sacral neural crest derived cells are contained within the Nerves of Remak, which is not part of the ENS. Is also Nerves of Remak (and even sacral ganglia) included in the sequencing? In this case the whole comparison is not between different ENS populations, but rather between the vagal-derived hindgut and Nerve of Remak (potentially consisting of both cells that will stay in this region, and that will migrate into the gut) in essence.

2) There are still a lot of assumptions of the functional neuron types based on neurotransmitters/receptors and other signaling pathways that are premature given the lack of basic characterization of the chicken neural composition. None of the below comments are corroborated in literature:

"Differential gene expression analysis revealed intriguing distinctions between vagal and sacral neural crest cells in the post-umbilical gut at the population level. Genes enriched in the sacral population include SST1/SSTR indicating interneuron cell fate, and DBH, TH, DDC, PNMT, and SLC18A2 which are present in catecholaminergic neurons and serotonergic neurons. GRM3 expression indicates that glutamatergic character is more abundant in the sacral population. In addition, we observed up-regulation of GFRA3 which is involved in the GDNF signaling pathway and CXCL12 which is related to signaling during cell migration (Figure 1C, D). Conversely, the vagal post-umbilical population expresses the adrenergic receptor ADRA1B, enzyme GAD1, CALB2, and NTS consistent with excitatory motor neuron fate."

a) Grm3 encodes the metabotrophic glutamate receptor. Hence it is true to say that the sacral population shows a higher ability to respond via Grm3 in the sacral population, however, it doesn't mean that it has a higher "glutamatergic character". Note that glutamate can be sensed by various receptor complexes (NMDA, AMDA, metabotrophic receptors), chances are therefore high that glutamate is sensed by other receptors in the vagal population.

b) SST1 and SSTR – there is no evidence for the linkage to chicken interneurons (please cite if this is the case). Receptors are found in various populations in the mouse and human, and while somatostatin is specific to interneurons in the mouse, somatostatin is expressed both in IPANs and interneurons in the human.

c) Gfra3 is the receptor for artemin. Note also that Gfra3 is associated with SCP in the developing mouse (Morarach et al., 2021)

It continues:

"Due to the expression of the secretomotor neuropeptide VIP and the tachykinin TAC1, we classified C2 as a motor neuron cluster that is predominantly derived from the vagal neural crest and is present in the pre-umbilical and post-umbilical gut (Figure 2C)."

d) VIP is expressed in several interneuronal subtypes in the mouse in addition to the motor neurons, and it is not only in secretomotor neurons, but also in inhibitory motor neurons of the myenteric plexus. Likewise, Tac1 is a very broadly expressed gene, also in interneurons not only motor neurons. In the human Tac1 is expressed in IPANs. Amongst motor neurons in the mouse, VIP is confined to inhibitory motor neurons, while Tac1 is expressed in excitatory motor neurons. Thus, you cannot conclude functional belonging based on these markers. You can speculate, but it would be inappropriate to reiterate this speculation in the abstract and major results.

And furthermore:

"DBH, TH, DDC, PNMT, and SLC18A2 which are present in catecholaminergic neurons and serotonergic neurons"

e) It is important to be aware of the large population of ENS cells in the intestine goes through a transient state where TH and to some extent DBH is expressed. It is thus unclear whether DBH and TH indeed are maintained in this neural population. If Nerve of Remak is also included in the sequencing these cells may represent sympathetic/parasympathetic neurons.

f) In general in order to compare subtypes across species it is not advisable to use the neurotransmitters/peptides only as these often vary substantially. Transcription factors that actually specify cell types are more likely to faithfully demarcate neuron types. As an example, Pbx3 expression is likely to indicate similarity to likely interneuron type (ENC12, Morarach et al., 2021), especially given the strong coexpression of Penk and Nefm. It would be advisable to screen and compare transcription factors if you would like to infer possible functional belongings.

3) The authors have not confirmed that non-ENS cells indeed stem from the labelled cells (question in previous round). Given that the fluorescent is rather low, it would be to expect that contaminating cells are captured during FACS. It is generally not possible to completely isolate a single cell population based on rather weak reporters used from the gut, which contains a large number of auto-flourescent cells. Moreover, consider that the enteric neural progenitors have migrated through the entire gut to reach the colonic region due to their native ability to do so. It sounds unreasonable that also for instance fibroblasts and epithelial cells would take the effort to migrate that far. It is highly uncertain that most of the indicated non-ENS cells actually are stemming from the vagal/sacral neural crest. It should be discussed in the manuscript that non-ENS cells may be contaminating cells, but that it cannot be excluded that some may be true traced cells. If you still believe that they are traced cells, please show pictures indicating co-expression between your reporter and markers of the various non-ENS cells.

4) Related to the question on whether Nerve of the Remak cells are included – if not included, and only ENS cells were captured, it also remains possible that the missing progenitor population resides within the pelvic ganglion. Sampling could lead to whether populations are included or not. Again, it will be important for the message and interpretation of data that readers understand which peripheral nervous system parts are included in the sequencing experiment.

5) As it takes several more days for sacral neural crest to reach the gut, and that may first differentiate somewhat (most likely undertaking a schwann-cell character) within the Nerve of Remak/Pelvic Ganglia, it is not surprising that the progenitor population express different genes in vagal versus sacral gut.

6) If Nerve of Remak is included, it is questionable how relevant the comparison is to ENS of humans and disease mechanisms of Hirschsprungs disease, as suggested in the discussion.

7) Please try to compress the results part. As the same datasets are analysed several times by different means it gets a bit repetitive, especially with regards to the neural markers, which are mentioned first as being expressed together, and then more refined in the second analysis. It is enough to talk about them in the more refined analysis (also in light of the comments above, with less functional attributes).

Reviewer #3 (Recommendations for the authors):

Abstract l33-34 'suggesting an important role for environmental factors promoting

differentiation of both vagal and sacral neural crest-derived cells in the post-umbilical gut' I am not quite clear of the emphasis the authors intend here. It is surely the case that local environments play a role at some level in the exact choice of individual cells, but the overlapping fates from vagal and sacral crest might reflect shared environment in gut, or simply overlap in endogenous potential. I wonder if this phrase is necessary – maybe consider deleting?

I consider that a number of speculative conclusions are phrased more strongly than appropriate based on the evidence presented. These include:

L38 'apparently lacks such a cluster' is too strong – cells of cluster 0 are present in sacral cells, just population is proportionately fewer. Suggest replace with 'is depleted in this cluster at this stage'.

And

L335 Can the authors distinguish developmental and timing differences? Could it be that potential is similar, but timing differences (perhaps driven by location differences, given that most sacral cells are found in Nerve of Remak?) explain the relative proportion of cells in the clusters, especially c0? Hence I suggest replacing 'and' with 'and/or'. Similarly, the conclusion in l409-413 may also be explained by the difference in location of most sacral cells. This possible caveat to these interpretations should be stated. Furthermore, the impact of this should be explored in the Discussion, as a caveat to any interpretations of the cell-types identified in each of the profiles.

L160 The authors state 26993 cells score positively for the RIA transcripts, but they should clarify whether all of these fulfil their criterion of >200 genes expressed i.e. what are the total numbers of vagal and sacral NCCs included in analysis? They should also add information on the numbers of genes identified per cell, e.g. what is the mean number of genes identified per single cell. The MandM indicate that cells with only 200 genes detected were included in the analysis (l561-562), but it is important to understand what the mean and variance was. This has implications for distinguishing putative cell-types (clusters) of different potency, as discussed recently by the Kelsh lab (see Kelsh et al., 2022, Development; Subkhankulova et al., 2023 Nature Comms.). For example, Figure 3 shows that C6 and C8, both classified by authors as glial, show low, but detectable levels of MITF, a key melanocyte determinant; this implies these cells retain melanocyte potential, although the authors do not comment on this. However, the extent to which other cells retain melanocyte or other fate potential is highly dependent upon the depth of sequencing (number of genes detected per single cell).

Figure 4 and 5 All colours should be shown independently, as well as merged, to clarify overlaps.

Figure 5 shows that most sacral cells reside in Nerve of Remak. Differences in relative contribution of sacral v vagal crest are clear in Figure 2C, but only absolute differences seem to be in small cluster 12 (?mesenchymal stem cell). To what extent does this bias in location of cells explain the bias in fates adopted? Put another way, if you consider the relative abundance of sacral versus vagal cells in the hindgut and Ganglion of Remak, are the apparent differences in certain cell-types (clusters) explained by sampling error? Likewise, in Figure 6, where the neuronal populations are analysed in more detail, there are some differences, but absolute differences are in relatively minor populations. This makes me hesitant to conclude that there are substantial differences in potential between the two populations, although this seems in places to be the authors' conclusion (e.g. l335, l484-486). The authors should add a discussion of this point, and soften their conclusions accordingly.

Figure 7 Authors should explain the meaning and significance of 'latent time' and 'absorption probabilities in panels B and C respectively.

[Editors' note: further revisions were suggested prior to acceptance, as described below.]

Thank you for resubmitting your work entitled "Single-cell profiling coupled with lineage analysis reveals vagal and sacral neural crest contributions to the developing enteric nervous system" for further consideration by eLife. Your revised article has been evaluated by Kathryn Cheah (Senior Editor) and a Reviewing Editor.

The manuscript has been improved but there are some remaining issues that need to be addressed, as outlined below:

Reviewer #2 (Recommendations for the authors):

The authors have been responsive to most concerns. However some remaining issues are listed below.

1) Introduction/Abstract: Trajectory analysis suggest that sacral neural crest has a predicted terminal fate of enteric glia.

What are the conclusions drawn from this? It likely means that neurons have already been generated and that progenitors only give rise to glia at E10 (which you allude to in the discussion). However, as it is phrased it sounds either like the conclusion is that all sacral cells eventually become glia (even the neurons), or that the sacral cells only become glia (which is not the case since you see neurons in the sacral population). Both the fact that you don't see the neurogenic population, and that the trajectory analysis points to glia indicate that at E10, sacral neurogenesis has ceased and progenitors that are present mainly give rise to glia. However, it is rather likely that this is the case for the Nerve of Remak, as the majority of cells that you capture from the sacral tracings are located here. An alternative conclusion could be that neurogenesis of sacral-derived ENS within the gut wall may occur later than E10 and have not yet started. A stringent comparison between cells that are only located in the post-umbilical gut wall that are either sacral or vagal in origin is evidently not performed. Please rephrase sections to make the message more clear on the glial destiny of sacral neural crest, as well as the comparison made in the current study being largely performed between NoR and postumbilical vagal NC. NoR may be equivalent to parts of ENS in other species, so the comparison is not irrelevant, but it needs to be more clearly explained so that no misunderstandings are created in the field.

2) On the same matter. That sacral neural crest can give rise to glia cells in NoR is not contrary to any conception in the field as far as this reviewer can tell. Therefore it is not clear why so much emphasis is placed on this observation (for instance "Validation of sacral contribution to glial fates" as its own subsection and Figure). It would be sufficient to conclude that no or only a small neuroblast cluster is captured from sacral-derived cells at E10, which could indicate that neurogenesis has ceased already. Clear sacral-derived glial clusters are presented in Figure 2, and validation could be presented in association with this figure 2. Remember also that an alternative conclusion could be that neurogenesis of sacral-derived ENS within the gut wall may occur later than E10 and have not yet started.

3) Row 262: These results confirm that a large proportion of the ENS in the colon and Nerve of Remak are derived from the sacral neural crest at E10.

Your data suggest that the cells in the NoR is generated from the sacral neural crest, however a very small proportion of the cells in the colon are generated from the sacral neural crest tracing. Please rephrase. Moreover, the sentence suggests that the ENS is located within the Nerve of Remak, is this nomenclature accepted?

4) Figure 7. RNA velocity analysis makes most sense if only cells are included that are clonally related. Thus, it is not clear why both sacral and vagal sources are included in a common analysis. Please perform individual analysis to understand if it is possible to see bridge populations between progenitor and neural populations (as you suggest there is no active neurogenesis) in the sacral-derived dataset.

5) On the issue of non-ENS cells being captured for sequencing, you have responded that while you cannot confidently confirm fluorescent non-ENS cells, the non-ENS cells have RIA transcripts. However for results presented in Figure 7 you state "In order to better identify potential differences in vagal and sacral contributions to neuronal and glial lineages within the E10 pre- and post-umbilical gut, we isolated cells from RIA+ neuronal (C2, C4), glial (C1, C6, C8), and progenitor clusters (C0, C7, C3) for RNA velocity analysis."

Does this means that the selected cells are the only cells with RIA, or you mean that you selected cells that are known to constitute ENS differentiation (progenitors, neurons and glia)? It would be helpful for this issue if you could present the average number of RIA for each cluster. This could also help guiding you in the conclusion whether the non-ENS cells are resulting from mere contamination (if the RIA is low or non-existing) or likely from true lineage-tracing (if the RIA is the same as the progenitor/neurons/glia). Note also that you in the results parts present the non-ENS cells as likely contaminants, while in the discussion it is more firmly concluded that the non-ENS cells are derived from the neural crest. Please be consistent in the message.

6) Discussion: "The enteric nervous system regulates critical gastrointestinal functions including digestion, hormone secretion and immune interactions."

Note that the role of the ENS in hormone secretion is not known in the gut. What you mean is probably luminal secretion from the epithelia. This is a fluid secretion, not hormones (at least this is what there is evidence for today)

7) Discussion: "Studies have suggested a critical role for the vagal neural crest development and disorders"

What does the sentence mean?

8) Figure 8: Glia in the Nerve of Remak is called "enteric glia" – is this correct, or should they be called schwann cells?

9) It would be advisable to review the figure legends. For instance the Figure 2 title does not reflect the contents and the description of Figure 3A is also not very well aligned with the contents.

10) Discussion: "Both species have Pmp22/Frzb/Cdh19+ glial clusters (C1/6/8), but only the embryonic chick glial clusters expressed Plp1 indicating potential species or stage (embryonic versus adult) differences."

It is not clear what study is referred to and if it is true that Plp1 is not expressed in the murine clusters. At least the juvenile scRNA-seq characterisation of enteric glia in mouse (Zeisel et al., 2018) does show massive expression of Plp1.

11) Discussion Row 479: "Compared with these studies done with postnatal and adult tissue, we observed more 479 clusters with progenitor/precursor identity (Figure 2A, Fig7A), which is not unexpected, given that our analysis utilizes the gut from embryonic stages."

Yes, perhaps not worthwhile mentioning.

12) In general the manuscript would win from a more concise writing, please go through and omit unnecessary information and lengthy description of clusters.

Reviewer #3 (Recommendations for the authors):

The authors have addressed in full all points I raised. In particular, the Discussion is now much-improved.

However, I think there is one place where the discussion still over-reaches slightly. At l427 the authors state 'RNA velocity analysis demonstrates that the vagal neural crest maintains a glial/neuronal bipotent progenitor pool while the sacral neural crest does not at E10. This may indicate potential differences in developmental potential, timing, and/or proliferative capacity.'

I think the description of Cluster C0 as a 'glial/neuronal bipotent progenitor pool' is flawed because the RNA velocity analysis only assessed (l308-9) 'neuronal (C2, C4), 309 glial (C1, C6, C8), and progenitor clusters (C0, C7, C3)'; thus, the analysis was restricted to identifying links to neuronal and glia fates only. Furthermore, the depth of sequencing (average <3000 genes/cell) is insufficient to show anything other than strong biases in differentiation state. Hence I would remove the word 'bipotent' which implies that the cells are partially-restricted, when that is simply not assessable from the data provided.

I think there is one key way in which the paper can be made more accessible to the reader. The Figureswith cluster diagrams are difficult for the reader to interpret due to a lack of a key to the clusters, giving their identifying number and their interpretation. A simple key repeated in each figure would solve this.

I noticed a couple of residual typos, e.g. l283 'interneruons', which should be carefully sort and corrected in Word.

In Discussion, I don't think this sentence makes sense: 'Studies have suggested a critical role for the vagal neural crest development and disorders11,18.'

https://doi.org/10.7554/eLife.79156.sa1

Author response

Essential revisions:

1) Extend the strategy of the scRNA study to stages when ENS neurons from both vagal and sacral origin are fully differentiated, compared to the current data set in which the expression profiles indicate that the differentiation states of both populations do not match, including relatively immature cell states. This will allow solid conclusions about cell types contributed by either source of neural crest, as well statements of relative proportions of the different populations.

We appreciate this comment and think it is an excellent suggestion that we definitely plan to do. This made us realize that we failed to clarify in the text why we chose this particular time point for our study, which is two-fold.

First, we are particularly interested in how neural crest cells choose their prospective fates. E10 (similar to E16 mouse and 8wk post-conception in human) is a time when the post-umbilical chick gut has been completely populated by both vagal and sacral neural crest cells for 2 days so cells are in the process of differentiation but not all cells are fully differentiated. For this reason, we can capture both precursors and some differentiated neuronal subtypes. We have clarified this point in the revised manuscript and now focus much more on the precursor population to identify both genes that are common to vagal and sacral neural crest cells as well as those that are distinct. This enables us to formulate testable hypotheses for the potential role of particular transcription factors is allocation of cell fate.

Second, in the US, chick embryos are not considered vertebrates until after E10. Thus, E10 represents the last timepoint we can raise embryos without animal approvals which are not currently in hand. We completely agree that performing experiments at later timepoints will be incredibly valuable and therefore are now applying for approvals. But realistically, these take considerable for approval and thus would delay publication of our datasets for at least another year. Therefore, we propose to publish the mature dataset as a Research Advance that would focus on differences between neuronal subtypes between pre-umbilical vagal, post-umbilical vagal and sacral datasets at more mature timepoints that would nicely complement the current work.

Rather than an additional time points, we have dived deeper into our analysis and discovered an interesting transition in the precursor population. We find that at E10, almost all the sacral precursors are glial in nature whereas the vagal populations (both pre- and post-umbilical) contain a bipotent progenitor pool that can give rise to neuronal or glial fates. Importantly, this suggests that the sacral crest cells are well on their way to differentiating whereas the vagal populations are continuing to maintain a more stem cell-like precursor pool. Second, we see a primary contribution of the vagal preumbilical population to motor neurons while sacral predominantly gives rise to cells with an IPAN/adrenergic signature. Third, this refocusing seems particularly important given that our original aim was to explore differences between vagal and sacral neural crest contributions to the gut. However, the single cell data reveals extensive overlap between sacral and vagal neural crest contributions to the post-umbilical gut, suggesting a strong environmental influence on cell fate decisions.

2) Validate the strategy for neural crest lineage tracing using the RIA retroviruses, given the substantial contribution of non-ENS cell types, such as hematopoietic cells, muscle, endothelial cells, etc.. This would be necessary to support the conclusion that the neural crest contributes to various gut cell types, which is potentially interesting given that multiple corresponding neural crest tracing studies in mouse label only limited or none of these cell populations.

Related to this point, it is important to show how far the injected virus spreads, given the viral labelling technique is new for avians.

Based on this question, we have reanalyzed our data including a quality control step that ensured only cells with successful infection were used for downstream analysis. The hematopoetic cells were eliminated in this way. However, neural crest contributions to muscle and melanocytes remained. Regarding validation that the RIA virus is limited to neural tube derived tissue, we are quite skilled at restricting our injections to the lumen of the neural tube given many years of experience performing these injections with many lineage labels. Using RIAs, we have already published four papers using this methodology, many of which are referred to in the manuscript (Li et al., 2019; Tang et al., 2019; 2019; 2021). In these papers, we clearly show that the virus only labels neural tube cells.

However, this was not previously validated for sacral injections. Unfortunately, it is not feasible to perform such an experiment with RIA retroviruses because it requires 48+ hours for integration and expression of the fluorescent protein. Therefore, to demonstrate specificity of our injections, we now include images showing sacral injection using an alternative lineage approach by injecting the vital lipophilic dye DiI into the sacral neural tube in exactly the same manner as we use for performing RIA injections. Embryos were fixed 1 hour or 48 hours post injection. At 1 hour, the DiI is confined to the lumen of the sacral neural tube. By 48 hrs, migrating neural crest cells are clearly visible migrating away from the labeled neural tube. No other tissue is labeled. These data are displayed in Supplemental Figure 1. We thank the reviewers for pointing out that this was not clear in the previous version of the manuscript.

3) Extending the bio-informatic analyses will be necessary to clarify cell type identities as well as the contribution of specific cell populations from the sacral and vagal neural crest, and thereby essential for supporting the conclusions of the study. Specifically, the following points should be addressed:

a) In addition of the merged datasets, provide separate analyses of the (pre- and postumbilical), and sacral (postumbilical) populations to elucidate the differentiation paths and relations of individual clusters with relevant methodologies (e.g. Velocity).

This is an excellent point. We now provide separate datasets for each condition as well as trajectory analysis using scVelo and CellRank.

b) Comparison to existing ENS data sets from other species, in particular mammals, will likely help to define the various ENS populations in the chicken and place the results and conclusions of the study into the larger context of the ENS field. (recommended).

Thank you for this excellent suggestion. We now use the markers presented in the dataset of Morarach et al. 2021 at E15.5 (which is close to age of our samples) for assigning gene signatures used for cluster identification. This has greatly aided our analysis and helps put our data in the broader context of regarding ENS development.

c) Related, this is the first scRNAseq study of the chicken ENS at this stage and will therefore represent a valuable resource to the ENS field across all species. Including for instance tables listing the DEG would be a highly useful supplement to the paper.

Thank you for the suggestion, which has been added as a supplement.

4) Validate the scRNAseq data and resulting conclusion by providing more extensive analyses of the different ENS populations employing immunohistochemistry similar to Figures 5 and 6, to i) define neuronal subpopulations and ii) quantify the size of the respective populations, which will support the comparison of the contributions from vagal and sacral neural neural crest and the conclusion that cells originating from different axial origin contribute different fates.

We appreciate this suggestion. Accordingly we now include further evidence using numerous antibody markers for glial cells (Plp1, P0, and GFAP), in addition to neuronal and neurotransmitter markers.

Central to this validation is to consider naming clusters systematically after prominent marker genes rather than possible function, since the physiology of the chicken ENS is currently not well characterised. This will allow for better comparison with other species.

[for detailed suggestions see comments by reviewer2]

Thank you for this suggestion. We have now renamed the clusters after prominent markers and agree that this is much more accurate. Indeed we previously struggled with appropriate names for these clusters so very much appreciate this input.

5) Depending on the outcome of the above points, it will be important for the authors to choose an appropriate hypothesis to address functionally to corroborate the implication(s) of the promising scRNAseq results.

Thank you for this suggestion. Our data suggest that there are differences in the neuronal precursor population between vagal and sacral neural crest cells. The new analysis demonstrates that the sacral neural crest cells contribute extensively to enteric glia. This leads us to hypothesize that this may be the reason that sacral neural crest cells appear unable to compensate for ablated vagal neural crest cells either in the intact animal or after transplantation in place of the vagal crest as shown by Burns and Le Douarin (2001). We now include a discussion of this point and recent data that has found aberrant enteric glia in Hirschsprung’s patients.

Secondly, we have included tables of putative genes that may be involved in receiving environmental signals as well as transcription factors within the glial clusters (C1, C6, C8). Thus, this Resource Paper provides an ample list of candidate factors for future functional experiments. However, we believe that the functional testing would take at least a year and is beyond scope of this Resource paper.

Additional points to be addressed:

i) Clarify whether there are true melanocytes in the GI-tract preparations or cell populations with a largely similar expression profile.

We observed expression of Mlana, Dct, and Mitf (cmi9) in our cluster and indeed see these genes in all of our neural crest datasets, consistent with the possibility that there are either neural crestderived melanocytes or progenitors present in the gut. These marker genes have been used by many other authors to indicate the presence of melanocytes including Chen et al., 2021 who profiled neural crest derived cells in the developing heart.

ii) Discuss the present results from chicken in the context of the work, with respect to neural crest-derived cell attaching to nerve bundles and obtaining a schwann cell precursor-like identity before migrating into the gut, from Uesaka et al. 2015 and Espinosa-Medina et al. 2017.

Thank you for pointing our attention to these very informative papers. We have included a discussion of our data in light of these findings.

iii) use distinguishable colours when representing more than one antibody staining in data panels showing immunohistochemistry. This is important when validating the scRNAseq results.

Thank you for pointing out this issue. Figures 4, 5 and 7 have been redone with better use of colors.

iv) Clarify information and name of the antibody denoted as AchR is nicotinic receptor β subunit. Does this correspond to Chrnb2? In this case, please state the full name of the antigen. AchR is a blunt denotation. Moreover, it is a rat antibody not a chicken antibody.

We appreciate this suggestion and have specified the full name of the antigen as Chrnb2 in the figures and Chrnb2 (Neuronal nicotinic acetylcholine receptor subunit β-2) as indicated in its reference. In the Materials and methods sections, we have modified the antibody as “rat anti chicken Neuronal nicotinic acetylcholine receptor subunit β-2” for clarification.

v) Ensure that information in the figures is accessible, in particular the size of text and labels.

Done.

vi) Assess whether neural-crest derived fibroblasts are present in your data sets, given that you find Col1a1, Acta, etc.).

Yes thank you for noting this. We do indeed see fibroblasts and have clarified this in the text.

Smaller corrections to the text and figures:

1) First sentence in the abstract – (describe also the origin from nerve-associated SCP-cells, and state gastrointestinal tract not "intestinal tract".

2) Second section introduction: "During development, these cells migrate from the neural tube (not "central nervous system" as neural crest by definition is part of neural tube but not central nervous system – it's a denotion of later structures).

3) p 3 para 2 line 4-5: Much of the ENS is derived…… , enter the foregut and migrate (remove from) caudally to populate…

4) p 3 para 2 line 12 – probably better to stick with caudally rather than switching to posteriorly.

5) For clarity in the last sentence of this section, please indicate what Embryonic day stage 17-18 correspond to.

6) Citation 19 should probably be 14 in last section on subtypes markers describing Figure 7.

7) Figures5 and 6 – In these figures the authors use pre.int and post.int rather than the pre- and post-umbilical used in the text and legend. Please clarify?

8) It is stated that neurons and secretomotor neurons are merged to generate Figure 7. As secretomotor neurons are neurons, it is not clear what is meant here?

9) p 5 para 1 line 1-2 – functionally distinct units have distinct functions by definition.

10) p 4 para 1 – The idea of differences between vagal and sacral crest contributions to ENS is inserted in the middle of this paragraph on enteric neurophathies, but whether the genes mentioned are differentially expressed in the two populations is not stated. This should be clarified.

11) p 11 para 2 line 7 – What is meant by intermediate levels of cells?

12) p 13 para 2 line 2 – These are neural clusters, but some of them are not neuronal, eg those that are glial or are precursors.

13) Figure 1 – Are both the vagal and sacral populations post-umbilical? In C it indicates that sacral is post, but that is not indicated for vagal.

Thank you for these suggestions changes all of which have been made.

Reviewer #1 (Recommendations for the authors):

This manuscript is generally well constructed, although I have a number of minor suggestions below. My major concern is that no functional data are included. The gene expression differences among specific enteric neural subtypes from vagal and sacral neural crest provides the authors with a multitude of hypotheses they could test to validate the many inferences they make throughout the manuscript. Testing at least one of these, for example showing whether a gene knockout has more of an effect on a sacral population or a vagal population would significantly enhance the importance of this manuscript for the field. This is especially the case since with the exception of vascular muscle and melanocytes, it seems like vagal and sacral crest can generate similar derivatives, although in very different proportions. So it is important to understand whether or not there is any compensation or regulation when a subset of one population is diminished. In addition, the authors suggest in a number of places that environmental cues influence cell fate decisions. However this remains untested in the context of these new lineage tracing studies.

We appreciate the reviewer’s suggestion to include functional experiments and we hope to conduct these in the future. Our main goal here was to generate a Resource that provides insights into possible driving factors that may influence differentiation and to establish a list of candidate factors for future testing. Performing long-term knockouts in the chick system requires CRISPR electroporation coupled with neural tube transplantation (as we recently published in Gandhi et al., 2021) which is quite an arduous and time-consuming experiment.

Reviewer #2 (Recommendations for the authors):

Suggestions to improve the manuscript:

Using regular immunohistochemistry – can you validate EGFP expression in the populations that may be arising from labelled cells? Another alternative explanation to the capture of non-ENS cells in the data could be (as you also mention) contamination during FACS, but also potential leakage of the virus when injecting into the neural tube. It is possible that small amounts of virus escape and infects the gut primordia (which is also easily accessible at this stage in the chicken). These alternative explanations should be tested and discussed.

Yes, we have done this as shown in Figure 5 and 6.

Please provide separate analysis of vagal (pre and postumbilical), and sacral (postumbilical) and analyse the differentiation paths with relevant methodologies (Velocity?) if possible. The UMAP in its current state is hard to interpret. As reviewer I cannot address the claims made on cellular identities without access to lists of differentially expressed genes.

Excellent suggestion. This is now included.

Clusters could be named after the most prominent marker, or only by numbers in figures. It is fine to speculate on which functional groups they may correspond to, but leave those out from the figures, or make sure it is clear that the functional names are presumed/potential/putative. Nomenclature is an alarming complication in the ENS field at the moment, where highly speculative names are given to scRNA-seq clusters – these tend to damage and confuse the field (seen in recent papers including Drokhylansky et al., 2020 and Fawkner-Corbett et al., 2021). For example, these publications include the errorness use of CGRP as sensory marker (it is expressed in at least 4 other functionally distinct ENS classes) and the made-up "neuroendocrine enteric neurons". It would be valuable to keep an open mind in the chicken system and allow the classification system to grow in a reasonable pace based on functional validations. I hope you agree!

Excellent suggestion. We have changed the nomenclature accordingly.

It would be advisable to use other colour combinations, and larger pictures displaying the cells in question. The zoomed-out gut with DAPI can be shown ones, as they don't add much information.

Excellent suggestion. We have changed the figures accordingly.

Related to issue 8: Marker analysis – please provide a more full-bodies analysis to support the claims that neural crest from different axial regions have different fate potencies. As an example, in Figure 6 it would be important to define the proportion of total neurons that expresses the marker genes. Please also define myenteric versus submucosal analysis. Make sure to align your validating IHC to the markers found in the scRNA-seq. In the case of AchR – why is this used? (it correspond to Chrnb2 which is not part of the scRNA-seq analysis). Other relevant markers such as Sst, Npy and more are however not shown. If adrenergic/dopaminergic/serotonergic neurons are claimed, please show IHC for these neurotransmitters (related to earlier comment most of these phenotypes aren't well validated in the mouse, and especially not in the chicken). An important extension of your work would be to analyse the markers in older chickens, this could partially resolve the issue you have in analysing the ENS with scRNA-seq at such an early stage of neuronal development.

We appreciate the helpful suggestions. Because we are using RIA labeling, we can only validate vagal vs sacral cells using antibodies to double-label individual cells and therefore must rely on available antibodies that work in the chick. Thus, we used Chrnb2 Ab according to its availability and that it would likely cross-react with Chrna5 and Chrnb4 which were abundantly expressed in the RIA labelled cells (C4, C5). We used TH and DBH as markers for adrenergic/dopaminergic/serotonergic neurons. We agree with the reviewer and examined Sst, but we could not obtain a reliable antibody for use in chicken.

Given the size of the chick gut and the fact that RIA does not stain the entire population, we are unable to accurately quantitate from our viral injection data. Thus, we have selected representative sections for illustrative purposes. Quantitative analysis can only be done from the single cell data since a few cross sections are not representative of the entire gut length and not all neural crest derived cells are labelled with the RIA.

Corrections on gene-cell-function correlations: Grm3 is the receptor for glutamate, not a sign of glutamate production. It is stated that excitatory motor neurons express Gad1, Calb2 and Nts, while Calretinin is expressed in excitatory motor neurons, there is no evidence that they would be GABAergic in other species. How Nts relates to this phenotype is also not clear. Gfra3 is the receptor for Artemin, not GDNF. Expression of neuroblast marker Hes5 could indicate differential maturity between vagal and sacral neural crest. Please state the paper showing connection between Myo9d and P75. Expression of Gal is not a sign of excitatory MN, rather inhibitory MN. Ntg1 is not a marker of IPAN, Noggin is correlated to IPANs in the mouse, but not a bona fide marker. How Calbindin relates to IPANs in the chicken is not clear, and in mouse it is also not a good marker, it is rather mostly not correlated (Morarach et al., 2021). Fut9 is in Morarach et al., a plausible marker of excitatory MN, not inhibitory MN, although not validated. Discussion: It is mentioned that Cck, Vip, Sst, Nog and Nmu are sensory markers. While Nmu was shown in Morarach et al., to specifically mark murine IPANs, Cck was shown to mark Dogiel Type 1 cells, and not classical IPANs. VIP and SST are excluded from IPANs in the mouse, while SST is expressed in human IPANs.

Thank you for this very helpful information. We have now used this information to assign our clusters and are extremely grateful to the reviewer for this comment, which has helped us to restructure our analysis. We heavily consulted the ENS single cell atlases published by Morarach et al. (mouse) and Drokhlyansky et al. (human) to postulate neuronal subtypes.

We have since re-analyzed the gene lists for all clusters and have included citations for gene markers. Upon this re-analysis, we have found better identification genes for our progenitor and stem cell clusters that are not Myo9d and P75.

Additional issues/questions:

1) Some markers found (Col1a1, Acta2 and more) are also associated with neural crest-derived fibroblasts (potential mesothelial cells according to Zeisel et al., 2018 or mesenchymal cells; Ling and Sauka-Spengler, Nat Cell Biol. 2019). Please compare gene expression patterns to explore if you can find the equivalent cells in your dataset.

Good point. We now have identified a neural crest-derived fibroblast cluster based on these previously published fibroblast markers.

2) Which is your definition of schwann cells? By definition, most ENS researchers tend to define enteric glia as those part of the ganglionic plexi or scattered within muscles/villi, while schwann cell precursors giving rise to ENS are nerve-attached. Mpz is found in developing ENS marking presumed schwann-cell precursors (Morarach et al., 2021). Perhaps calling them schwann cell precursors would be ok, although if Mpz expression is found within ganglia and not attached to nerves?

We agree that we have clusters that could either be called Schwann cell precursors or enteric glia given that there is heterogeneity in these populations and we identify three clusters that are characterized by these markers. We now call these Enteric Glia 1, 2 and 3 and posit that they may be differentially localized but cannot really parse them based on the single cell data.

3) It is highly advisable to perform scRNA-sequencing at a stage when the ENS is mature, or if not possible, at the very least at a stage when the full plethora of ENS neuron classes have differentiated from both sacral and vagal origins. Only then would you be able to evaluate the difference in fate between the two axial regions of the neural crest.

We agree and hope to do this at a later time but it is beyond the scope of our current study.

4) A recommendation would also be to scrutinize the scRNA-seq dataset from the human ENS (Elmentaite et al., Nature 2021). Markers conserved between the mouse (Morarach et al., 2021) and human may be the most relevant to assess in your scRNA-seq chicken dataset.

Thank you for this suggestion. We now link the markers we use to those in mouse and chick.

5) Figures need more attention. Figure 2: A – instead of displaying all genenames (which are unreadable), consider to name examples of genes instead. B – Again, the text is unreadable, please make bigger C- The C0-12 index needs to be larger and please put in the numbering in the UMAP. Comments on Figure 5-6 are already provided above. Figure 7: Images are stretched in general. Provide larger numbering in A, and bigger index.

Thank you for bringing this to our attention. We have now improved the figures.

[Editors’ note: what follows is the authors’ response to the second round of review.]

The manuscript has been improved but there are some remaining issues that need to be addressed, as outlined below:

Reviewer #2 (Recommendations for the authors):

Several concerns have been dealt with. Outlined below are the remaining or new issues:

1) In light of Figures 4 and 5, I wonder which part of the peripheral nervous system that actually has been included in the single cell RNA-sequencing experiment. The validating figures indicate that most sacral neural crest derived cells are contained within the Nerves of Remak, which is not part of the ENS. Is also Nerves of Remak (and even sacral ganglia) included in the sequencing? In this case the whole comparison is not between different ENS populations, but rather between the vagal-derived hindgut and Nerve of Remak (potentially consisting of both cells that will stay in this region, and that will migrate into the gut) in essence.

We are grateful that the reviewer brought this to our attention as this raises an important point. The reviewer rightly points out that the Nerve of Remak is closely associated with the hindgut in the chick embryo. It is the staging ground for many sacral neural crest-derived cells to migrate to the gut. According to Burns and LeDouarin, sacral neural crest cells from the Nerve of Remak and pelvic plexus migrate into the hindgut along extrinsic axons and colonize the hindgut in large number by E10. Therefore, we did include both the Nerve of Remak and the caudal part of the gut which contains the pelvic plexus in our dissections.

We now clarify the dissection process in the Materials and methods and have amended the Figure description to make this more clear. We also include an image of the dissected gut for clarification in supplemental figure 2.

2) There are still a lot of assumptions of the functional neuron types based on neurotransmitters/receptors and other signaling pathways that are premature given the lack of basic characterization of the chicken neural composition. None of the below comments are corroborated in literature:

Thank you for pointing out the over-interpretations below. Accordingly, we have tried to remove all speculation and changed the sentences as described below:

"Differential gene expression analysis revealed intriguing distinctions between vagal and sacral neural crest cells in the post-umbilical gut at the population level. Genes enriched in the sacral population include SST1/SSTR indicating interneuron cell fate, and DBH, TH, DDC, PNMT, and SLC18A2 which are present in catecholaminergic neurons and serotonergic neurons. GRM3 expression indicates that glutamatergic character is more abundant in the sacral population. In addition, we observed up-regulation of GFRA3 which is involved in the GDNF signaling pathway and CXCL12 which is related to signaling during cell migration (Figure 1C, D). Conversely, the vagal post-umbilical population expresses the adrenergic receptor ADRA1B, enzyme GAD1, CALB2, and NTS consistent with excitatory motor neuron fate."

Changed to: "Differential gene expression analysis revealed intriguing distinctions between vagal and sacral neural crest cells in the post-umbilical gut at the population level. Genes enriched in the sacral population include SST1/SSTR, DBH, TH, DDC, PNMT, and SLC18A2. GRM3 expression is more abundant in the sacral population. In addition, we observed up-regulation of GFRA3, the receptor for artemin, and CXCL12 which is related to signaling during cell migration (Figure 1C, D).

Conversely, the vagal post-umbilical population expresses the adrenergic receptor ADRA1B, enzyme GAD1, CALB2, and NTS."

"Due to the expression of the secretomotor neuropeptide VIP and the tachykinin TAC1, we classified C2 as a motor neuron cluster that is predominantly derived from the vagal neural crest and is present in the pre-umbilical and post-umbilical gut (Figure 2C)."

Changed to: "C2 expresses the neuropeptide VIP and tachykinin (TAC1) and is predominantly vagal-derived with 81% of cells contributed from the post-umbilical population and 16% from the pre-umbilical."

"DBH, TH, DDC, PNMT, and SLC18A2 which are present in catecholaminergic neurons and serotonergic neurons"

Changed to: "Genes enriched in the sacral population include SST1/SSTR, DBH, TH, DDC, PNMT, and SLC18A2. GRM3 expression is more abundant in the sacral population, which may reflect a transient state of differentiation."

e) It is important to be aware of the large population of ENS cells in the intestine goes through a transient state where TH and to some extent DBH is expressed. It is thus unclear whether DBH and TH indeed are maintained in this neural population. If Nerve of Remak is also included in the sequencing these cells may represent sympathetic/parasympathetic neurons.

Thank you for this suggestion. We have now gone back over our data to associate clusters with transcription factors in addition to neurotransmitters (Supplemental Table 4). Of note, there is not much literature regarding the role of TFs in particular lineage allocation in the developing ENS with the exception of Pbx3

3) The authors have not confirmed that non-ENS cells indeed stem from the labelled cells (question in previous round). Given that the fluorescent is rather low, it would be to expect that contaminating cells are captured during FACS. It is generally not possible to completely isolate a single cell population based on rather weak reporters used from the gut, which contains a large number of auto-flourescent cells. Moreover, consider that the enteric neural progenitors have migrated through the entire gut to reach the colonic region due to their native ability to do so. It sounds unreasonable that also for instance fibroblasts and epithelial cells would take the effort to migrate that far. It is highly uncertain that most of the indicated non-ENS cells actually are stemming from the vagal/sacral neural crest. It should be discussed in the manuscript that non-ENS cells may be contaminating cells, but that it cannot be excluded that some may be true traced cells. If you still believe that they are traced cells, please show pictures indicating co-expression between your reporter and markers of the various non-ENS cells.

Point taken. In the revised version, we have erred on the side of caution as the reviewer suggests regarding the non-ENS cells, which indeed represent relatively minor populations. We tried to verify these with antibody markers but were unable to find antibodies (e.g. Mitf) that cross-reacts for immunohistochemistry in the chick. Therefore, we now state that although the non-ENS cell clusters were identified based on detectable RIA transcript levels, we cannot rule out the possibility that some may be contaminating cells captured due to autofluorescence.

4) Related to the question on whether Nerve of the Remak cells are included – if not included, and only ENS cells were captured, it also remains possible that the missing progenitor population resides within the pelvic ganglion. Sampling could lead to whether populations are included or not. Again, it will be important for the message and interpretation of data that readers understand which peripheral nervous system parts are included in the sequencing experiment.

We agree and it may be very interesting in the future to attempt to separately isolate and sequence cells from the Nerve of Remak and the Pelvic ganglion. We now clarify our dissection procedure which includes both of the Nerve of Remak and the caudal part of the gut.

5) As it takes several more days for sacral neural crest to reach the gut, and that may first differentiate somewhat (most likely undertaking a schwann-cell character) within the Nerve of Remak/Pelvic Ganglia, it is not surprising that the progenitor population express different genes in vagal versus sacral gut.

Perhaps but it’s interesting that the vagal pre- and vagal post- populations are quite different whereas the sacral and vagal post-umbilical populations resemble each other quite closely. This speaks to the importance of the local environment in differentiation of both populations of neural crest-derived cells, which is a point we now try to emphasize.

6) If Nerve of Remak is included, it is questionable how relevant the comparison is to ENS of humans and disease mechanisms of Hirschsprungs disease, as suggested in the discussion.

We have removed this discussion point.

7) Please try to compress the results part. As the same datasets are analysed several times by different means it gets a bit repetitive, especially with regards to the neural markers, which are mentioned first as being expressed together, and then more refined in the second analysis. It is enough to talk about them in the more refined analysis (also in light of the comments above, with less functional attributes).

Point taken. The results have been shortened accordingly.

Reviewer #3 (Recommendations for the authors):

Abstract l33-34 'suggesting an important role for environmental factors promoting

differentiation of both vagal and sacral neural crest-derived cells in the post-umbilical gut' I am not quite clear of the emphasis the authors intend here. It is surely the case that local environments play a role at some level in the exact choice of individual cells, but the overlapping fates from vagal and sacral crest might reflect shared environment in gut, or simply overlap in endogenous potential. I wonder if this phrase is necessary – maybe consider deleting?

The reviewer raises a good point and we have amended the abstract to be more circumspect.

I consider that a number of speculative conclusions are phrased more strongly than appropriate based on the evidence presented. These include:

L38 'apparently lacks such a cluster' is too strong – cells of cluster 0 are present in sacral cells, just population is proportionately fewer. Suggest replace with 'is depleted in this cluster at this stage'.

Changed to: “cells of cluster 0 are depleted in the sacral population at this stage'

And

L335 Can the authors distinguish developmental and timing differences? Could it be that potential is similar, but timing differences (perhaps driven by location differences, given that most sacral cells are found in Nerve of Remak?) explain the relative proportion of cells in the clusters, especially c0? Hence I suggest replacing 'and' with 'and/or'. Similarly, the conclusion in l409-413 may also be explained by the difference in location of most sacral cells. This possible caveat to these interpretations should be stated. Furthermore, the impact of this should be explored in the Discussion, as a caveat to any interpretations of the cell-types identified in each of the profiles.

This is an excellent point. We now add discussion of this possibility as suggested and are more circumspect about the differences in the progenitor population.

L160 The authors state 26993 cells score positively for the RIA transcripts, but they should clarify whether all of these fulfil their criterion of >200 genes expressed i.e. what are the total numbers of vagal and sacral NCCs included in analysis? They should also add information on the numbers of genes identified per cell, e.g. what is the mean number of genes identified per single cell. The MandM indicate that cells with only 200 genes detected were included in the analysis (l561-562), but it is important to understand what the mean and variance was. This has implications for distinguishing putative cell-types (clusters) of different potency, as discussed recently by the Kelsh lab (see Kelsh et al., 2022, Development; Subkhankulova et al., 2023 Nature Comms.). For example, Figure 3 shows that C6 and C8, both classified by authors as glial, show low, but detectable levels of MITF, a key melanocyte determinant; this implies these cells retain melanocyte potential, although the authors do not comment on this. However, the extent to which other cells retain melanocyte or other fate potential is highly dependent upon the depth of sequencing (number of genes detected per single cell).

Thank you for raising this point of confusion. We have now clarified in the material and methods that the RIA+ analyzed cells fulfill the criterion of >200 genes expressed. We have additionally updated the material and methods to reflect the total number of cells analyzed for each sample, alongside the mean and median of the counts and genes identified per cell.

Figure 4 and 5 All colours should be shown independently, as well as merged, to clarify overlaps.

Thank you for this suggestion which is now included. We have generated supplemental figures showing all channels separately.

Figure 5 shows that most sacral cells reside in Nerve of Remak. Differences in relative contribution of sacral v vagal crest are clear in Figure 2C, but only absolute differences seem to be in small cluster 12 (?mesenchymal stem cell). To what extent does this bias in location of cells explain the bias in fates adopted? Put another way, if you consider the relative abundance of sacral versus vagal cells in the hindgut and Ganglion of Remak, are the apparent differences in certain cell-types (clusters) explained by sampling error? Likewise, in

Thank you for raising this interesting point. To address differences in relative contributions of sacral versus vagal crest-derived cells in various clusters, we now include Supplemental Figures that report the proportion of sacral and vagal-derived cells across the clusters (Supplemental Figure 3) and neuronal subclusters (Supplemental Figure 8).

We have also clarified that the single cell sequencing dataset does include the Nerve of Remak and caudal hindgut which contains the pelvic plexus. Thus we do not believe there is a sampling error that would bias the overrepresentation of one cell type over another based on physical location in the gut.

Figure 6, where the neuronal populations are analysed in more detail, there are some differences, but absolute differences are in relatively minor populations. This makes me hesitant to conclude that there are substantial differences in potential between the two populations, although this seems in places to be the authors' conclusion (e.g. l335, l484-486). The authors should add a discussion of this point, and soften their conclusions accordingly.

We agree and have softened our conclusions regarding difference in developmental potential. It seems that spatial localization may be the most important determining factor for cell fate.

Figure 7 Authors should explain the meaning and significance of 'latent time' and 'absorption probabilities in panels B and C respectively.

Thank you for this suggestion. We have subsequently described in greater detail the meaning and significance of these terms in RNA velocity analysis.

[Editors’ note: what follows is the authors’ response to the third round of review.]

The manuscript has been improved but there are some remaining issues that need to be addressed, as outlined below:

Reviewer #2 (Recommendations for the authors):

The authors have been responsive to most concerns. However some remaining issues are listed below.

1) Introduction/Abstract: Trajectory analysis suggest that sacral neural crest has a predicted terminal fate of enteric glia.

What are the conclusions drawn from this? It likely means that neurons have already been generated and that progenitors only give rise to glia at E10 (which you allude to in the discussion). However, as it is phrased it sounds either like the conclusion is that all sacral cells eventually become glia (even the neurons), or that the sacral cells only become glia (which is not the case since you see neurons in the sacral population). Both the fact that you don't see the neurogenic population, and that the trajectory analysis points to glia indicate that at E10, sacral neurogenesis has ceased and progenitors that are present mainly give rise to glia. However, it is rather likely that this is the case for the Nerve of Remak, as the majority of cells that you capture from the sacral tracings are located here. An alternative conclusion could be that neurogenesis of sacral-derived ENS within the gut wall may occur later than E10 and have not yet started. A stringent comparison between cells that are only located in the post-umbilical gut wall that are either sacral or vagal in origin is evidently not performed. Please rephrase sections to make the message more clear on the glial destiny of sacral neural crest, as well as the comparison made in the current study being largely performed between NoR and postumbilical vagal NC. NoR may be equivalent to parts of ENS in other species, so the comparison is not irrelevant, but it needs to be more clearly explained so that no misunderstandings are created in the field.

Thank you for pointing out that this wording is confusing. Accordingly, we have modified the abstract and introduction to clarify our interpretation. We speculate that the sacral progenitors at this time point, many of which reside within the Nerve of Remak, have glial characteristics that may reflect an enteric glia/Schwann cell precursor state; we speculate that these cells can differentiate into either neuronal or glial cell types at later time points. This is consistent with a recent publication (Nat Commun 14, 5904 2023) suggesting that enteric glia may reflect a transitional cell state that retains neurogenic potential. However, other sacral neural crest-derived cells at E10 have already differentiated within the gut walls into neurons of the submucosal and myenteric plexuses (Figure 5E), some of which at are ACHR+ cells (Figure 5A”). Given these findings, we agree that it is likely that many sacral-derived enteric glia from the Nerve of Remak may invade the gut wall and differentiate at later time points. These points are further discussed in the introduction and discussion.

2) On the same matter. That sacral neural crest can give rise to glia cells in NoR is not contrary to any conception in the field as far as this reviewer can tell. Therefore it is not clear why so much emphasis is placed on this observation (for instance "Validation of sacral contribution to glial fates" as its own subsection and Figure). It would be sufficient to conclude that no or only a small neuroblast cluster is captured from sacral-derived cells at E10, which could indicate that neurogenesis has ceased already. Clear sacral-derived glial clusters are presented in Figure 2, and validation could be presented in association with this figure 2. Remember also that an alternative conclusion could be that neurogenesis of sacral-derived ENS within the gut wall may occur later than E10 and have not yet started.

Thank you for this comment, with which we agree. There is established literature demonstrating sacral crest contribution to glia of the Nerve of Remak. We have adjusted the text accordingly and included the possibility that the sacral-derived ENS within the gut wall may develop later than E10. Since the glial clusters were so prominent, we included Figure 8 to further validate glial marker expression. However, we would be happy to move Figure 8 as a supplemental figure to the in vivo validation of Figure 2 if the reviewer and/or senior editor would prefer that arrangement.

3) Row 262: These results confirm that a large proportion of the ENS in the colon and Nerve of Remak are derived from the sacral neural crest at E10.

Your data suggest that the cells in the NoR is generated from the sacral neural crest, however a very small proportion of the cells in the colon are generated from the sacral neural crest tracing. Please rephrase. Moreover, the sentence suggests that the ENS is located within the Nerve of Remak, is this nomenclature accepted?

Thank you for this comment. Accordingly, we have rephrased it as: “These results confirm that the sacral neural crest contributes both to a large portion of the Nerve of Remak as well as a subset of neurons in the colon at E10.”

4) Figure 7. RNA velocity analysis makes most sense if only cells are included that are clonally related. Thus, it is not clear why both sacral and vagal sources are included in a common analysis. Please perform individual analysis to understand if it is possible to see bridge populations between progenitor and neural populations (as you suggest there is no active neurogenesis) in the sacral-derived dataset.

Thank you for your suggestion. We have removed the combined analysis from Figure 7 and have further highlighted the analysis of individual populations.

5) On the issue of non-ENS cells being captured for sequencing, you have responded that while you cannot confidently confirm fluorescent non-ENS cells, the non-ENS cells have RIA transcripts. However for results presented in Figure 7 you state "In order to better identify potential differences in vagal and sacral contributions to neuronal and glial lineages within the E10 pre- and post-umbilical gut, we isolated cells from RIA+ neuronal (C2, C4), glial (C1, C6, C8), and progenitor clusters (C0, C7, C3) for RNA velocity analysis."

Does this means that the selected cells are the only cells with RIA, or you mean that you selected cells that are known to constitute ENS differentiation (progenitors, neurons and glia)? It would be helpful for this issue if you could present the average number of RIA for each cluster. This could also help guiding you in the conclusion whether the non-ENS cells are resulting from mere contamination (if the RIA is low or non-existing) or likely from true lineage-tracing (if the RIA is the same as the progenitor/neurons/glia). Note also that you in the results parts present the non-ENS cells as likely contaminants, while in the discussion it is more firmly concluded that the non-ENS cells are derived from the neural crest. Please be consistent in the message.

Thank you for pointing out that this description was still confusing. We now clarify in the text and methods that Figure 7 was generated only from RIA+ cells (Figure 2). To make this more clear, we have included a table (supplemental table 5) that provides the average number of RIA transcripts for each cluster. Since only cells with a minimum of 2 RIA transcripts were clustered and further analyzed in our study, we believe that these are truly lineage traced neural crest-derived cells. We have adjusted the text of the paper accordingly.

6) Discussion: "The enteric nervous system regulates critical gastrointestinal functions including digestion, hormone secretion and immune interactions."

Note that the role of the ENS in hormone secretion is not known in the gut. What you mean is probably luminal secretion from the epithelia. This is a fluid secretion, not hormones (at least this is what there is evidence for today)

Thank you for clarifying. We have adjusted the text accordingly.

7) Discussion: "Studies have suggested a critical role for the vagal neural crest development and disorders"

What does the sentence mean?

Apologies that this sentence was unclear. We meant to say that the majority of papers on ENS developmental disorders have focused on the role of the vagal neural crest and did not distinguish between potentially unique contributions from either vagal or sacral neural crest.

8) Figure 8: Glia in the Nerve of Remak is called "enteric glia" – is this correct, or should they be called schwann cells?

Thank you for this comment. Given the markers used to identify these cells, we do not believe we can distinguish between a glial or Schwann cell fate. Therefore we have now refer to them as “enteric glia/Schwann cell precursors”. According to Pachnis and colleagues (Nat Commun 14: 5904, 2023) these “enteric glia” may represent a transitional cell state that retains neurogenic potential.

9) It would be advisable to review the figure legends. For instance the Figure 2 title does not reflect the contents and the description of Figure 3A is also not very well aligned with the contents.

Thank you for highlighting this concern. We have updated the figure legends accordingly.

10) Discussion: "Both species have Pmp22/Frzb/Cdh19+ glial clusters (C1/6/8), but only the embryonic chick glial clusters expressed Plp1 indicating potential species or stage (embryonic versus adult) differences."

It is not clear what study is referred to and if it is true that Plp1 is not expressed in the murine clusters. At least the juvenile scRNA-seq characterisation of enteric glia in mouse (Zeisel et al., 2018) does show massive expression of Plp1.

Thank you for pointing out this mistake. We have corrected the sentence to reflect the presence of Plp1+ glia in mice (Drokhlyansky et al., 2020).

11) Discussion Row 479: "Compared with these studies done with postnatal and adult tissue, we observed more 479 clusters with progenitor/precursor identity (Figure 2A, Fig7A), which is not unexpected, given that our analysis utilizes the gut from embryonic stages."

Yes, perhaps not worthwhile mentioning.

We appreciate this suggestion and have removed the sentence.

12) In general the manuscript would win from a more concise writing, please go through and omit unnecessary information and lengthy description of clusters.

Thank you for this suggestion. We have condensed the paper accordingly. Reviewer #3 (Recommendations for the authors):

The authors have addressed in full all points I raised. In particular, the Discussion is now much-improved.

However, I think there is one place where the discussion still over-reaches slightly. At l427 the authors state 'RNA velocity analysis demonstrates that the vagal neural crest maintains a glial/neuronal bipotent progenitor pool while the sacral neural crest does not at E10. This may indicate potential differences in developmental potential, timing, and/or proliferative capacity.'

I think the description of Cluster C0 as a 'glial/neuronal bipotent progenitor pool' is flawed because the RNA velocity analysis only assessed (l308-9) 'neuronal (C2, C4), 309 glial (C1, C6, C8), and progenitor clusters (C0, C7, C3)'; thus, the analysis was restricted to identifying links to neuronal and glia fates only. Furthermore, the depth of sequencing (average <3000 genes/cell) is insufficient to show anything other than strong biases in differentiation state. Hence I would remove the word 'bipotent' which implies that the cells are partially-restricted, when that is simply not assessable from the data provided.

Thank you for this suggestion. We have condensed the paper accordingly.

I think there is one key way in which the paper can be made more accessible to the reader. The Figureswith cluster diagrams are difficult for the reader to interpret due to a lack of a key to the clusters, giving their identifying number and their interpretation.

Thank you for the insight. We have updated the cluster diagrams to include the interpretation of each cluster.

A simple key repeated in each figure would solve this.

I noticed a couple of residual typos, e.g. l283 'interneruons', which should be carefully sort and corrected in Word.

Thank you for the suggestion. We have corrected the typos.

In Discussion, I don't think this sentence makes sense: 'Studies have suggested a critical role for the vagal neural crest development and disorders11,18.'

Thank for your suggestion. This sentence has been expanded to emphasize the point that a majority of papers on ENS developmental disorders have focused on the role of the vagal neural crest in ENS development or have been unable to parse out the potentially unique contributions of the vagal versus sacral neural crest.

https://doi.org/10.7554/eLife.79156.sa2

Article and author information

Author details

  1. Jessica Jacobs-Li

    Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, United States
    Contribution
    Data curation, Formal analysis, Validation, Writing - review and editing
    Contributed equally with
    Weiyi Tang
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-1339-3555
  2. Weiyi Tang

    Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, United States
    Contribution
    Conceptualization, Data curation, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing - original draft, Writing - review and editing
    Contributed equally with
    Jessica Jacobs-Li
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-1279-1001
  3. Can Li

    Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, United States
    Contribution
    Formal analysis, Methodology
    Competing interests
    No competing interests declared
  4. Marianne E Bronner

    Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, United States
    Contribution
    Conceptualization, Resources, Funding acquisition, Writing - original draft, Project administration, Writing - review and editing
    For correspondence
    mbronner@caltech.edu
    Competing interests
    Senior editor, eLife
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-4274-1862

Funding

National Institute of Diabetes and Digestive and Kidney Diseases (R01DK13348)

  • Marianne E Bronner

Eunice Kennedy Shriver National Institute of Child Health and Human Development (F31HD11128)

  • Jessica Jacobs-Li

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Acknowledgements

This work was supported by 1R01DK133480 to MEB and F31 HD111287 to JLL We thank Drs. Igor Antoshechkin and Vijaya Kumar and the Millard and Muriel Jacobs Genetics and Genomics Laboratory at California Institute of Technology for their guidance and support in bulk RNA-sequencing. We thank Jamie Tijerina and Rochelle Diamond from the Beckman Institute Flow Cytometry Facility for their help with the FACS. We thank Dr. Sisi Chen, Jeff Park, Prof. Matt Thomson, and SPEC at Caltech for their dedicated support in optimization and guidance in single-cell RNA-sequencing. We thank Dr. Fan Gao and Bioinformatics Resource Center in the Beckman Institute at Caltech for guiding us through single-cell transcriptomic analysis. We appreciate the help from Prof. Carlos Lois for kindly sharing equipment with us to perform RIA concentration. We thank Dr. Michael L Piacentino, Dr. Erica J Hutchins, and Prof. Angelike Stathopoulos for the helpful discussion on the manuscript.

Senior and Reviewing Editor

  1. Kathryn Song Eng Cheah, University of Hong Kong, Hong Kong

Reviewer

  1. Ulrika Marklund, Karolinska Institutet, Sweden

Version history

  1. Received: April 1, 2022
  2. Preprint posted: May 9, 2022 (view preprint)
  3. Accepted: October 23, 2023
  4. Accepted Manuscript published: October 25, 2023 (version 1)
  5. Version of Record published: November 6, 2023 (version 2)

Copyright

© 2023, Jacobs-Li, Tang et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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  1. Jessica Jacobs-Li
  2. Weiyi Tang
  3. Can Li
  4. Marianne E Bronner
(2023)
Single-cell profiling coupled with lineage analysis reveals vagal and sacral neural crest contributions to the developing enteric nervous system
eLife 12:e79156.
https://doi.org/10.7554/eLife.79156

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