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).

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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.

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.

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

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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

   "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

   "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

   "This ORCID iD identifies the author of this article:" 0000-0003-4274-1862

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