Neural tube-associated boundary caps are a major source of mural cells in the skin

  1. Gaspard Gerschenfeld
  2. Fanny Coulpier
  3. Aurélie Gresset
  4. Pernelle Pulh
  5. Bastien Job
  6. Thomas Topilko
  7. Julie Siegenthaler
  8. Maria Eleni Kastriti
  9. Isabelle Brunet
  10. Patrick Charnay
  11. Piotr Topilko  Is a corresponding author
  1. Institut de Biologie de l'Ecole normale supérieure (IBENS), Ecole normale supérieure, CNRS, Inserm, Université PSL, France
  2. Sorbonne Université, Collège Doctoral, France
  3. nstitut Mondor de Recherche Biomédicale, Inserm U955-Team 9, France
  4. Genomic facility, Ecole normale supérieure, PSL Research University, CNRS, Inserm, Institut de Biologie de l'Ecole normale supérieure (IBENS), France
  5. Inserm US23, AMMICA, Institut Gustave Roussy, France
  6. Laboratoire de Plasticité Structurale, Sorbonne Université, ICM Institut du Cerveau et de la Moelle Epinière, Inserm U1127, CNRS UMR7225, France
  7. Department of Pediatrics Section of Developmental Biology, University of Colorado Anschutz Medical Campus, United States
  8. Department of Physiology and Pharmacology, Karolinska Institutet, Sweden
  9. Department of Neuroimmunology, Center for Brain Research, Medical University Vienna, Austria
  10. Inserm U1050, Centre Interdisciplinaire de Recherche en Biologie (CIRB), Collège de France, France

Abstract

In addition to their roles in protecting nerves and increasing conduction velocity, peripheral glia plays key functions in blood vessel development by secreting molecules governing arteries alignment and maturation with nerves. Here, we show in mice that a specific, nerve-attached cell population, derived from boundary caps (BCs), constitutes a major source of mural cells for the developing skin vasculature. Using Cre-based reporter cell tracing and single-cell transcriptomics, we show that BC derivatives migrate into the skin along the nerves, detach from them, and differentiate into pericytes and vascular smooth muscle cells. Genetic ablation of this population affects the organization of the skin vascular network. Our results reveal the heterogeneity and extended potential of the BC population in mice, which gives rise to mural cells, in addition to previously described neurons, Schwann cells, and melanocytes. Finally, our results suggest that mural specification of BC derivatives takes place before their migration along nerves to the mouse skin.

Editor's evaluation

The authors show that Krox20 positive boundary cap cells travel along the nerves to the dermis and become incorporated into the vascular plexus to form dermal mural cells, confirmed by a cluster in single cell RNA sequencing. This provides the first evidence of a boundary cap contribution to a portion of mural cells in the trunk dermis.

https://doi.org/10.7554/eLife.69413.sa0

Introduction

Blood vessels and nerves are branched structures that are established in parallel during development to supply almost every organ in the body. Their patterning is achieved through the coordinated action of a common set of factors (Carmeliet and Tessier-Lavigne, 2005). Indeed, recent studies have shown that in addition to using similar signals to differentiate, grow, and navigate, the vascular and nervous systems maintain a cross-talk that plays a key role in their mutual development. For instance, sympathetic nerves follow specific guidance cues produced by the vasculature to reach their appropriate targets (Glebova and Ginty, 2005). Reciprocally, studies by Mukouyama and colleagues have demonstrated that nerves and Schwann cell precursors (SCPs) play a decisive role in the development of the vascular network, as they provide essential diffusible factors, drive the alignment of blood vessels with nerves, and promote arteriogenesis (Li et al., 2013; Mukouyama et al., 2005; Mukouyama et al., 2002). Using Neurogenin mutant mice, which lack cutaneous nerves, they showed that both blood vessel patterning and arterial differentiation are prevented (Mukouyama et al., 2002). Furthermore, using in vitro and in vivo approaches, they demonstrated that two proteins secreted by cutaneous nerves, CXCL12 and VEGF-A, act as key factors to trigger the remodeling and nerve alignment of cutaneous blood vessels and arterial differentiation, respectively (Li et al., 2013; Mukouyama et al., 2005).

Over the last decade, numerous studies have revealed an unexpected plasticity of SCPs, which give rise, apart from a plethora of glial derivatives, to skin melanocytes, adrenal medulla chromaffin cells, tooth mesenchymal stem cells, osteocytes, chondrocytes, and enteric and parasympathetic neurons (Adameyko et al., 2009; Dyachuk et al., 2014; Espinosa-Medina et al., 2014; Furlan et al., 2017; Kaucka et al., 2016; Uesaka et al., 2015; Xie et al., 2019). However, a cellular contribution from SPCs to the endothelial or mural components of the vascular plexus has not been reported so far. Mural cells (MCs) include pericytes and vascular smooth muscle cells (vSMCs) that cover capillaries and larger vessels, respectively, and participate in blood vessel remodeling and stabilization (Holm et al., 2018). MCs have been shown to have multiple embryonic origins, depending on their maturation location: hence, facial and thymic MCs derive from the neural crest (NC), whereas gut, lung, and liver MCs are mesoderm-derived (Asahina et al., 2011; Etchevers et al., 2001; Que et al., 2008; Wilm et al., 2005). In the skin, although an MC subset has been recently reported to originate from myeloid progenitors, the major source(s) of MCs remain(s) to be unraveled (Yamazaki et al., 2017).

Boundary cap (BC) cells form transient aggregates during embryogenesis at the central/peripheral nervous system (CNS/PNS) interface, at the levels of dorsal entry and ventral exit points of all cranial and spinal nerves. Most BC cells express the genes Egr2 (also known as Krox20) and/or Prss56 that have been used as molecular markers in functional and tracing studies (Coulpier et al., 2009; Niederländer and Lumsden, 1996). Fate mapping experiments using a murine Prss56Cre allele have recently revealed that Prss56-positive BC cell derivatives migrate between embryonic day (E) 11.5 and E13.5 along nerve roots into dorsal root ganglia (DRGs), where they give rise to Schwann cells (SCs) and a subpopulation of sensory neurons, and along spinal nerves to reach the skin, where they differentiate into SCs, terminal glia, and melanocytes (Gresset et al., 2015; Radomska et al., 2019). Tracing experiments performed with an Egr2Cre allele led to similar conclusions concerning nerve roots and DRGs (Maro et al., 2004). However, in this case, peripheral migration was not explored. Here, we show that Egr2-positive BC cell derivatives migrate, during the same time period, along spinal nerves to reach the skin. In the skin, most of them detach from nerves, integrate the vascular plexus, and provide the major source of MCs to the adult skin vasculature.

Results

Derivatives of Egr2-positive BC cells join the skin vascular plexus after migration along the nerves

To investigate the contribution of Egr2-positive BC cells to the developing trunk skin, we crossed mice carrying Egr2Cre and Rosa26RTom alleles and traced derivatives, on the basis of tdTomato (Tom) expression in Egr2Cre/+,Rosa26RTom embryos. As previously reported, we found numerous Tom-positive cells in the dorsal and ventral roots at E11.5 (Figure 1A), as well as in the DRGs at E12.5 (Figure 1B). In addition, traced cells were observed in the proximal spinal nerve segments at E11.5 (Figure 1A and D), in the intermediate segments of dorsal and ventral rami at E12.5 (Figure 1B and E) and E13.5 (Figure 1C), and finally at dorsal and ventral skin nerve terminals from E13.5 (Figure 1C and F). This dynamic time-course of proximo-distal distributions strongly suggests a migration of derivatives of Egr2-positive BC cells along nerves. We also noticed that some of the traced cells were not in contact with nerves, near the ventral roots at E11.5–12.5 (Figure 1A and B, arrows) and later in the skin (Figure 1C and F, arrows). To ensure that all traced cells originate from Krox-20-positive BC cells, i.e., that Tom expression does not result from de novo activation of Egr2 in other cells populations, we searched for Egr2 expression at E12.5 by in situ hybridization on whole embryos (Figure 1—figure supplement 1A), RT-PCR analysis of total RNA extracted skin (Figure 1—figure supplement 1B), and RNAscope on whole embryos (Figure 1—figure supplement 1C–J). These experiments did not reveal the presence of Egr2 mRNA apart from BC cells. Furthermore, single-cell transcriptomic analysis of Tom-positive skin cells in E12.5 Egr2Cre/+,Rosa26RTom embryos did not reveal any expression of Cre (see below). Together these data indicate the absence of de novo Egr2 expression in traced cells outside of the BC. In conclusion, these analyses establish that derivatives from Egr2-expressing BC cells migrate along spinal nerves into the skin, and that some of them detach from the nerves during their journey.

Figure 1 with 1 supplement see all
Boundary cap (BC) cell derivatives migrate along the nerves and incorporate into the vascular plexus.

(A–F) Trunk transverse sections from Egr2Cre/+,Rosa26RTom embryos at the indicated stages, and labeled with antibodies against Tomato (magenta) and Tuj1 (orange). Cells detached from the nerves (arrows) appear first in the vicinity of the ventral roots (A,B) and later close to the skin, as indicated by the dotted line (C,F). (G–J) Whole-mount dorsal skin from Egr2Cre/+,Rosa26RTom embryos at the indicated stages, labeled with antibodies against Tomato (magenta), Tuj1 (orange), and PECAM (blue). (K,L) Higher magnifications showing cells in contact with both nerve and vessel at embryonic day (E) 13.5. (M) Quantification of the number of labeled cells associated with nerve or vessel per field at the indicated stages (n=3 embryos per stage). Statistical analyses of the “on nerve”/“on vessels” ratio between time points were carried out using a Mann-Whitney U test. Scale bars, 100 μm (A–C,G–J) and 20 μm (D–F,K–L). Error bars, one standard deviation (M). ***=p < 0.001.

To further characterize derivatives of Egr2-positive BC cell in the skin, we performed whole-mount immunohistochemistry on embryonic skin between E12.5 and E15.5, staining for axons (Tuj1), blood vessels (PECAM), and traced cells (Tom). Whereas 89.9% (SD 6.5%) of the traced cells were in contact with nerves at E12.5, this proportion rapidly diminished to reach 9.4% (SD 6.3%) at E15.5 (Figure 1G–J and M). Conversely, traced cells were found in contact with blood vessels in increasing proportions, from 10.1% (SD 6.4%) at E12.5 up to 90.6% (SD 6.3%) at E15.5 (Figure 1G–J and L). Traced cells in contact with capillaries and larger vessels did not express the endothelial marker PECAM, raising the possibility that they might have acquired an MC identity (Figure 1H–K). Finally, a few traced cells were observed straddling both nerves and vessels (Figure 1K and L). Together these data suggest that most traced cells, after having traveled from the BCs to the skin along nerves, detach from nerves and are recruited within the vascular plexus. Their behavior dramatically differs from that of derivatives of Prss56-positive BC cells (Figure 1—figure supplement 1K–M and N–P), which migrate along the same nerves over the same time period, but remain associated with nerves in their vast majority (Gresset et al., 2015).

To investigate a possible role of Egr2 in the behavior of derivatives of Egr2-positive cells, we generated and analyzed Egr2Cre/Cre,Rosa26RTom mutant embryos, in which the tracing system is combined with the inactivation of Egr2. We did not observe any obvious difference in the pattern and number of Tom-positive cells in contact with blood vessels at E14.5, as compared to Egr2Cre/+,Rosa26RTom control embryos (Figure 1—figure supplement 1K–M and Q–S). These latter data do not support a role of Egr2 in the migration of BC cell derivatives and in their contribution to the vascular plexus.

In the skin vasculature, BC cell derivatives express perivascular markers and adopt mural characteristics

To characterize the derivatives of Egr2-positive BC cells in the skin vasculature, we performed immunolabeling analyses using a panel of MC markers. We found that at E14.5, traced cells associated with the vascular network showed characteristic morphologies compatible with pericyte or vSMC identities, as they covered small capillaries or wrapped around larger vessels, both arteries and veins (Figure 2A–D). Consistently, these cells expressed several MC markers, such as ABCC9, NG2, PDGFRβ, and smooth muscle actin (SMA) (Figure 2A–D). Quantification studies of traced cells in E15.5 skin whole-mounts and in postnatal day 1 (P1) skin sections revealed that they represented respectively 68% (SD 4.6%) and 66% (SD 8.1%) of the NG2-positive MCs (Figure 2E–G). Whole-mount analysis of newborn and P30 dorsal skin indicated the persistence of numerous BC cell-derived MCs on capillaries, arteries, and veins (Figure 2—figure supplement 1A–E).

Figure 2 with 2 supplements see all
Boundary cap (BC) cell derivatives give rise to the majority of skin mural cells (MCs).

Whole-mount embryonic dorsal skin at the indicated stages (A–E) or transverse section of newborn skin (F), labeled with antibodies against Tomato (magenta), PECAM (blue), and ABCC9, NG2, PDGFRβ, or smooth muscle actin (SMA) (yellow). (A–D) In the vascular plexus, Tomato-positive cells express MC markers: ABCC9 (A), NG2 (B), PDGFRβ (C), and SMA (D). (E–G) Tomato-positive cells account for approximately two thirds of NG2-positive MCs in the skin at embryonic day (E) 15.5 (E,G ; n=3)and postnatal day (P) 1 (F,G; n=3). Tomato labeling is omitted in the lower images (A–F). Statistical analysis of the “NG2 and Tomato-positive”/“NG2-positive” ratio between time points was carried out using a Mann-Whitney U test. Scale bars, 20 μm. ns, non-significant (G).

Overall, these data indicate that, in the skin, derivatives of Egr2-positive BCs detach from nerves, adopt mural characteristics, and constitute the major part of the embryonic and newborn MC component. Furthermore, this BC cell-derived MC population is maintained up to P30 at least.

A subpopulation of Egr2-expressing BC cells acquires mesenchymal identity before their emigration to the periphery

The immunolabeling analysis presented above revealed that derivatives of Egr2-expressing BC cells migrate along peripheral nerves, detach from them, and contribute to the major part of pericytes and vSMCs in the skin. Notably, this population differs from Prss56-expressing BC cells, which never give rise to such derivatives. Two main hypotheses can be proposed to explain this process. First, a subpopulation of Egr2-expressing BC cells is already specified as MPs before migration to the skin and their derivatives detach from nerves and differentiate into MCs once reaching their target. Second, Egr2-expressing BC cell-derived glial progenitors migrate along nerves to the skin and undergo a glial-to-vascular transition that allows them to detach from nerves and mature into MCs. In both scenarios, nerve detachment would likely be dictated by the microenvironment through the influence of locally secreted factors.

To further explore the identity of Egr2-expressing BC cells and their derivatives, we first analyzed E12.5 ventral nerve roots to assess whether all Tom-positive cells were expressing the glial marker Sox10, at the protein or mRNA levels (Figure 3A–C). We observed a majority of cells positive for both tomato and the Sox10, protein, but also some nerve-attached Tom-positive/Sox10-negative cells (Figure 3A–C). Similar results were obtained by RNAscope, combining Tomato and Sox10 probes (Figure 3D–F). However, since Sox10 activation occurs progressively in early-stage glial progenitors, we could not exclude that Tom-positive/Sox10-negative cells could correspond to early glial progenitors that have not yet activated this marker. This led us to repeat this analysis on skin nerve endings at E12.5 (Figure 3G and H). Similarly, we found among nerve-attached Tom-positive cells a majority of Sox10-positive cells, but also some Sox10-negative cells. An additional argument in favor of an early commitment of some ventral root cells toward a Sox-10-negative fate came from the analysis of the dorsal root (data not shown). In this case, we never observed Tom-positive/Sox10-negative cells. This is consistent with the fact that Tom-positive cells do not detach from the dorsal root.

An Egr2-expressing boundary cap (BC) cell subpopulation displays mesenchymal identity before migrating to the periphery.

Trunk transverse sections (A–F, I–N) and skin sections (G,H) from Egr2Cre/+,Rosa26RTom embryos at embryonic day (E) 12.5. Sections were labeled with antibodies (A–C) against Tomato (magenta) and Sox10 (yellow), or analyzed by RNAscope in situ hybridization (D–H) for Tomato (magenta) and Sox10 (yellow). Detailed analysis revealed that some Tomato-positive cells on ventral roots did not express Sox10 at the protein (arrows) and RNA level (arrowheads). RNAscope in situ hybridization of Tbx18 (I,J), Col1a1 (K,L), and Pdgfra (M,N) expression in ventral roots from E12.5 embryos. Note that some tomato-positive cells on ventral roots express these markers (empty arrowheads). Scale bars, 25 μm (A,D,G,I,K,M) or 10 μm (B,C,E,F,H,J,L,N).

To determine whether the presence of Sox10-negative cells would be accompanied by the expression of MP markers on ventral nerve roots, we used RNAscope to assess the expression of Tbx18, Col1a1, and Pdgfra. As triple RNA labeling was technically challenging, we could only perform double labeling, with Tomato and one marker at a time. We found that each of these three markers was co-expressed with Tomato in some cells (Figure 3I–N).

In conclusion, in the ventral root, these findings support the existence of cells of mesenchymal identity derived from Egr2-expressing BC cells. These derivatives further migrate along the nerves to reach the skin, then detach and differentiate into MCs. In the dorsal root, such cells would not exist, consistent with the absence of cell detachment from the nerve.

Ventral BCs are comprised of cells from distinct embryonic origins

Although fate mapping studies performed in chick and mice point to the NC as the population at the origin of dorsal root BC cells, the situation appears more complex in the case of the ventral root population (George et al., 2007; Kucenas et al., 2008; Yaneza et al., 2002). Moreover, only cranial NC was shown to give rise to MCs in the head vasculature and similar potential for thoracic NC was never reported, despite efforts from many teams. To identify the embryonic origin of ventral root the Egr2-expressing BC cells that give rise to skin MCs, we performed genetic fate mapping analyses using Cre drivers targeting the NC (Wnt1Cre, Sox10Cre-Ert2) and the ventral neural tube (Olig2Cre), which are in the vicinity of BCs, in combination with Rosa26RTom or Rosa26RYFP Cre-inducible reporters. In the case of Sox10Cre-Ert2, tamoxifen induction was performed at E9.5, which corresponds to the period of NC delamination. Embryos carrying the different Cre drivers were collected between E12.5 and E14.5 and analyzed by immunohistochemistry. In all cases, we were not able to identify any traced MCs, either near ventral roots or in the skin (Figure 4A–F), in accordance with previous studies that have shown that the NC and neural tube do not contribute to the trunk vasculature (Etchevers et al., 2019). We noticed that a subpopulation of cells derived from BC cells co-express Tbx18 in the ventral root (Figure 3G–L). Furthermore, at E9.5, we detected Tbx18 expression in the vicinity of the neural tube, in a layer of MPs that gives rise to fibroblasts in the meninges (Figure 4—figure supplement 1), consistent with a previous report (DeSisto et al., 2020). We therefore performed a genetic fate mapping of Tbx18 lineage using a Tbx18 Cre driver line (TbxCre-ERT2). Tamoxifen was administrated to pregnant females at E9.5 and embryos were analyzed at E11.5 and E14.5. At E11.5, we observed Tom-positive/Sox10-negative cells lining the ventral root and the spinal nerve with some of them closely attached to nerves (Figure 4G–L). At E14.5, we observed numerous Tom-positive cells along blood vessels co-expressing NG2 or/and SMA (Figure 4—figure supplement 2A–F, J–L). None of them express Sox10 (Figure 4—figure supplement 2G–I, M–O).

Figure 4 with 2 supplements see all
Ventral boundary caps (BCs) have a dual embryonic origin.

Trunk transverse sections (A,C,E–L), dorsal skin whole-mount (B), and skin section (D) at the indicated embryonic stages. Genetic fate mapping of neural crest (NC) using Wnt1Cre/+,Rosa26RTom (A,B) and Sox10Cre-Ert2/+,Rosa26RTom (C,D) embryos and of neuroepithelial cells and their derivatives using Olig2Cre/+,Rosa26RTom embryos (E,F) did not reveal any tomato-positive cells attached to the vascular plexus, neither close to ventral roots nor to nerves in the skin. (G,L) Genetic fate mapping of developing pia matter using Tbx18Cre-Ert2/+,Rosa26RTom embryos revealed the presence of numerous tomato-positive, Sox10-negative cells in close contact with the nerve (asterisks, I–L). Tamoxifen was delivered to pregnant females at embryonic day (E) 9.5 (C,D,G–L). Scale bars, 50 μm.

Together our observations suggest a dual embryonic origin of ventral root BCs expressing Egr2: part of this population originate from the NC, while another may originate from the pial cells. These latter population, once in contact with the ventral root, activate Egr2, do not express Sox10, and then undergo migration along the nerves to the skin and maturate into perivascular derivatives.

BCs are comprised of cells with distinct glial and mesenchymal signatures

The above analyses support the existence of a subpopulation of Egr2-expressing BC cells already specified as MPs, before migrating into the skin. To further characterize the molecular identity and the fate of their derivatives, we performed a single-cell transcriptomic analysis (single-cell RNA sequencing [scRNA-seq]) of traced Egr2-positive cells. As the quantification of traced cells in contact with nerves or blood vessels indicated that detachment was initiated at E12.5 (Figure 1M), we focused on this stage, on Tom-positive cells isolated by fluorescence-activated cell sorting (FACS) from dissociated skin. Using a customized pipeline (Materials and methods), we were able to analyze 2209 single-cell transcriptomes with a mean number of expressed genes per cell of 5424 (SD 1491). As indicated above, to ensure that tomato expression was not due to ectopic Cre expression, we inserted the Cre sequence in our scRNA-seq analysis. We did not observe any Cre expression. Using the Seurat software (Stuart et al., 2019), we identified 10-cell clusters, based on their expression patterns (Figure 5A). To determine their molecular identities, we overlaid plotted cells with levels of expression of well-characterized marker genes (Figure 5B–D). This approach allowed us to regroup the initial clusters into three main cell type-defined supra-clusters: an SCP supra-cluster, a mesenchymal progenitor (MP) supra-cluster, and a smaller, isolated MC cluster (Figure 5A). The SCP supra-cluster contained 1031 cells (47%) that expressed high levels of SCP markers, such as Sox10, Erbb3, Fabp7, Plp1, Ngfr, and Cdh19 (Figure 5B). The MP supra-cluster contained 1056 cells (48%) that expressed a combination of mesenchymal markers, such as Pdgfra, Pdgfrb, Dlk1, Tbx18, Igf1, and Col1a1 (Figure 5C). Interestingly, whereas most traced cells (90%) were in contact with nerves at E12.5 (Figure 1G and M), 54% of them showed either low or undetectable levels of expression of glial markers, such as Sox10, and significant expression of mesenchymal markers (Figure 4B–C). This finding is in line with our previous observation that a subpopulation of Tom-positive cells lining the nerves do not express Sox10 (Figure 3A–D’). Finally, the isolated MC cluster contained 122 cells (6%) that expressed high levels of Pdgfrβ, Abcc9, Acta2, Cspg4, and Rgs5 (Figure 5C and D), which collectively constitute a signature for pericytes and vSMCs. This percentage is similar to the proportion of traced cells attached to blood vessels at E12.5 (Figure 1M). Therefore, the latter cells may have achieved their maturation into MCs.

Figure 5 with 1 supplement see all
Numerous molecular features reveal diversity in cell identity in embryonic day (E) 12.5 boundary cap (BC) derivatives.

(A–D) 10 clusters can be delineated on the basis of single-cell RNA sequencing (scRNA-seq) experiments performed on traced cells from E12.5 dorsal skin. They can be organized in three supra-clusters based on their molecular signatures: Schwann cell precursors, mesenchymal progenitors, and mural cells. In (B–D), cells are color-coded, with color intensities (from gray to red) reflecting the relative levels of expression of each gene.

Further analyses using additional markers revealed a significant heterogeneity within the supra-clusters, in line with the identification of the 12 original clusters (Figure 5—figure supplement 1A–C). Hence, the SCP supra-cluster regroups four clusters (SCP1–4) that differ in the expression of SCP markers, such as Gap43, Pou3f1, and Pax3 (Figure 5—figure supplement 1B). Similarly, the MP supra-cluster regroups five clusters: four of them (MP1–4) differentially express some MC progenitor genes, including Tbx18, Pax9, Kcnj8, or Acta2 (Figure 5C and D and Figure 5—figure supplement 1C), whereas MP5 displays a transcriptomic signature of chondroprogenitors with markers such as Sox9, Dlx5, Col2a1, and Fgfr2 (Figure 5—figure supplement 1D). These results suggest that in addition to mural progenitors, Egr2-positive BC cells may contribute to chondroprogenitors in the trunk.

In conclusion, the single-cell RNA analysis corroborates and extends the immunolabeling data presented above, and establish that Egr2-positive BC cells consist of two major subpopulations: a first one which evolves into SCP and is likely to derive from the NC, and a second one, which gives rise to derivatives with an MC identity and may originate from the mesenchymal precursors of meningeal fibroblasts.

Derivatives of Egr2-positive BC cells contribute to the adult vasculature of several serous membranes

As indicated above, in Egr2Cre/+,Rosa26RTom embryos, we observed Tom-positive cells without nerve contact, near the ventral roots at E11.5 and at the level of the developing pulmonary artery at E12.5 (Figure 1A and B, arrows). These findings suggested a broader implication of BC cells in trunk vasculature development, and led us to search more systematically for Tom-positive MCs. We performed a detailed study of two adult Egr2Cre/+,Rosa26RTom mice and analyzed various organs (thyroid gland, lungs, heart, esophagus, stomach, intestines, liver, spleen, and kidneys) and serous membranes (pre-tracheal fasciae, pericardium, and peritoneum). Whereas we did not observe traced MCs in these organs, we found numerous Tom-positive MCs in the serous membranes that ensheath some of them, as revealed by co-labeling with PECAM of NG2 (Figure 2—figure supplement 2A–C). As described in the skin, Tom-positive MCs do not form a continuum and are separated by non-traced MCs, suggesting at least another embryonic origin for MCs in serous membranes. While these latter data demonstrate that serous membrane MCs derive from Egr2-expressing cells, in the absence of additional data on earlier Egr2 expression, it remains to be established whether they actually originate from BC cells. Nevertheless, the latter hypothesis is attractive, considering the precedent of the skin and the presence of such cells at the level of the pulmonary artery at E12.5. In this situation, BC cells would provide a widespread contribution to the adult vasculature.

BC cell ablation results in a depletion in skin MCs

As about two thirds of newborn skin MCs originate from Egr2-expressing BC cells (Figure 3F and G), we questioned the functional importance of this population in blood vessel development. To address this issue, we performed a genetic ablation of Egr2-expressing BC cells. In Egr2Cre/GFP(DT),Rosa26RTom embryos, Cre-inducible diphtheria toxin (DT) expression is restricted to Egr2-expressing cells, including BC cells, leading to their rapid elimination (Vermeren et al., 2003). We noticed that genetic ablation of Egr2-expressing cells induced embryonic lethality at around E15.5, as we were unable to obtain mutant embryos beyond that stage, and we often observed sites of necrosis in the uterus when collecting embryos at E15.5. At E15.5, among the 60 collected embryos, issued from Egr2Cre/+,Rosa26RTom × Egr2GFP(DT) breeding, 10 were Egr2Cre/GFP(DT),Rosa26RTom based on PCR genotyping. We analyzed these embryos for tomato fluorescence using a stereomicroscope. Unexpectedly, we observed traced cells in the skin in six of these embryos, suggesting that BC cell ablation was not complete. The four others displayed a complete loss of traced cells and were used for further analyses of the vascular network. Skin whole-mount immunohistochemistry for blood vessels (PECAM) and MCs (NG2) were performed on these latter mutants and three littermate controls (Egr2Cre/+,Rosa26RTom) embryos (Figure 6A–F). The skin vascular network appeared grossly similar in both types of embryos, with a dense network of arterioles, venules, and capillaries (Figure 6A–C, E, and G). However, when we compared the fraction of the vascular area covered by NG2-positive MCs (Figure 6D, F, and H), we observed a significant reduction in the mutants (6.5%, SD 4.3%), as compared to the controls (17.9%, SD 2.0%). We also explored whether Egr2-expressing cell ablation impacted nerve development and performed skin whole-mount immunohistochemistry for nerves (Tuj1) and SCPs (Sox10) at E15.5 in two controls and two mutants with complete loss of traced cells. There was no significant difference between littermate controls (Egr2Cre/+,Rosa26RTom) and mutants in terms of Tuj1-positive area fraction and Sox10-positive cell count (Figure 6—figure supplement 1A–C).

Figure 6 with 1 supplement see all
Ablation of Egr2-expressing boundary caps (BCs) impacts mural cell (MC) development in the skin.

Whole-mount embryonic dorsal skin at embryonic day (E) 15.5, labeled with antibodies against PECAM (blue) and NG2 (yellow). Compared with littermate Egr2Cre/+,Rosa26RTom control mice (A), vascular network development appeared normal at low magnification in mutant mice (B), in which BC cells and their derivatives were eliminated (arterioles and venules are indicated by full and empty arrowheads, respectively). Capillaries from mutant mice skin (E,F) showed a significant decrease in NG2-positive MCs as compared to controls (C,D). Vessel area fractions were similar in controls and mutants (G), but the NG2-positive area fraction was significantly decreased in the mutants (H), indicating a depletion of MCs. Statistical analyses of the PECAM and NG2-positive surface areas between controls (n=3) and mutants (n=4) were carried out using a Mann-Whitney U test. Scale bars, 500 μm (A,B), 50 μm (C–F). Error bars, one standard deviation (G,H). ****=p < 0.0001. ns, non-significant.

In conclusion, we find that the targeted ablation of Egr2-expressing cells does not appear to impact nerve development, but leads to a significant depletion of NG2-positive MCs in developing capillaries in the skin. This indicates that MCs from myeloid origin cannot fully compensate this defect. It remains to be determined whether this vascular phenotype is at the origin of embryonic lethality, as ablation targets all Egr2-positive cells and is therefore not limited to BC cells.

Discussion

This study uncovers a dual embryonic origin for MCs in mouse skin: while approximately one third of this population derives from myeloid precursors (Yamazaki et al., 2017), about two thirds originate from BCs. Hence, we identified a specific subpopulation derived from Egr2-expressing, ventral BCs that migrate along nerves into the skin, then detach and give rise to pericytes and vSMCs. The remaining BC-derived cells follow the same path, but remain attached to nerves and give birth to SCPs. Whereas these latter cells express the typical NC lineage marker Sox10, our observations suggest that the former population giving rise to MCs do not express this marker and originates from the developing pia matter. In accordance with our tracing studies, genetic ablation of Egr2-expressing BC cells results in a depletion of the skin MC compartment. Finally, we found that numerous MCs in serous membranes protecting the trachea, heart, and digestive tract are derived from Egr2-expressing cells, suggesting a broader implication of BCs in the development of the systemic vasculature.

Our observations highlight a novel role for BCs in peripheral vascular development and further expand peripheral glia recently reported broad potential (Adameyko et al., 2009; Dyachuk et al., 2014; Espinosa-Medina et al., 2014; Furlan et al., 2017; Kaucka et al., 2016; Uesaka et al., 2015; Xie et al., 2019). While virtually all BC cells at the dorsal root express Sox10 and give rise to either SCP or DRG sensory neurons, the situation appears more complex in the ventral roots. We discovered that ventral roots encompass two populations of Egr2-expressing BCs with distinct molecular and functional profiles. In addition to the Sox10-positive BCs at the origin of SCPs that migrate along nerve roots and spinal nerves, we identified a population of Sox10-negative BC-derived cells that express mesenchymal markers such as Tbx18, Col1a1, and Pdgfra. Our scRNA-seq analysis of traced cells extracted from E12.5 skin, at a time when the majority of BC-derived cells are still in contact with nerves, further supports the presence of nerve-attached BC derivatives with mesenchymal signature. Interestingly, at the same time, myeloid precursors are recruited by the primitive vascular plexus to form MCs (Yamazaki et al., 2017). In both systems, traced cells along blood vessels do not form continuous blocks, but are interrupted by blocks of non-traced cells. This suggests the existence of an attraction mechanism operated by the developing vascular plexus and acting on neighboring cell populations of at least two distinct origins, which have the potential to mature into MCs. In this scenario, nerves would act as a track for the migration of one of these populations from the ventral BCs to the skin and probably some deeper structures in the developing embryo. Since this migration of BC derivatives was observed along all spinal nerves, this mechanism is likely to provide an important source of MCs to the developing embryonic skin. Notably, Egr2 inactivation does not appear to have any obvious effect on the migration and differentiation of this population. Nevertheless, more detailed analyses will be required to exclude any later function in MCs.

BCs are cell clusters, defined by their location at the CNS/PNS boundary and which were shown to express specific markers such as Egr2 and Prss56 (Coulpier et al., 2009). Using genetic tracing with the Prss56 gene, we have previously established that a population of BC-derived traced cells give rise to SCs, DRG sensory neurons, and, in the skin, to terminal glia and melanocytes (Gresset et al., 2015; Radomska et al., 2019). Genetic tracing performed with the Egr2, also revealed a BC cell population that gives rise to DRG neurons and SCs (Maro et al., 2004). In this study, we establish that the ventral BC cell population traced with the Egr2 marker also gives rise to skin MCs, derivatives that are never observed in the case of Prss56 tracing. The common derivatives observed in the two tracing studies are likely to reflect an already noticed, partial overlap between Prss56- and Egr2-positive BC subpopulations (Gresset et al., 2015). In contrast, the differences in fates observed in the skin points to the existence of a functional heterogeneity within BC cells. The developmental fates of BC cells might be determined by the encounter with environmental cues during their migration or, in contrast, by the existence of blueprints established at or even before the BC cell stage. Our studies support such an early specification in the development of ventral BC cells; genetic tracing of cells expressing Tbx18 indicate that some of them attach to ventral roots before E11.5, where they do not express Sox10 and may activate Egr2. It is likely that these cells contribute to the Egr2-traced subpopulation that migrates along nerves to the skin, detaches and differentiates into MCs.

In accordance, our findings require to revisit the simplistic concept of BCs as transient and homogenous clusters of stem-like cells, characterized by co-expression of a panel of markers including Egr2 and Prss56 and initially thought to be entirely derived from the NC. The present data suggest that ventral exit point BCs possess mixed NC and mesenchymal origins. In particular, we show that the Egr2-expressing BC cells that give rise to MCs are neither derived from the NC nor from the neural tube. They are likely to originate from Tbx18-expressing cells that are also at the origin of developing meningeal fibroblasts (DeSisto et al., 2020). Actually, molecular heterogeneity was also observed within NC-derived BCs, as they contain populations expressing either Egr2 or Prss56, or co-expressing both markers (Gresset et al., 2015 and unpublished results). This heterogeneity in the embryonic origin of BC cells is likely to explain another observation: whereas targeted ablation of NC-derived cells expressing Egr2, obtained by combining Wnt1Cre and Egr2GFP/DT alleles, is effective, but not lethal before birth (Odelin et al., 2018), targeted ablation of all Egr2-expressing cells does lead to embryonic death at around E15.5 (this study). This may be explained by the fact that the populations ablated in those experiments do not completely overlap.

Our demonstration of a novel source of MCs in the skin is of interest in the context of recent advances in understanding MC lineages. Based on their location, MCs have been divided into pericytes that ensheath capillaries, and vSMCs that cover arterioles, venules, and larger vessels (Armulik et al., 2011). However, this simplistic view has been recently challenged by growing evidence supporting a continuum of MC lineages with intermediate types associated to specific functions (Holm et al., 2018). For instance, molecular analysis of CNS MCs at the single-cell level revealed the existence of subpopulations specifically involved in processes such as inflammation (Duan et al., 2018) or fibrosis (Göritz et al., 2011; Soderblom et al., 2013). A similar heterogeneity among NG2-positive MCs has recently been reported in the skin, with different subpopulations at specific locations and with differing roles in wound repair (Goss et al., 2021). Our study reveals the existence of at least two embryonic origins for both skin pericytes and vSMCs, BCs derivatives and myeloid precursors, which are recruited during the same period to form MCs. Each population appears to be unable to fully compensate the other upon depletion: we report that targeted ablation of BC cells results in a significant decrease in NG2-positive MCs at E15.5 and, conversely, Yamazaki and colleagues have shown that in PU.1 (also known as Spi1) mutants, depleted of tissue myeloid cells, there is also a loss of MCs at E15.5 (Yamazaki et al., 2017). In addition, our data show molecular differences between MP clusters susceptible to give rise to MCs in the peripheral vasculature, with most MP clusters (1–4) sharing molecular markers (Pdgfrα and Col1a1) with CNS MC subtypes involved in fibrosis. Whether these differences in embryonic origin or marker expression identify distinct functional lineages corresponding to those reported at later stages remains an open question.

Materials and methods

Mouse lines, genotyping, and ethical considerations

Request a detailed protocol

Mice used in this study were housed in a temperature- and humidity-controlled vivarium on a 12 hr dark-light cycle with free access to food and water. All mouse lines were maintained in a mixed C57BL/6-DBA2 background. We used the following alleles or transgenes that were genotyped as indicated in the original publications: Egr2Cre(Voiculescu et al., 2000), Prss56Cre (Gresset et al., 2015), Wnt1cre (Lewis et al., 2013), Sox10XCre-Ert2 (Simon et al., 2012), Olig2Cre (Zawadzka et al., 2010), Tbx18Cre-Ert2 (Guimarães-Camboa et al., 2017), Rosa26RYFP (Srinivas et al., 2001), Rosa26RTom (Madisen et al., 2010), and Egr2GFP(DT) (Vermeren et al., 2003). Day of plug was considered E0.5. Animals were sacrificed by decapitation (newborn) or cervical dislocation (adult) unless indicated otherwise. All animal manipulations were performed according to French and European Union regulations. According to these regulations, no ethics committee approval was required for this study which only used mouse embryos and newborns.

In situ hybridization and immunofluorescence

Request a detailed protocol

In situ hybridization on embryo sections was performed as previously described (Gresset et al., 2015). Briefly, samples were fixed overnight (o/n) in 4% paraformaldehyde (PFA; Electron Microscopy Science) in 0.1 M phosphate buffer (PBS) before being serially sectioned (150 μm) and processed for in situ hybridization. Embryonic immunohistochemical analysis was performed either on 50 μm transverse cryosections or embryonic skin whole-mounts, both performed as previously described (Gresset et al., 2015; Radomska et al., 2019). Sections and embryonic skin whole-mounts were stored at –20°C in 0.1 M PBS with 30% glycerol and 30% ethylene glycol. Briefly, for whole-mount immunolabeling, dorsal skins of E12.5–15.5 embryos were dissected after o/n fixation in 4% PFA. Samples were blocked o/n in 4% bovine serum albumin (BSA, Sigma-Aldrich) in PBS containing 0.3% Triton X-100 (PBST, Sigma-Aldrich), then incubated for 3 days with the primary antibody/BSA/PBST solution at 4°C. After rinsing, secondary antibodies were applied o/n at room temperature. Samples were then washed and flat-mounted in Fluoromount-G (Southern Biotech). Antibodies are described below. Nuclei were counterstained with Hoechst (Life Technologies). Whole-mount immunostaining and clarification of newborn skin was performed using the iDISCO+ method (Renier et al., 2016). Z-stacks were acquired using Leica TCS SP5 and TCS SP8 laser-scanning confocal microscopes and assembled in ImageJ.

RNAscope in situ hybridization

Request a detailed protocol

We used an RNAscope Multiplex Fluorescent Reagent Kit V2 and Target Probes (ACD Biotechne) for mouse high-resolution in situ hybridization detection of Collagen1a1, Egr2, Pdgfra, Sox10, Tbx18, and Tom mRNAs (ACD Biotechne). The experiment was performed according to the manufacturer’s instructions. In an RNase-free environment, 14-μm-thick slices of fixed WT embryos at E9.5 and E10.5, and Egr2Cre/+,Rosa26RTom embryos at E12.5, were sectioned and mounted on a glass slides. Sections were baked in a 60°C dry oven for 30 min, post-fixed in 4% PFA for 2 hr, then dehydrated in 100% ethanol. Sections were incubated for 10 min with a 30% H2O2 solution before a 5 min incubation in a 1× target retrieval solution at 90–95°C. Sections were then rinsed with distilled water, followed by 100% ethanol. Subsequently, sections were incubated with the following solutions in a HybEZ humidified oven at 40°C with rinsing steps in between: protease III, 30 min; target probes, 2 hr; amplification (Amp), 30 min; Amp 2, 30 min; Amp 3, 15 min; HRP-OPAL Dye (HRP-C1), 60 min; HRP-C2, 60 min; HRP-C3, 60 min. All reagents were from the RNAscope Multiplex Fluorescent Reagent kit.

Antibodies

For immunofluorescence, the following primary antibodies were used: rabbit anti-Tomato (1:500, Rockland #600-401-379), goat anti-Tomato (1:500, Sicgen, #AB0040-200), mouse biotinylated anti-Tuj1 (1:800, R&D Systems, #BAM1195), rabbit anti-Tuj1 (1:1000, BioLegend, #802001), rat anti-PECAM (1:100, BD Pharmingen, #553370), goat anti-PECAM (1:1000, R&D Systems, #AF3628), goat anti-Sox10 (1:200, SantaCruz Biotechnology, #sc-17342), rabbit anti-NG2 (1:200, Merck, #AB5320), mouse anti-SMA-Cy3 (1:400, Merck, #C6198), goat anti-PDGFRβ (1:500, R&D Systems, #AF1042), rabbit anti-Abcc9 (1/100, ThermoFisher Scientific, #PA5-52413), and rabbit anti-Desmin (Abcam, #ab15200). Fluorophore-conjugated secondary antibodies were from Jackson ImmunoResearch.

Cell quantification

Request a detailed protocol

Quantification of Tomato-positive cells was performed on whole-mount preparations of embryonic skin from three Egr2Cre/+,Rosa26RTom embryos from each stage (E12.5, E13.5, E14.5, and E15.5), labeled for Tomato, Tuj1, and PECAM. For each embryo, five z-stacks of different and non-overlapping fields of view were selected randomly and acquired using a Leica TCS SP5 laser-scanning confocal microscope. The scanned surface area per field corresponds to 0.38 µm2. All Tomato-positive cells were counted in each stack on the ImageJ software, and among them cells in contact with nerves were counted to calculate the proportion of cells ‘on nerve’. Cells not in contact with nerves were on the vascular plexus. Statistical analyses of the ‘on nerve’/‘on vessels’ ratio between time points were carried out using a Mann-Whitney U test.

Quantifications of Tomato-positive cells among NG2-positive cells in Egr2Cre/+,Rosa26RTom mice were performed on whole-mount skin preparations at E15.5 and transverse skin sections at P1, labeled for Tomato, PECAM, and NG2. At E15.5, four non-overlapping z-stacks of a scanned surface area of 0.10 µm2 each were analyzed. At P1, five sections were analyzed, each with two non-overlapping z-stacks of a scanned surface area of 0.10 µm2. All NG2-positive cells and among them Tomato-positive cells were counted in each stack on the ImageJ software. Statistical analysis of the ‘NG2 and Tomato-positive’/‘NG2-positive’ ratio between time points was carried out using a Mann-Whitney U test.

Quantifications of Sox10-positive cells in mutant Egr2Cre/GFP(DT),Rosa26RTom (n=2) and control (n=2) mice were performed on whole-mount skin preparations at E15.5, labeled for Sox10 and Tuj1. For each embryo, three (controls) or four (mutants) non-overlapping z-stacks of a scanned surface area of 0.20 µm2 were acquired using a Zeiss LSM 900 laser-scanning confocal microscope. All Sox10-positive cells were counted in each stack on the ImageJ software. Statistical analysis of Sox10-positive cell counts in mutants and controls was carried out using a Mann-Whitney U test.

NG2, PECAM, and Tuj1-positive surface area quantification

Request a detailed protocol

NG2-positive surface area quantifications in mutant Egr2Cre/GFP(DT),Rosa26RTom (n=4) and control (n=3) mice were performed on whole-mount skin preparations at E15.5, labeled for PECAM and NG2. For each embryo, three (controls) or six (mutants) z-stacks of different and non-overlapping fields of view were acquired using a Leica TCS SP5 (mutants) or Zeiss LSM 900 (controls) laser-scanning confocal microscope. The scanned surface area per field corresponds to 0.10 µm2. A similar approach was used for Tuj1-positive surface area quantifications in mutant Egr2Cre/GFP(DT),Rosa26RTom (n=2) and control (n=2) mice, which were also performed on whole-mount skin preparations at E15.5, labeled for Sox10 and Tuj1. For each embryo, three (controls) or four (mutants) z-stacks of different and non-overlapping fields of view were acquired using a Zeiss LSM 900 laser-scanning confocal microscope. The algorithm to determine the area coverage of the NG2, PECAM, and Tuj1 signals was implemented in Python using the OpenCV (https://opencv.org/) library. Briefly, for both channels, a maximum z-projection of each stack was generated to simplify the subsequent analysis. Images were then pre-processed using histogram equalization, only for the PECAM channel, and Gaussian blurring to respectively enhance contrast and reduce high-frequency noise. They were then thresholded using an adaptive mean filter with a block size of 101 pixels for PECAM and 501 pixels for Tuj1 and NG2. Small objects were removed based on their area size (inferior to 300, 100, and 50 pixels for PECAM, Tuj1, and NG2, respectively) to clean up the masks. Finally, the percentage of coverage of each channel was defined as the number of positive pixels in the mask divided by the total number of pixels in the image multiplied by 100. Statistical analyses of the PECAM, NG2, and Tuj1-positive surface areas between controls and mutants were carried out using a Mann-Whitney U test.

Statistical analyses

Request a detailed protocol

Statistical analyses were carried out using a Mann-Whitney U test using the R software version 3.6.1 (http://www.r-project.org). Non-significant p-values are marked ‘ns’. p-Values considered significant are indicated by asterisks as follows: *, p<0.05; **, p<0.01; ***, p<0.001. Data are represented as mean values ± standard deviation.

Semi-quantitative RT-PCR

Request a detailed protocol

Total RNA (100 ng) was isolated from E12.5 embryonic skin, reverse transcribed using the pSuperscript III RNAse H reverse transcriptase (Invitrogen), and a mix of oligo-dT and random primers (Invitrogen), according to the manufacturer’s instructions. PCR was performed as follows: 2 min at 94°C; 35 cycles of 2 min at 94°C, 1 min at 58°C, 30 s at 72°C. Egr2 primer sequences were the following: (5’–3’) GCAGAAGGAACGGAAGAGC; (3’–5’) ACTGGTGTGTCAGCCAGAGC.

Skin dissection and FACS

Request a detailed protocol

E12.5 Egr2Cre/+,Rosa26RTom embryos were identified on the basis of Tomato expression under fluorescent stereomicroscope (Leica, Nussloch, Germany), and their head and viscera were removed. The back skin was dissected and then digested with collagenase/dispase type I (Merck/Roche) for 15 min at 37°C. Digestion was stopped by addition of 0.1 ml of fetal calf serum. Skin samples were then mechanically dissociated and the cell suspension was filtered. Dissociated cells were then resuspended in PBS, 1% BSA, and subjected to FACS. Tomato-positive cells were isolated, while dead cells and doublets were excluded by gating on a forward-scatter and side-scatter area versus width. Log RFP fluorescence was acquired through a 530/30 nm band pass. Internal Tomato-negative cells served as negative controls for FACS gating. Tomato-positive cells were sorted directly into lysis buffer for RT-PCR, or into PBS, 0.04% BSA for scRNA-seq experiments. To evaluate the purity of sorted cells, aliquots of the positive and negative fractions were sorted by FACS again with similar gating parameters, seeded onto coverslips and analyzed by immunohistochemistry with an anti-Tomato antibody.

scRNA-seq and data analysis

Request a detailed protocol

Tomato-positive cells were isolated by FACS from E12.5 embryonic skin. Around 10,000 cells were loaded into one channel of the Chromium system using the V3 single-cell reagent kit (10X Genomics). Following capture and lysis, cDNAs were synthesized, then amplified by PCR for 12 cycles as per the manufacturer’s protocol (10X Genomics). The amplified cDNAs were used to generate Illumina sequencing libraries that were each sequenced on one flow cell NextSeq500 Illumina. Cell Ranger v3.0.2 (10X Genomics) was used to process raw sequencing data. This pipeline converted Illumina base call files into Fastq format, aligned sequencing reads to a custom mm10 transcriptome with added sequences for the Tomato and Cre transgenes using the STAR aligner (Li et al., 2009), and quantified the expression of transcripts in each cell using Chromium barcodes. The count matrix generated by Cell Ranger v3.0.2 was loaded into R, thanks to the Seurat package v3.1.2 (Stuart et al., 2019) for R 3.6.1 (http://www.r-project.org). Empty droplets were filtered out using the EmptyDrops method implemented into the droplet Utils package v1.6.1 (Lun et al., 2019), with an FDR-adjusted p-value cut-off of 1E-03. Cells were selected if they expressed at least 500 genes including Tom, and if mitochondrial genes accounted for at most 20% of the total, and ribosomal genes for at least 7.5% of the total. Genes expressed in less than 10 cells were filtered out. Doublet cells were identified by a combination of two methods: the hybrid method implemented in the scds v1.2.0 package (Bais et al., 2020) and the method from the scDblFinder v1.0.0 package (McGinnis et al., 2019). This resulted in a dataset of 2672 single-cell transcriptomes, among which three contaminating cell types were detected: Myod1-positive myoblasts (n=64), PECAM-positive endothelial cells (n=22), Neurod1-positive sensory neurons (n=5), and C1qb-positive immune cells (n=3). These cell types, accounting for 3% of the total cell number, were removed from the raw dataset for the following analysis. Cells for which no counts were measured for Tomato (n=377) were also removed. Cell cycle scores and class prediction were performed with the cyclone method implemented in the scran v1.14.5 package (Lun et al., 2016). Count values were normalized using the LogNormalize (Hafemeister and Satija, 2019) method implemented in Seurat, regressing out cell cycle (S minus G2M) as covariates. Principal component analysis (PCA)-based dimensionality reduction, graph-based cell clustering, and uniform manifold approximation and projection embedding were performed with Seurat, using 19 PCA dimensions with a 0.9 resolution and all the other parameters to default values.

Materials availability

Request a detailed protocol

Mouse strains carrying the Egr2Cre and Rosa26RTom alleles are freely available from The Jacksons Laboratory (USA). Prss56Cre mice will require an MTA for future users.

Data availability

Single-cell RNA-seq data have been deposited in the ArrayExpress database at EMBL-EBI under accession number E-MTAB-8972.

The following data sets were generated

References

Decision letter

  1. Marianne E Bronner
    Senior and Reviewing Editor; California Institute of Technology, United States
  2. Igor Adameyko
    Reviewer; Karolinska Institutet, Sweden
  3. Tatiana Solovieva
    Reviewer; California Institute of Technology, United States

In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.

Decision letter after peer review:

Thank you for submitting your article "Neural tube-associated boundary caps are a major source of mural cells in the skin" for consideration by eLife. Your article has been reviewed by 3 peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Marianne Bronner as the Senior Editor. The following individuals involved in review of your submission have agreed to reveal their identity: Igor Adameyko (Reviewer #1); Tatiana Solovieva (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:

The reviewers find the paper potentially very interesting but also raise a major important question regarding the neural crest origin or lack thereof of these perivascular cells.

1) It is essential to provide further data regarding whether or not the boundary cap cells producing mural cells are of neural crest origin, as promised. To this end, you should perform additional lineage tracing with a Sox10-Cre line since boundary cap cells are Sox10 and Krox20 double positive. Wnt1-Cre would also be an appropriate line to use.

2) If the lineage tracing does not label mural cells, then the conclusion that these are neural crest derived should be revised and ideally it would be nice to show where they come from.

The full reviews are included below to help with your revision.

Reviewer #1 (Recommendations for the authors):

The manuscript by Gerschenfeld and co-authors is an interesting study focused around the question of a potential contribution from the trunk neural crest cells to the lineages of perivascular cells. I really enjoyed reading it, and I believe this question deserves a serious attention. Although the authors performed a lot of accurate experiments, the current results do not come together with the current knowledge in the field, mostly because other Cre strains recombining in the neural crest populations do not recapitulate trunk perivascular cells, which the authors find to be traced with Krox20 Cre line.

The key would be to rule out the neural crest (classical Sox10+/Wnt1+) or non-neural crest origin of these perivascular cells. It might be that the embryonic nerves contain non-neural crest-derived cells with the plasticity discovered by the authors.

Below I list my suggestions for the critical improvements:

1. Other NCC and SCP-specific Cre lines should be used in parallel. The authors shall try Sox10-CreERT2, PLP1-CReERT2, Wnt1-Cre or similar.

2. The authors should check the expression of Krox20-driven Cre mRNA throughout all investigated stages for ruling out any ectopic recombination.

3. The in situ for Krox20 in the entire embryo and mostly in skin should be done for addressing possible expression outside of the boundary cap.

4. In the case, where the authors will not be able to confirm the neural crest-derived nature of the pervascular cells in the trunk, the authors will need to re-write the manuscript suggesting an alternative origin. Indeed, it might be possible that ventral boundary cap is a heterogeneous populations, and is only partly derived from the classic neural crest. The rest of this population, in theory, might come from the ventral neural tube (Sox2 Cre lines should clarify it) or from mesoderm (feels unlikely), and in that case Mesp1 Cre or similar might be helpful. Of note: the paper will appear even more interesting to me in case when non-neural crest progenitors are associated with the peripheral nerves and give rise to the perivascular component.

5. The transitions should be validated in single cell data with RNA velocity. Krox20 expression needs to be shown on UMAPs. It is well known that Schwann cell precursors give rise to endoneurial fibroblasts. Those can be similar in their transcriptomics profile to the collected perivascular populations, leading to the false idea of connectedness. This needs to be investigated deeper.

Reviewer #2 (Recommendations for the authors):

Krox20 is a marker for boundary cap (BC) cells. The authors trace BC derivatives in the mouse using Krox20Cre/+, Rosa26RTom embryos, where Tom-positive cells represent Krox20 derivatives. The authors identify BC derivatives that migrate along nerves towards the skin where a large proportion of these then detach from the nerves and instead attach to blood vessels. While these BC derived cells express glial markers for Schwann cell precursors (SCPs) (Sox10, BLBP) during their migration along the nerves, expression of these markers is reduced in some cells when they reach the skin. BC derived cells that detach from the nerves and instead associate with vessels express markers of mural cells (ABCC9, NG2, PDGFR β, SMA). At postnatal day 1, two-thirds of NG2-positive cells in the skin were Tom-positive, suggesting that two thirds of mural cells were BC-derived at this stage. Analysis of cell transcriptomes (from scRNAseq) of Tom-positive cells, at the stage when these cells begin to detach from the nerve (E12.5), revealed four general cell clusters; SCP-like, mesenchymal progenitor-like, transition-like cells, and a mural cell-like cluster. Inferred lineages from this data were used to suggest that SCPs go through a transition phase; from SCPs to mesenchymal progenitor like cells, before becoming mural cells.

Beautiful immunohistochemistry images provide strong support for the authors' claims through most of the paper. However, there are a couple more images that would be nice to have, to visualise some of the conclusions that authors derived from the scRNAseq data.

1. Authors say that for some cells, the transition from SCP to mural identity initiates while the cell is still attached to the nerve. The authors show antibody staining for SCP markers but not for mural cell markers at these stages (E12.5-E13.5, Figure 2). Antibody staining for both SCP and mesenchymal progenitor and/or mural cell markers in the same section showing positive signal for both in the same cell while detaching from a nerve would reinforce and visualise the authors' claims from the scRNAseq data.

2. Analysis of the scRNAseq data lead authors to suggest that BC derivatives also give rise to chondroprogenitors in the trunk. Antibody staining for chondroprogenitor markers (e.g. Sox9, Dlx5, Col2a1, Fgfr2) in the traced (Tom-positive) cells would reinforce the authors' claims from the inferred lineages from scRNAseq.

This is a great paper. My only recommendation to the authors would be to add a couple of antibody stainings. The most crucial would be for both SCP markers and mesenchymal progenitor and/or mural cell markers in the same section at E12.5, showing Tom-positive cells that co-express glial and mesenchymal and/or mural molecular markers in the same cell. Showing this in a Tom-positive cell that is detaching from a nerve would reinforce the authors' claims from the scRNAseq data for a 'transition' phase.

Reviewer #3 (Recommendations for the authors):

Gerschenfeld et al. investigate offspring of boundary cap (BC) cells in the embryonic dermis using lineage tracing in mice. They report that Krox20 positive BC cells travel along the nerves to the dermis, where they undergo a glial to vascular identity switch, detach from nerves and incorporate into the vascular plexus to become mural cells. Knockout of Krox20 does not affect cell migration, suggesting this gene is a marker but not involved in cell fate switch. Quantifications of immune-labelled sections showed that up to 60% of neonatal dermal mural cells are derived from Krox20+ BC precursors. Single cell RNA sequencing confirms heterogeneity of Krox20+ cells, including a cluster of cells with mural cell identity.

Overall, this manuscript is well written and illustrated and provides what constitutes to my knowledge the first evidence of a neural crest origin of a portion of mural cells in the dermis of the trunk, thereby adding to the multiple known origins of vascular mural cells.

I have just a few comments that should be addressed before publication can be recommended.

Figure 3 Quantification of PdgfrB+Tom+ double positive cells would strengthen the quantification and the authors conclusion and should be added.

Figure 3 and S5a-c show that Tom-positive MCs do not form a continuum and are separated by non-traced MCs, suggesting that multiple sources of mural cells contribute to the dermal vessel wall. Why do the authors think that is?

Figure 4 and 5: can the scRNA seq analysis be further exploited towards more mechanistic understanding of the transdifferentiation. It would also be interesting to compare mural cells from different origins ie BC derived versus other sources.

Figure 6 Ablation of Krox20+ cells using DTR led to embryonic lethality, and analysis of a few surviving embryos revealed incomplete ablation of BC progeny in 4/6 embryos, while two showed compete loss of BCs and were chosen for analysis of the vascular network. The authors measured vascular area in those embryos and found no differences. This result is surprising and deserves some consideration. One would expect lack of mural cell coverage to lead to microaneurysms and hemorrhage, yet this was apparently not observed? The vasculature in Figure 6 panels C and F does look different between controls and mutants, I would encourage the authors to further characterize the vascular defects in these mice. Also, presence and absence of Schwann cells in these mice should be shown and discussed.

Figure S2c: please label the two circular NG2+ structures.

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

Thank you for resubmitting your work entitled "Neural tube-associated boundary caps are a major source of mural cells in the skin" for further consideration by eLife. Your revised article has been evaluated by Marianne Bronner (Senior Editor) and a Reviewing Editor.

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

One of the main concerns from the original submission was the origin of the perivascular cells. The authors have addressed this and tested for possible neural crest (Sox10Cre-Ert2 and Wnt1Cre lines), neural tube (Olig2Cre line), and pial (Tbx18Cre-Ert2 line) origins. Sox10Cre-Ert2, Wnt1Cre lines, and Olig2Cre lines did not give rise to traced mural cells. With the Tbx18Cre-Ert2 line, tamoxifen was administered to pregnant females at E9.5, and embryos analyzed at E11.5. SOX10 negative traced cells were identified at E11.5 along the ventral root and spinal nerve. This was used to suggest a pial cell origin of perivascular cells. If possible, it would be beneficial to have a visual of the expression of Tbx18 at E9.5, to visualize the origin of the traced cells. It is also unclear why traced cells from the TBX18 line were not followed through to later stages (E14.5) where cells could be more stringently verified as mural cells by immunostaining for some mural cell markers (as in Figure 2A-D) and their morphology and migratory behavior could also be better verified as 'mural cell'.

https://doi.org/10.7554/eLife.69413.sa1

Author response

Essential revisions:

The reviewers find the paper potentially very interesting but also raise a major important question regarding the neural crest origin or lack thereof of these perivascular cells.

1) It is essential to provide further data regarding whether or not the boundary cap cells producing mural cells are of neural crest origin, as promised. To this end, you should perform additional lineage tracing with a Sox10-Cre line since boundary cap cells are Sox10 and Krox20 double positive. Wnt1-Cre would also be an appropriate line to use.

As indicated in detail below in the specific responses to the referees, we have performed additional tracing experiments designed to investigate the origin of the perivascular cells. Using Sox10Cre-Ert2 and Wnt1Cre lines, as well as a neural tube tracing line (Olig2Cre), we show that the ventral BC subpopulation that gives rise to mural cells does not originate from the neural crest or from the neural tube.

Our new findings are detailed in a new subsection in the Results on page 8 (lines 1 to 28), with its associated Figure 4. The Discussion was also substantially modified to reflect these findings from pages 12 to 14. The Methods were also updated on page 15 (lines 2 to 12).

2) If the lineage tracing does not label mural cells, then the conclusion that these are neural crest derived should be revised and ideally it would be nice to show where they come from.

This is indeed what we found. On this basis, we have chosen to explore whether the developing pia matter could contribute to BCs. Using a Tbx18Cre-Ert2 line, we show that traced cells are found in close contact with the ventral root, and that they migrate along the nerves on top of Schwann cell precursors. In addition, we performed RNA scope in situ hybridization and show that some Krox20-positive BC cell derivatives along the ventral root do not express Sox10, but are positive for mesenchymal markers such as Tbx18, Col1a1 and Pdgfra. Finally, after upgrading our scRNA-seq analysis pipeline, we observe two separate superclusters of Tomato-positive cells with glial and mesenchymal signatures, further supporting a dual origin of ventral BCs, with a neural crest-derived population at the origin of SCPs and a mesenchymal-like population at the origin of mural cells.

Our new findings are detailed in two new subsections in the Results from page 6 (line 30) to page 10 (line 5), with their associated Figures 3 to 5. The Discussion was also substantially modified to reflect these findings from page 12 to 14. The Methods were updated on page 16 (lines 1 to 17) and from page 19 (line 8) to 20 (line 5).

Reviewer #1 (Recommendations for the authors):

The manuscript by Gerschenfeld and co-authors is an interesting study focused around the question of a potential contribution from the trunk neural crest cells to the lineages of perivascular cells. I really enjoyed reading it, and I believe this question deserves a serious attention. Although the authors performed a lot of accurate experiments, the current results do not come together with the current knowledge in the field, mostly because other Cre strains recombining in the neural crest populations do not recapitulate trunk perivascular cells, which the authors find to be traced with Krox20 Cre line.

The key would be to rule out the neural crest (classical Sox10+/Wnt1+) or non-neural crest origin of these perivascular cells. It might be that the embryonic nerves contain non-neural crest-derived cells with the plasticity discovered by the authors.

Below I list my suggestions for the critical improvements:

1. Other NCC and SCP-specific Cre lines should be used in parallel. The authors shall try Sox10-CreERT2, PLP1-CReERT2, Wnt1-Cre or similar.

We have performed additional lineage tracing studies using neural crest (Sox10Cre-Ert2 and Wnt1Cre) as well as neural tube (Olig2Cre) tracing lines. None of them labeled mural cells in the skin indicating that ventral BC subpopulation at their origin does not derive from the neural crest or neural tube.

Our new findings are detailed in a new subsection in the Results on page 8 (lines 1 to 28), with its associated Figure 4. The Discussion was also substantially modified to reflect these findings from pages 12 to 14. The Methods were also updated on page 15 (lines 2 to 12).

2. The authors should check the expression of Krox20-driven Cre mRNA throughout all investigated stages for ruling out any ectopic recombination.

We agree that exploring possible ectopic Cre recombination is of importance. To investigate this issue, we attempted to reveal Cre mRNA using RNAscope and Cre protein with immunostainings. However, both techniques are known to be delicate with Cre and this actually did not work. To tackle the issue in another way, we included the Cre sequence in our scRNA-seq analysis. We did not observe any Cre expression either at E12.5 (in our published dataset, which included 2209 cells), or at E13.5, using another unpublished dataset (1029 cells, data not shown). Although this analysis does not cover earlier stages, it demonstrates that from E12.5 no ectopic expression can be detected.

We have mentioned the absence of Cre expression in the scRNA-seq dataset in the Results on page 9 (lines 5 to 7).

3. The in situ for Krox20 in the entire embryo and mostly in skin should be done for addressing possible expression outside of the boundary cap.

This constitutes a related, important issue and we have performed additional analyses on ventral roots and the skin from E12.5 embryos. We have found that at that stage Krox20 expression is restricted to BCs and is totally absent from skin. These data reinforce our previous observations obtained by in situ hybridization and RT-PCR data to exclude the possibility of Krox20 expression in other sites than BCs.

Our new findings are detailed in the first subsection in the Results on page 5 (lines 14 to 20), with its associated Figure 1—figure supplement 1 (C-J). The Methods were also updated on page 16 (lines 1 to 17).

4. In the case, where the authors will not be able to confirm the neural crest-derived nature of the pervascular cells in the trunk, the authors will need to re-write the manuscript suggesting an alternative origin. Indeed, it might be possible that ventral boundary cap is a heterogeneous populations, and is only partly derived from the classic neural crest. The rest of this population, in theory, might come from the ventral neural tube (Sox2 Cre lines should clarify it) or from mesoderm (feels unlikely), and in that case Mesp1 Cre or similar might be helpful. Of note: the paper will appear even more interesting to me in case when non-neural crest progenitors are associated with the peripheral nerves and give rise to the perivascular component.

We have explored whether the neural tube or the developing pia matter (future meninges) might contribute to the ventral to BC cells population. Using an Olig2Cre line, we have found no traced cells in the periphery, excluding a neural tube contribution. In contrast, using a Tbx18Cre-Ert2 line, we have identified traced cells on the ventral roots, within the BCs and migrating along the nerves on top of Schwann cell precursors. In addition, we performed RNAscope in situ hybridization analyses that revealed the presence on the ventral root of Krox20-positive BC derivatives negative for Sox10 and displaying mesenchymal markers such as Tbx18, Col1a1 and Pdgfra. Finally, upon upgrading our scRNA-seq analysis pipeline we have observed two distinct superclusters of Tomatopositive cells that further support the idea that ventral BCs are composed of two subpopulations: neural crest-derived cells that give rise to SCPs and cells with a mesenchymal identity that will give rise to mural cells and may be pia matter-derived.

Our new findings are detailed in two new subsections in the Results from page 6 (line 30) to page 10 (line 5), with their associated Figures 3 to 5. The Discussion was also substantially modified to reflect these findings from page 12 to 14. The Methods were updated on page 16 (lines 1 to 17) and from page 19 (line 8) to 20 (line 5).

5. The transitions should be validated in single cell data with RNA velocity. Krox20 expression needs to be shown on UMAPs. It is well known that Schwann cell precursors give rise to endoneurial fibroblasts. Those can be similar in their transcriptomics profile to the collected perivascular populations, leading to the false idea of connectedness. This needs to be investigated deeper.

To address this issue, we have upgraded our scRNA-seq analysis pipeline and found that most of the Tom-positive cells that connected the two super-clusters did not reach a proper quality and should be excluded from the analysis. In these conditions, it appears that the two super-clusters are clearly separated, which is consistent with the observations presented above. Together our data support the existence of a major heterogeneity in ventral BCs, both in terms of identity and origin as discussed above.

Our new scRNA-seq analysis is detailed in the Results from page 8 (line 29) to page 10 (line 5), with its associated Figure 5 and Figure 5—figure supplement 1. The Discussion was also substantially modified to reflect these findings from page 12 to 14. The Methods were also updated from page 19 (line 8) to 20 (line 5).

Reviewer #2 (Recommendations for the authors):

Krox20 is a marker for boundary cap (BC) cells. The authors trace BC derivatives in the mouse using Krox20Cre/+, Rosa26RTom embryos, where Tom-positive cells represent Krox20 derivatives. The authors identify BC derivatives that migrate along nerves towards the skin where a large proportion of these then detach from the nerves and instead attach to blood vessels. While these BC derived cells express glial markers for Schwann cell precursors (SCPs) (Sox10, BLBP) during their migration along the nerves, expression of these markers is reduced in some cells when they reach the skin. BC derived cells that detach from the nerves and instead associate with vessels express markers of mural cells (ABCC9, NG2, PDGFR β, SMA). At postnatal day 1, two-thirds of NG2-positive cells in the skin were Tom-positive, suggesting that two thirds of mural cells were BC-derived at this stage. Analysis of cell transcriptomes (from scRNAseq) of Tom-positive cells, at the stage when these cells begin to detach from the nerve (E12.5), revealed four general cell clusters; SCP-like, mesenchymal progenitor-like, transition-like cells, and a mural cell-like cluster. Inferred lineages from this data were used to suggest that SCPs go through a transition phase; from SCPs to mesenchymal progenitor like cells, before becoming mural cells.

Beautiful immunohistochemistry images provide strong support for the authors' claims through most of the paper. However, there are a couple more images that would be nice to have, to visualise some of the conclusions that authors derived from the scRNAseq data.

We thank the referee for his/her encouraging comments. Please note that the manuscript has been substantially modified following additional analyses detailed in the answers to the editor and to the other referees.

1. Authors say that for some cells, the transition from SCP to mural identity initiates while the cell is still attached to the nerve. The authors show antibody staining for SCP markers but not for mural cell markers at these stages (E12.5-E13.5, Figure 2). Antibody staining for both SCP and mesenchymal progenitor and/or mural cell markers in the same section showing positive signal for both in the same cell while detaching from a nerve would reinforce and visualise the authors' claims from the scRNAseq data.

To look for nerve-attached cells with SCP or mesenchymal identities, we have performed immunolabeling and RNAscope in situ hybridization analyses on Krox20-traced ventral roots and subcutaneous nerves. Using neural crest/glial (Sox10) and mesenchymal (Tbx18, Col1a1 and Pdgfra) probes, we have identified traced cells on ventral roots that do not express Sox10 and also observed traced cells on nerves that express the mesenchymal markers. On subcutaneous nerves, we have identified Krox20-traced Sox10-negative cells. Although we could not perform double-labeling analyses, these data strongly support the presence on nerves of two subpopulations of BC-derived cells with glial and mesenchymal identities.

Our new findings are detailed in a new subsection in the Results from page 6 (line 30) to page 7 (line 34), with its associated Figure 3. The Discussion was also substantially modified to reflect these findings from page 12 to 14. The Methods were updated on page 16 (lines 1 to 17).

2. Analysis of the scRNAseq data lead authors to suggest that BC derivatives also give rise to chondroprogenitors in the trunk. Antibody staining for chondroprogenitor markers (e.g. Sox9, Dlx5, Col2a1, Fgfr2) in the traced (Tom-positive) cells would reinforce the authors' claims from the inferred lineages from scRNAseq.

We agree that immunostainings would be potentially of interest to support the scRNAseq data. However, given the known expression of Krox20 at E14 in chondroprogenitors, we felt that the time window to perform such an analysis was too narrow and that additional controls would be required to exclude late ectopic Krox20 activation. This did not appear to be feasible in a reasonable time, as we had to focus on other aspects more central to the major messages of the paper.

Reviewer #3 (Recommendations for the authors):

Gerschenfeld et al. investigate offspring of boundary cap (BC) cells in the embryonic dermis using lineage tracing in mice. They report that Krox20 positive BC cells travel along the nerves to the dermis, where they undergo a glial to vascular identity switch, detach from nerves and incorporate into the vascular plexus to become mural cells. Knockout of Krox20 does not affect cell migration, suggesting this gene is a marker but not involved in cell fate switch. Quantifications of immune-labelled sections showed that up to 60% of neonatal dermal mural cells are derived from Krox20+ BC precursors. Single cell RNA sequencing confirms heterogeneity of Krox20+ cells, including a cluster of cells with mural cell identity.

Overall, this manuscript is well written and illustrated and provides what constitutes to my knowledge the first evidence of a neural crest origin of a portion of mural cells in the dermis of the trunk, thereby adding to the multiple known origins of vascular mural cells.

I have just a few comments that should be addressed before publication can be recommended.

Figure 3 Quantification of PdgfrB+Tom+ double positive cells would strengthen the quantification and the authors conclusion and should be added.

We agree with the reviewer that such a quantification would be of interest. However, it is not technically feasible on wholemount immunostaining. Pdgfrb immunostaining presents a higher background noise and is more difficult to attribute to a specific cell than NG2, for which we have performed quantifications, which were already challenging. An alternative way would be to use flow-cytometry, but it would be also challenging to restrict the analysis to cells attached to the vasculature.

Figure 3 and S5a-c show that Tom-positive MCs do not form a continuum and are separated by non-traced MCs, suggesting that multiple sources of mural cells contribute to the dermal vessel wall. Why do the authors think that is?

Our study and the one from Yamazaki and colleagues suggest a dual origin for mural cells in the skin vasculature. Approximately one-third of them originate from myeloid progenitors and the remaining ones derive from a subpopulation of ventral root BCs. The fact that each model of mural cell tracing shows a non continuous distribution of traced cells along blood vessels, while we know that the distribution of mural cells is homogeneous and continuous suggest that the two populations are intermingled. This might results from a simultaneous colonization of the vascular plexus by both populations. Interestingly, the genetic ablation of one population leads only to a partial compensation by the cells from the other origin.

We have updated the Discussion to address this question on page 12 (lines 25 to 30).

Figure 4 and 5: can the scRNA seq analysis be further exploited towards more mechanistic understanding of the transdifferentiation. It would also be interesting to compare mural cells from different origins ie BC derived versus other sources.

Additional analyses were performed and, as mentioned above, strongly suggest the existence of at least two subpopulations of ventral BCs with glial and mesenchymal characteristics instead of bi-potent glial progenitors migrating along the nerves to the skin and changing their fate into mural derivatives.

Our new scRNA-seq analysis is detailed in the Results from page 8 (line 29) to page 10 (line 5), with its associated Figure 5 and Figure 5—figure supplement 1. The Discussion was also substantially modified to reflect these findings from page 12 to 14. The Methods were also updated from page 19 (line 8) to 20 (line 5).

Whether the dual origin of trunk skin mural cells is accompanied by molecular and especially functional differences is indeed a primary question. However, this is a topic in itself that has not been explored in the present study.

Figure 6 Ablation of Krox20+ cells using DTR led to embryonic lethality, and analysis of a few surviving embryos revealed incomplete ablation of BC progeny in 4/6 embryos, while two showed compete loss of BCs and were chosen for analysis of the vascular network. The authors measured vascular area in those embryos and found no differences. This result is surprising and deserves some consideration. One would expect lack of mural cell coverage to lead to microaneurysms and hemorrhage, yet this was apparently not observed? The vasculature in Figure 6 panels C and F does look different between controls and mutants, I would encourage the authors to further characterize the vascular defects in these mice.

Regarding embryonic lethality at E15.5 following the targeted ablation of Krox20expressing BC cells, we strongly suspect that it is the ablation of the BC-derived MC component that is responsible for two reasons:

1. We have shown that targeted ablation of rhombomeres 3 and 5, where Krox20 is first expressed between E8.5 and E9.5, leads to death within two weeks after birth (SchneiderMaunoury et al., 1993);

2. We also know that the targeted ablation of neural crest-derived cells which express Krox20, by combining Wnt1Cre and Krox20GFP/DT mice, also leads to death within two weeks after birth (Odelin et al., 2018 and unpublished data).

In conclusion, while the accumulated evidence points to the role of BC-derived MCs, their implication remains to be formally demonstrated. We mention this point in the Discussion from page 13 (line 32) to page 14 (line 3).

We agree that the vasculature in panels C and F looks different. However, we have found that skin wholemount samples at the same stage can differ greatly in terms of aspect, even between littermates, likely because of factors not taken into account such as the exact skin location. Hence, caution is warranted when comparing these two pictures.

Overall, further investigations are needed to prove this hypothesis and study the vascular phenotype appropriately, but we felt that this would not be feasible in a reasonable time for two main reasons:

1. When these embryos are retrieved at E15.5 they usually are already moribund, which may induce post mortem changes in the vascular plexus that are not linked directly to the lack of MCs.

2. Generating embryos with complete ablation proved to be difficult with only 4 embryos on 60 collected embryos.

Also, presence and absence of Schwann cells in these mice should be shown and discussed.

Regarding the impact of the ablation of Krox20-expressing BCs on Schwann cells and nerve development, we have generated two additional mutants and performed immunostainings on them as well as two controls with neural (TuJ1) and glial (Sox10) markers. As a result, we did not observe any alteration in the innervation pattern of the skin nor the distribution of Schwann cell precursors along these nerves.

We have detailed this analysis in the relevant subsection in the Results on page 11 (lines 12 to 17), with its associated Figure 6—figure supplement 1. The Methods were also updated from page 17 (line 16) to page 18 (line 12).

Figure S2c: please label the two circular NG2+ structures.

These two circular structures are hair follicles. We have labelled them in panel C of Figure 2—figure supplement 1 of the Supplementary Materials.

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

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

One of the main concerns from the original submission was the origin of the perivascular cells. The authors have addressed this and tested for possible neural crest (Sox10Cre-Ert2 and Wnt1Cre lines), neural tube (Olig2Cre line), and pial (Tbx18Cre-Ert2 line) origins. Sox10Cre-Ert2, Wnt1Cre lines, and Olig2Cre lines did not give rise to traced mural cells. With the Tbx18Cre-Ert2 line, tamoxifen was administered to pregnant females at E9.5, and embryos analyzed at E11.5. SOX10 negative traced cells were identified at E11.5 along the ventral root and spinal nerve. This was used to suggest a pial cell origin of perivascular cells. If possible, it would be beneficial to have a visual of the expression of Tbx18 at E9.5, to visualize the origin of the traced cells.

To address the question about the origin of traced cells, we performed RNAscope in

situ hybridization with the Tbx18 probe on E9.5 and E10.5 embryos, as tamoxifen-induced recombination is considered to take around 12-24h to occur. On these sections, we found Tbx18-expressing cells located around the neural tube, dorsal roots and dorsal root ganglia, in the area that corresponds to the developing pia matter.

We have added a supplemental figure (Figure 4—figure supplement 1) that contains the

RNAscope data at E9.5 and E10.5 with its corresponding legend on page 29. We have also updated the Results section to mention these results on Page 8 lines 22 to 25.

“Furthermore, at E9.5, we detected Tbx18 expression in the vicinity of the neural tube, in a layer of mesenchymal progenitors that gives rise to fibroblasts in the meninges (Figure 4-figure supplement 1), consistent with a previous report (DeSisto et al., 2020).”

It is also unclear why traced cells from the TBX18 line were not followed through to later stages (E14.5) where cells could be more stringently verified as mural cells by immunostaining for some mural cell markers (as in Figure 2A-D) and their morphology and migratory behavior could also be better verified as 'mural cell'.

We agree that additional fate mapping of the Tbx18 lineage at a later stage was

necessary, and as suggested we performed immunostaining on sections of Tbx18Cre-

Ert2/+,Rosa26RTom embryos at E14.5 that had received tamoxifen at E9.5. Near the neural tube, we found numerous traced cells around blood vessels that expressed mural cell markers such as NG2 and SMA, as well as cells around and within the developing spinal nerve that did not express Sox10. In the skin, we also found that traced cells did not express Sox10 and that some of them were in contact with blood vessels and expressed NG2. Overall, although caution is warranted given the preliminary nature of our analyses on the Tbx18 line, our observations are in line with the hypothesis of a pial origin of mural cells.

We have added a supplemental figure (Figure 4—figure supplement 2) that contains the

immunostaining data at E14.5 with its corresponding legend on page 29. We have also

updated the Results section to mention these data on Page 8 lines 22 to 25:

“At E14.5, we observed numerous Tom-positive cells along blood vessels co-expressing NG2 or/and SMA (Figure 4—figure supplement 2A-F, J-L). None of them express Sox10 (Figure 4-figure supplement 2G-I, M-O).”

https://doi.org/10.7554/eLife.69413.sa2

Article and author information

Author details

  1. Gaspard Gerschenfeld

    1. Institut de Biologie de l'Ecole normale supérieure (IBENS), Ecole normale supérieure, CNRS, Inserm, Université PSL, Paris, France
    2. Sorbonne Université, Collège Doctoral, Paris, France
    Contribution
    Conceptualization, Data curation, Software, Formal analysis, Investigation, Writing - original draft
    Contributed equally with
    Fanny Coulpier
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-2456-704X
  2. Fanny Coulpier

    1. Institut de Biologie de l'Ecole normale supérieure (IBENS), Ecole normale supérieure, CNRS, Inserm, Université PSL, Paris, France
    2. nstitut Mondor de Recherche Biomédicale, Inserm U955-Team 9, Créteil, France
    3. Genomic facility, Ecole normale supérieure, PSL Research University, CNRS, Inserm, Institut de Biologie de l'Ecole normale supérieure (IBENS), Paris, France
    Contribution
    Conceptualization, Data curation, Software, Formal analysis, Investigation, Methodology, Project administration, Writing – review and editing
    Contributed equally with
    Gaspard Gerschenfeld
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0009-0007-7638-3676
  3. Aurélie Gresset

    Institut de Biologie de l'Ecole normale supérieure (IBENS), Ecole normale supérieure, CNRS, Inserm, Université PSL, Paris, France
    Contribution
    Investigation
    Competing interests
    No competing interests declared
  4. Pernelle Pulh

    1. Institut de Biologie de l'Ecole normale supérieure (IBENS), Ecole normale supérieure, CNRS, Inserm, Université PSL, Paris, France
    2. nstitut Mondor de Recherche Biomédicale, Inserm U955-Team 9, Créteil, France
    Contribution
    Investigation
    Competing interests
    No competing interests declared
  5. Bastien Job

    Inserm US23, AMMICA, Institut Gustave Roussy, Villejuif, France
    Contribution
    Data curation, Software, Formal analysis, Investigation
    Competing interests
    No competing interests declared
  6. Thomas Topilko

    Laboratoire de Plasticité Structurale, Sorbonne Université, ICM Institut du Cerveau et de la Moelle Epinière, Inserm U1127, CNRS UMR7225, Paris, France
    Contribution
    Software, Formal analysis, Investigation
    Competing interests
    No competing interests declared
  7. Julie Siegenthaler

    Department of Pediatrics Section of Developmental Biology, University of Colorado Anschutz Medical Campus, Aurora, United States
    Contribution
    Investigation, Writing – review and editing
    Competing interests
    No competing interests declared
  8. Maria Eleni Kastriti

    1. Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden
    2. Department of Neuroimmunology, Center for Brain Research, Medical University Vienna, Vienna, Austria
    Contribution
    Investigation, Writing – review and editing
    Competing interests
    No competing interests declared
  9. Isabelle Brunet

    Inserm U1050, Centre Interdisciplinaire de Recherche en Biologie (CIRB), Collège de France, Paris, France
    Contribution
    Resources, Writing – review and editing
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-5490-2937
  10. Patrick Charnay

    Institut de Biologie de l'Ecole normale supérieure (IBENS), Ecole normale supérieure, CNRS, Inserm, Université PSL, Paris, France
    Contribution
    Supervision, Funding acquisition, Writing – review and editing
    Competing interests
    No competing interests declared
  11. Piotr Topilko

    1. Institut de Biologie de l'Ecole normale supérieure (IBENS), Ecole normale supérieure, CNRS, Inserm, Université PSL, Paris, France
    2. nstitut Mondor de Recherche Biomédicale, Inserm U955-Team 9, Créteil, France
    Contribution
    Conceptualization, Supervision, Funding acquisition, Investigation, Methodology, Writing - original draft, Writing – review and editing
    For correspondence
    piotr.topilko@inserm.fr
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-7381-6770

Funding

Agence Nationale de la Recherche (ANR-10-LABX-54 MEMOLIFE)

  • Patrick Charnay

Agence Nationale de la Recherche (ANR-11-IDEX-0001-02 PSL* Research University)

  • Patrick Charnay

Institut National de la Santé et de la Recherche Médicale

  • Patrick Charnay
  • Piotr Topilko

Centre National de la Recherche Scientifique

  • Patrick Charnay
  • Piotr Topilko

Institut National Du Cancer

  • Patrick Charnay
  • Piotr Topilko

Ministère de l'Enseignement Supérieur et de la Recherche Scientifique

  • Patrick Charnay
  • Piotr Topilko

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

Acknowledgements

We are grateful to the IBENS animal, Imaging and Genomic facilities. The IBENS imaging facility is a member of the national infrastructure of France-BioImaging, supported by the ANR (ANR-10-INBS-04, ANR-10-LABX-54 MEMO LIFE, ANR-11-IDEX-0001-02 PSL 'Investments for the future') and by the 'Région Ile-de-France' (NERF N°2011-45, DIM Cerveau et Pensée 'Alpins'). The IBENS Genomic Facility was supported by France Génomique, managed by the ANR (ANR-10-INBS-09). Funding: The Charnay laboratory was financed by INSERM, CNRS, MESRI, and INCa. It has received support under the program 'Investissements d’Avenir' with the references: ANR-10-LABX-54 MEMOLIFE and ANR-11-IDEX-0001-02 PSL* Research University.

Ethics

All animal manipulations were performed according to French and European Union regulations. According to these regulations, no ethics committee approval was required for this study which only used mouse embryos and newborns.

Senior and Reviewing Editor

  1. Marianne E Bronner, California Institute of Technology, United States

Reviewers

  1. Igor Adameyko, Karolinska Institutet, Sweden
  2. Tatiana Solovieva, California Institute of Technology, United States

Version history

  1. Received: April 14, 2021
  2. Accepted: December 12, 2023
  3. Accepted Manuscript published: December 14, 2023 (version 1)
  4. Version of Record published: January 12, 2024 (version 2)

Copyright

© 2023, Gerschenfeld, Coulpier 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.

Metrics

  • 291
    Page views
  • 83
    Downloads
  • 0
    Citations

Article citation count generated by polling the highest count across the following sources: Crossref, PubMed Central, Scopus.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

  1. Gaspard Gerschenfeld
  2. Fanny Coulpier
  3. Aurélie Gresset
  4. Pernelle Pulh
  5. Bastien Job
  6. Thomas Topilko
  7. Julie Siegenthaler
  8. Maria Eleni Kastriti
  9. Isabelle Brunet
  10. Patrick Charnay
  11. Piotr Topilko
(2023)
Neural tube-associated boundary caps are a major source of mural cells in the skin
eLife 12:e69413.
https://doi.org/10.7554/eLife.69413

Share this article

https://doi.org/10.7554/eLife.69413

Further reading

    1. Developmental Biology
    2. Evolutionary Biology
    Paul Knabl, Alexandra Schauer ... Grigory Genikhovich
    Research Article

    BMP signaling has a conserved function in patterning the dorsal-ventral body axis in Bilateria and the directive axis in anthozoan cnidarians. So far, cnidarian studies have focused on the role of different BMP signaling network components in regulating pSMAD1/5 gradient formation. Much less is known about the target genes downstream of BMP signaling. To address this, we generated a genome-wide list of direct pSMAD1/5 target genes in the anthozoan Nematostella vectensis, several of which were conserved in Drosophila and Xenopus. Our ChIP-seq analysis revealed that many of the regulatory molecules with documented bilaterally symmetric expression in Nematostella are directly controlled by BMP signaling. We identified several so far uncharacterized BMP-dependent transcription factors and signaling molecules, whose bilaterally symmetric expression may be indicative of their involvement in secondary axis patterning. One of these molecules is zswim4-6, which encodes a novel nuclear protein that can modulate the pSMAD1/5 gradient and potentially promote BMP-dependent gene repression.

    1. Biochemistry and Chemical Biology
    2. Developmental Biology
    Sima Stroganov ,  Talia   Harris  ... Michal Neeman
    Research Article

    Background: Fetal growth restriction (FGR) is a pregnancy complication in which a newborn fails to achieve its growth potential, increasing the risk of perinatal morbidity and mortality. Chronic maternal gestational hypoxia, as well as placental insufficiency are associated with increased FGR incidence; however, the molecular mechanisms underlying FGR remain unknown.

    Methods: Pregnant mice were subjected to acute or chronic hypoxia (12.5% O2) resulting in reduced fetal weight. Placenta oxygen transport was assessed by blood oxygenation level dependent (BOLD) contrast magnetic resonance imaging (MRI). The placentae were analyzed via immunohistochemistry and in situ hybridization. Human placentae were selected from FGR and matched controls and analyzed by immunohistochemistry (IHC). Maternal and cord sera were analyzed by mass spectrometry.

    Results: We show that murine acute and chronic gestational hypoxia recapitulates FGR phenotype and affects placental structure and morphology. Gestational hypoxia decreased labyrinth area, increased the incidence of red blood cells (RBCs) in the labyrinth while expanding the placental spiral arteries (SpA) diameter. Hypoxic placentae exhibited higher hemoglobin-oxygen affinity compared to the control. Placental abundance of Bisphosphoglycerate mutase (BPGM) was upregulated in the syncytiotrophoblast and spiral artery trophoblast cells (SpA TGCs) in the murine gestational hypoxia groups compared to the control. Hif1a levels were higher in the acute hypoxia group compared to the control. In contrast, human FGR placentae exhibited reduced BPGM levels in the syncytiotrophoblast layer compared to placentae from healthy uncomplicated pregnancies. Levels of 2,3 BPG, the product of BPGM, were lower in cord serum of human FGR placentae compared to control. Polar expression of BPGM, was found in both human and mouse placentae syncytiotrophoblast, with higher expression facing the maternal circulation. Moreover, in the murine SpA TGCs expression of BPGM was concentrated exclusively in the apical cell side, in direct proximity to the maternal circulation.

    Conclusions: This study suggests a possible involvement of placental BPGM in maternal-fetal oxygen transfer, and in the pathophysiology of FGR.

    Funding: This work was supported by the Weizmann Krenter Foundation and the Weizmann - Ichilov (Tel Aviv Sourasky Medical Center) Collaborative Grant in Biomedical Research, and by the Minerva Foundation (to MN), by the ISF KillCorona grant 3777/19 (to MN, MK).