NC-derived Cells Research Articles

IN VITRO PROPERTIES OF NEURAL CREST-DERIVED MULTIPOTENT STEM CELLS FROM A BULGE REGION OF WHISKER FOLLICLE

71 The neural crest (NC), a transient structure in embryo development, is unique to vertebrates. NC is derived from the ectoderm and formed at the border of neuroectoderm and somatic ectoderm. Originally NC is represented by a cell population lying in the form of epithelial cords emerging from both sides of the neural tube. At the neurulation process end the NC cells undergo an epithelialmesenchymal transition (EMT) and migrate distantly in various tissues and organs of the developing embryo, where they give rise to the multiple differentiated cell types [1, 2]. In adult vertebrate organism the NC derivatives are represented by the following cell types: neurons and glial cells of the peripheral nervous system, the majority of sensory neurons, melanocytes, Merkel cells, odontoblasts, stromal and endothelial cells of cornea, C cells of thyroid gland, the adrenal medulla, a part of thymic stroma [1, 2]. The most plastic are the cells of cranial NC. In addition to the above mentioned differentiated cell types, they form a part of the bone, cartilage and connective tissues of the face, forehead and ventral neck [3, 4]. Unusually wide differentiation potential of the NC cells suggests an existence of multipotent stem cells. Stemple and Anderson firstly revealed the existence of multipotent stem cells in the NC in early embryonic stage of mammalian development [5]. A long time it was thought that, like NC per se, neural crestderived multipotent stem cells (NC-MSCs) have a transient nature. Consequently, the NCMSCs exist only at the premigratory (from the NC specification moment until the beginning of its cell migration) and migratory stages (from the NC cell delamination until their incoming into the sites of final destination), and further, within migration to sites of its final localization, undergo a process of commitment, turning into the progenitor cells with limited proliferative and/or differentiation potential [6, 7]. An example is the transition of NC-MSCs into the melanocyte precursors — melanoblasts. In the last decades, NC-derived cells have been isolated from a number of tissues and organs of the adult mammalians. These cells demonstrate the ability to self-renewal and multilineage differentiation, at least in vitro, i.e., they are multipotent stem cells [8–11]. The origin of these cells from NC was proved by creating special lines of transgenic mice [12, 13], as well as by means of their transplantation in the developing chick embryo, where the NC-MSCs migrated via the UDC 576.3+612.014.2/3

Neural crest‐derived cells sustain their multipotency even after entry into their target tissues

Neural crest cells (NC cells) are highly migratory multipotent cells. Their multipotency is transient at the early stage of their generation; soon after emerging from the neural tube, these cells turn into lineage-restricted precursors. However, recent studies have disputed this conventionally believed paradigm. In this study, we analyzed the differentiation potency of NC-derived cells after their arrival at target tissues. Using Sox10-IRES-Venus mice, we found that the NC-derived cells in the skin, DRG, and inner ear could be divided into two populations: Sox10-positive/Kit-negative cells (Sox10+/Kit- cells) and Sox10- and Kit-positive cells (Sox10+/Kit+ cells). Only the Sox10+/Kit- cells were detected in the intestines. Unexpectedly, the Sox10+/Kit+ cells differentiated into neurons, glial cells, and melanocytes, showing that they had maintained their multipotency even after having entered the target tissues. The Sox10+/Kit+ cells in the DRG maintained their multipotency for a restricted period during the earlier embryonic stages, whereas those in the skin and inner ear were multipotent yet even in later embryonic stages. We showed that NC-derived Sox10+/Kit+ cells maintained their multipotency even after entry into the target tissues. This unexpected differentiation potency of these cells in tissues seems to have been strictly restricted by the tissue microenvironment.

Human Adult Periodontal Ligament-Derived Cells Integrate and Differentiate after Implantation into the Adult Mammalian Brain

Previous studies suggest that neural crest (NC)-derived stem cells may reside in NC derivatives including the human periodontal ligament (hPDL). The isolation and manipulation of autologous NC-derived cells could be an accessible source of adult neural stem cells for their use in cell replacement and gene transfer to the diseased central nervous system. In this study, we examined the expression of NC markers and neural differentiation potential of hPDL-derived cells both in vitro and in vivo. In vitro we found that hPDL-derived cells expressed stem cell markers (Oct3/4, Nestin, Sox2, and Musashi-1) and a subset of NC cell markers (Slug, p75(NTR), Twist, and Sox9). hPDL-derived cells differentiated into neural-like cells based on cellular morphology and neural marker expression (TUJ1, MAP2, MAP1b, GAD65/67, GABA, NeuN, ChAT, GAT1, synaptophysin, GFAP, NG2, and O4). In vivo, hPDL-derived cells survive, migrate, and give rise to DCX(+), NF-M(+), GABA(+), GFAP(+), and NG2(+) cells after grafting the adult mouse brain. Some of the grafted hPDL-derived cells were located in stem cell niches such as the ventricular epithelium and the subventricular zone of the anterolateral ventricle wall as well as in the subgranular zone of the hippocampal dentate gyrus. Thus, the hPDL contains stem cells that originate from the NC and can differentiate into neural cells. The engraftment and differentiation properties of hPDL-derived cells in the adult brain indicate that they are a potential stem cell source to be used in neuroregenerative and/or neurotrophic medicine.

Origins and Properties of Dental, Thymic, and Bone Marrow Mesenchymal Cells and Their Stem Cells

Mesenchymal cells arise from the neural crest (NC) or mesoderm. However, it is difficult to distinguish NC-derived cells from mesoderm-derived cells. Using double-transgenic mouse systems encoding P0-Cre, Wnt1-Cre, Mesp1-Cre, and Rosa26EYFP, which enabled us to trace NC-derived or mesoderm-derived cells as YFP-expressing cells, we demonstrated for the first time that both NC-derived (P0- or Wnt1-labeled) and mesoderm-derived (Mesp1-labeled) cells contribute to the development of dental, thymic, and bone marrow (BM) mesenchyme from the fetal stage to the adult stage. Irrespective of the tissues involved, NC-derived and mesoderm-derived cells contributed mainly to perivascular cells and endothelial cells, respectively. Dental and thymic mesenchyme were composed of either NC-derived or mesoderm-derived cells, whereas half of the BM mesenchyme was composed of cells that were not derived from the NC or mesoderm. However, a colony-forming unit-fibroblast (CFU-F) assay indicated that CFU-Fs in the dental pulp, thymus, and BM were composed of NC-derived and mesoderm-derived cells. Secondary CFU-F assays were used to estimate the self-renewal potential, which showed that CFU-Fs in the teeth, thymus, and BM were entirely NC-derived cells, entirely mesoderm-derived cells, and mostly NC-derived cells, respectively. Colony formation was inhibited drastically by the addition of anti-platelet–derived growth factor receptor-β antibody, regardless of the tissue and its origin. Furthermore, dental mesenchyme expressed genes encoding critical hematopoietic factors, such as interleukin-7, stem cell factor, and cysteine-X-cysteine (CXC) chemokine ligand 12, which supports the differentiation of B lymphocytes and osteoclasts. Therefore, the mesenchymal stem cells found in these tissues had different origins, but similar properties in each organ.

Identification and characterization of neural crest-derived cells in adult periodontal ligament of mice

ObjectiveCells derived from the neural crest (NC) contribute to the development of several adult tissues, including tooth and periodontal tissue. Here, two transgenic lines, Wnt1-Cre/ZEG and P0-Cre/ZEG, were analysed to determine the fate and distribution of neural crest cells (NCCs) in adult mouse periodontal ligament (PDL). DesignParaffin-embedded and decalcified histology samples were prepared from Wnt1-Cre/ZEG and P0-Cre/ZEG mice that were 4-, 8-, or 12-weeks old. Expression of GFP (NC-derived cells), NC-markers (Slug, AP-2 alpha, HNK-1, p75NTR and Nestin), and mesenchymal stem cell markers (CD29 and STRO-1) were examined using immunohistochemistry. ResultsIn four-week-old Wnt1-Cre/ZEG mice, GFP(+) NC-derived cells were specifically detected in the mid-zone of PDL. The GFP(+) cells constituted 1.4% of all cells in PDL, and this percentage decreased as the mice aged. The distribution and prevalence of GFP(+) cells were comparable between Wnt1-Cre/ZEG and P0-Cre/ZEG mice. NC-marker(+) cells were expressed only in GFP(+) cells while MSC markers were detected only in GFP(−) cells. ConclusionThe prevalence and specific distribution of NC-derived cells in adult PDL of Wnt1-Cre/ZEG and P0-Cre/ZEG mouse were examined. Interestingly, various NC markers, including markers for undifferentiated NCCs, were still expressed at high levels in GFP(+) cells. These observations may indicate that labelled cells in the Wnt1-Cre/ZEG and P0-Cre/ZEG mice did not constituted all NC-derived cells, but rather an interesting subset of NC-derived cells. These findings may be useful in understanding the homeostatic character of the PDL and contribute to establishing successful periodontal tissue maintenance.

Neural crest contribution to lingual mesenchyme, epithelium and developing taste papillae and taste buds

The epithelium of mammalian tongue hosts most of the taste buds that transduce gustatory stimuli into neural signals. In the field of taste biology, taste bud cells have been described as arising from “local epithelium”, in distinction from many other receptor organs that are derived from neurogenic ectoderm including neural crest (NC). In fact, contribution of NC to both epithelium and mesenchyme in the developing tongue is not fully understood. In the present study we used two independent, well-characterized mouse lines, Wnt1-Cre and P0-Cre that express Cre recombinase in a NC-specific manner, in combination with two Cre reporter mouse lines, R26R and ZEG, and demonstrate a contribution of NC-derived cells to both tongue mesenchyme and epithelium including taste papillae and taste buds. In tongue mesenchyme, distribution of NC-derived cells is in close association with taste papillae. In tongue epithelium, labeled cells are observed in an initial scattered distribution and progress to a clustered pattern between papillae, and within papillae and early taste buds. This provides evidence for a contribution of NC to lingual epithelium. Together with previous reports for the origin of taste bud cells from local epithelium in postnatal mouse, we propose that NC cells migrate into and reside in the epithelium of the tongue primordium at an early embryonic stage, acquire epithelial cell phenotypes, and undergo cell proliferation and differentiation that is involved in the development of taste papillae and taste buds. Our findings lead to a new concept about derivation of taste bud cells that include a NC origin.

Reduced bone morphogenetic protein receptor type 1A signaling in neural-crest-derived cells causes facial dysmorphism.

SUMMARYBone morphogenetic protein (BMP) receptor type 1A (BMPR1A) mutations are associated with facial dysmorphism, which is one of the main clinical signs in both juvenile polyposis and chromosome 10q23 deletion syndromes. Craniofacial development requires reciprocal epithelial/neural crest (NC)-derived mesenchymal interactions mediated by signaling factors, such as BMP, in both cell populations. To address the role of mesenchymal BMP signaling in craniofacial development, we generated a conditional knockdown mouse by expressing the dominant-negative Bmpr1a in NC-derived cells expressing the myelin protein zero(Mpz)-Cre transgene. At birth, 100% of the conditional mutant mice had wide-open anterior fontanelles, and 80% of them died because of cleft face and cleft palate soon after birth. The other 20% survived and developed short faces, hypertelorism and calvarial foramina. Analysis of the NC-derived craniofacial mesenchyme of mutant embryos revealed an activation of the P53 apoptosis pathway, downregulation of both c-Myc and Bcl-XL, a normal growth rate but an incomplete expansion of mesenchymal cells. These findings provide genetic evidence indicating that optimal Bmpr1a-mediated signaling is essential for NC-derived mesenchymal cell survival in both normal nasal and frontal bone development and suggest that our model is useful for studying some aspects of the molecular etiology of human craniofacial dysmorphism.

The dual origin of the peripheral olfactory system: placode and neural crest

BackgroundThe olfactory epithelium (OE) has a unique capacity for continuous neurogenesis, extending axons to the olfactory bulb with the assistance of olfactory ensheathing cells (OECs). The OE and OECs have been believed to develop solely from the olfactory placode, while the neural crest (NC) cells have been believed to contribute only the underlying structural elements of the olfactory system. In order to further elucidate the role of NC cells in olfactory development, we examined the olfactory system in the transgenic mice Wnt1-Cre/Floxed-EGFP and P0-Cre/Floxed-EGFP, in which migrating NC cells and its descendents permanently express GFP, and conducted transposon-mediated cell lineage tracing studies in chick embryos.ResultsExamination of these transgenic mice revealed GFP-positive cells in the OE, demonstrating that NC-derived cells give rise to OE cells with morphologic and antigenic properties identical to placode-derived cells. OECs were also positive for GFP, confirming their NC origin. Cell lineage tracing studies performed in chick embryos confirmed the migration of NC cells into the OE. Furthermore, spheres cultured from the dissociated cells of the olfactory mucosa demonstrated self-renewal and trilineage differentiation capacities (neurons, glial cells, and myofibroblasts), demonstrating the presence of NC progenitors in the olfactory mucosa.ConclusionOur data demonstrates that the NC plays a larger role in the development of the olfactory system than previously believed, and suggests that NC-derived cells may in part be responsible for the remarkable capacity of the OE for neurogenesis and regeneration.

Novel Technique for Inducing Neural Crest Fate in Embryonic Stem Cells (Stem Cells 2009;27:2896-2905)

Hotta et al1 has reported a study entitled Small-molecule induction of neural crest-like cells derived from human neural progenitors in the December 2009 issue of Stem Cells. Neural crest (NC) cells are ectoderm-derived multipotent migratory cells that give rise to diverse cell lineages in a developing embryo. Migratory or proliferative deficit of NC cells results in many developmental disorders. Stem cell-based therapies are potential treatment options for such disorders. The enteric nervous system (ENS) of vertebrates predominantly develops from NC cells originating from the vagal end of the neural tube. In Hirschsprung's disease, the distal bowel lacks an ENS caused by a failure of colonization by NC-derived cells. This study shows a simple method of coculture with mouse embryonic fibroblasts (mEFs) for generating NC-like cells from human embryonic stem cells (hESCs). The authors also demonstrated that subsequent exposure to Y27632, a selective inhibitor of the Rho-associated coiled kinase (ROCK), significantly increased the efficiency of differentiation into NC-like cells. These hESC-derived NC-like cells expressed NC markers including p75 (p75NTR low affinity neurotrophin receptor), SoxE (Sox8, Sox9 and Sox10) and HNK1 (human natural killer-1 antigen). The mEFs induced NC-like cells were also able to migrate along NC pathways in avian embryos and differentiate into cells expressing neuronal and glial markers. The hESC-derived NC-like cells also demonstrated migration in mouse gut explants, however, Y27632 treatment was required for differentiation into neuronal and glial cells.

SOX10 directly modulates ERBB3 transcription via an intronic neural crest enhancer

BackgroundThe ERBB3 gene is essential for the proper development of the neural crest (NC) and its derivative populations such as Schwann cells. As with all cell fate decisions, transcriptional regulatory control plays a significant role in the progressive restriction and specification of NC derived lineages during development. However, little is known about the sequences mediating transcriptional regulation of ERBB3 or the factors that bind them.ResultsIn this study we identified three transcriptional enhancers at the ERBB3 locus and evaluated their regulatory potential in vitro in NC-derived cell types and in vivo in transgenic zebrafish. One enhancer, termed ERBB3_MCS6, which lies within the first intron of ERBB3, directs the highest reporter expression in vitro and also demonstrates epigenetic marks consistent with enhancer activity. We identify a consensus SOX10 binding site within ERBB3_MCS6 and demonstrate, in vitro, its necessity and sufficiency for the activity of this enhancer. Additionally, we demonstrate that transcription from the endogenous Erbb3 locus is dependent on Sox10. Further we demonstrate in vitro that Sox10 physically interacts with that ERBB3_MCS6. Consistent with its in vitro activity, we also show that ERBB3_MCS6 drives reporter expression in NC cells and a subset of its derivative lineages in vivo in zebrafish in a manner consistent with erbb3b expression. We also demonstrate, using morpholino analysis, that Sox10 is necessary for ERBB3_MCS6 expression in vivo in zebrafish.ConclusionsTaken collectively, our data suggest that ERBB3 may be directly regulated by SOX10, and that this control may in part be facilitated by ERBB3_MCS6.

Ovo1 links Wnt signaling with N-cadherin localization during neural crest migration

A fundamental issue in cell biology is how migratory cell behaviors are controlled by dynamically regulated cell adhesion. Vertebrate neural crest (NC) cells rapidly alter cadherin expression and localization at the cell surface during migration. Secreted Wnts induce some of these changes in NC adhesion and also promote specification of NC-derived pigment cells. Here, we show that the zebrafish transcription factor Ovo1 is a Wnt target gene that controls migration of pigment precursors by regulating the intracellular movements of N-cadherin (Ncad). Ovo1 genetically interacts with Ncad and its depletion causes Ncad to accumulate inside cells. Ovo1-deficient embryos strongly upregulate factors involved in intracellular trafficking, including several rab GTPases, known to modulate cellular localization of cadherins. Surprisingly, NC cells express high levels of many of these rab genes in the early embryo, chemical inhibitors of Rab functions rescue NC development in Ovo1-deficient embryos and overexpression of a Rab-interacting protein leads to similar defects in NC migration. These results suggest that Ovo proteins link Wnt signaling to intracellular trafficking pathways that localize Ncad in NC cells and allow them to migrate. Similar processes probably occur in other cell types in which Wnt signaling promotes migration.

PTH-046 Enteric nervous system stem cell-containing neurospheres: all that glitters is not gold?

Introduction Enteric nervous system (ENS) stem cells provide potential therapeutic tools for hitherto incurable gut neuropathies. Recent progress confirms they can be generated from human gut in cell aggregates or neurospheres (NS), but numerous challenges remain. Although previous studies have suggested that NS are a source of putative stem cells, can be successfully propagated and expanded in culture, and transplanted, few have assessed their purity and implications for clinical use. The aim of this study was to characterise the cellular content of NS generated and propagated in culture and identify putative cellular markers for better selective isolation or enrichment of ENS stem cells for therapy. Methods Transgenic Wnt1-Cre/Floxed-YFP mice (express yellow fluorescent protein (YFP) in all Neural Crest (NC)-derived and ENS cells) were used. FACS-selection of YFP-positive cells from dissociated gut was used to isolate NC-derived cells to generate NS, which were then compared to those from YFP-negative (non-NC) or unsorted (mixed – akin to current protocols for human ENS stem cells) gut cells. Using immunostaining and clonogenic studies putative markers were then quantitatively assessed across all cultures for their ability to enrich for ENS stem cells, and studies applied to human tissue. Result All cell fractions (unsorted (US), sorted YFP-positive (SoP) and sorted YFP-negative (SoN)) contained cells capable of generating NS. US NS showed partial YFP fluorescence unlike those from SoP (pure YFP) and SoN (no YFP). SoP NS appeared perfectly spherical unlike the others which were irregular and formed faster. NS characterisation showed that US NS contained both NC cells (YFP positive+any of-Tuj1, GFAP, p75, Sox10) and significant numbers of non-NC cells (positive for any of SMA, c-kit, cytokeratin). SoP NS contained NC cells only and SoN NS non-NC cells only. Both the precursor marker Nestin and proliferation marker PH3 were positive in both NC and non-NC cells although PH3 showed higher expression in non-NC cells. NS from all sources could be propagated and expanded in culture over multiple passages, but this did not alter the mixed (NC/non-NC) cellular content of US NS. The low affinity neurotrophin receptor p75 could be used to FACS-sort YFP-positive cells from dissociated gut (mixed NC/non-NC), and was also expressed in NS generated from human gut. Conclusion Current protocols to generate ENS stem cell-containing NS for transplantation need refinement to reduce contamination with proliferating non-NC cells, which may carry safety implications if inadvertently transplanted in conditions of specific neuronal loss (eg, Hirschsprung9s disease). Putative markers (eg, p75) may enable selective isolation or enrichment of ENS stem cells for clinical use.

SOX10 structure-function analysis in the chicken neural tube reveals important insights into its role in human neurocristopathies

The HMG-domain containing transcription factor Sox10 is essential for neural crest (NC) development and for oligodendrocyte differentiation. Heterozygous SOX10 mutations in humans lead to corresponding defects in several NC-derived lineages and to leukodystrophies. Disease phenotypes range from Waardenburg syndrome and Waardenburg-Hirschsprung disease to Peripheral demyelinating neuropathy, Central dysmyelination, Waardenburg syndrome and Hirschsprung disease (PCWH). The phenotypic variability can partly be explained by the action of modifier genes, but is also influenced by the mutation that leads to haploinsufficiency in some and to mutant SOX10 proteins with altered properties in other cases. Here, we used in ovo electroporation in the developing neural tube of chicken to determine which regions and properties of SOX10 are required for early NC development. We found a strict reliance on the DNA-binding activity and the presence of the C-terminal transactivation domain and a lesser influence of the dimerization function and a conserved domain in the center of the protein. Intriguingly, dominant-negative effects on early NC development were mostly observed for truncated SOX10 proteins whose production in patients is probably prevented by nonsense-mediated decay. In contrast, mutant SOX10 proteins that occur in patients were usually inactive. Any dominant negative activity which some of these mutants undoubtedly possess must, therefore, be restricted to single NC-derived cell lineages or oligodendrocytes at later times. This contributes to the phenotypic variability of human SOX10 mutations.

Melanocytes don’t always take the high road

A central dogma of pigment cell development has been that the truncal neural crest (NC) cells that will ultimately give rise to skin melanocytes migrate under the epidermis along a dorsolateral route in developing embryos, while NC cells migrating along the ventral pathway between the neural tube and the dermamyotome contribute to the neurons and glia of the peripheral nervous system (Figure 1A) (Le Douarin and Kalcheim, 1999). The seminal work by Adameyko et al., recently published in Cell (Adameyko et al., 2009), challenges this simplistic dichotomy by providing strong evidence that NC derivatives in the ventral pathway are also an important origin of melanocytes in the skin.

Small-Molecule Induction of Neural Crest-like Cells Derived from Human Neural Progenitors

Neural crest (NC) cells are stem cells that are specified within the embryonic neuroectodermal epithelium and migrate to stereotyped peripheral sites for differentiation into many cell types. Several neurocristopathies involve a deficit of NC-derived cells, raising the possibility of stem cell therapy. In Hirschsprung's disease the distal bowel lacks an enteric nervous system caused by a failure of colonization by NC-derived cells. We have developed a robust method of producing migrating NC-like cells from human embryonic stem cell-derived neural progenitors using a coculture system of mouse embryonic fibroblasts. Significantly, subsequent exposure to Y27632, a small-molecule inhibitor of the Rho effectors ROCKI/II, dramatically increased the efficiency of differentiation into NC-like cells, identified by marker expression in vitro. NC-like cells derived by this method were able to migrate along NC pathways in avian embryos in ovo and within explants of murine bowel, and to differentiate into cells with neuronal and glial markers. This is the first study to report the use of a small molecule to induce cells with NC characteristics from embryonic stem cells that can migrate and generate neurons and support cells in complex tissue. Furthermore, this study demonstrates that small-molecule regulators of ROCKI/II signaling may be valuable tools for stem cell research aimed at treatment of neurocristopathies.

Stage-Specific Control of Neural Crest Stem Cell Proliferation by the Small Rho GTPases Cdc42 and Rac1

The neural crest (NC) generates a variety of neural and non-neural tissues during vertebrate development. Both migratory NC cells and their target structures contain cells with stem cell features. Here we show that these populations of neural crest-derived stem cells (NCSCs) are differentially regulated by small Rho GTPases. Deletion of either Cdc42 or Rac1 in the NC results in size reduction of multiple NC target structures because of increased cell-cycle exit, while NC cells emigrating from the neural tube are not affected. Consistently, Cdc42 or Rac1 inactivation reduces self-renewal and proliferation of later stage, but not early migratory NCSCs. This stage-specific requirement for small Rho GTPases is due to changes in NCSCs that, during development, acquire responsiveness to mitogenic EGF acting upstream of both Cdc42 and Rac1. Thus, our data reveal distinct mechanisms for growth control of NCSCs from different developmental stages.

Lumbo-sacral neural crest derivatives fate mapped with the aid of Wnt-1 promoter integrate but are not essential to kidney development

Neural crest (NC) cells may be involved in kidney organogenesis by providing inductive signals and contributing to cells of the renal stroma. We show here that the lumbo-sacral NC cells fate mapped with the aid of Wnt-1 promoter in the mouse migrate close to the metanephros at the initiation of organogenesis but these cells remain superficial to the condensed Pax2-expressing mesenchymal cells. NC-derived cells enter later into the kidney proper from the midline region. The NC cells contribute also to development of the extra-adrenal para-aortic bodies, Zuckerkandl's bodies and the nerve cord of the sympathetic nervous system. Splotch ( Sp 2H / Sp 2H ) embryos, having a NC defect in the lumbo-sacral region, develop a normal metanephros even though the kidney does not express the NC markers Sox10, Phox2b and tyrosine hydroxylase. Consistent with the histological findings, the kidneys of Sp 2H / Sp 2H embryos also express the stromal genes Foxd1, Hoxa10 and RARβ normally. Wnt-1 promoter-marked wild-type LacZ NC cells migrate intensely from the heterologous inducer tissue of the embryonic dorsal spinal cord (SPC) to the kidney mesenchyme, but tubule induction does not depend on NC migration, since the Sp 2H / Sp 2H SPC also induces tubulogenesis. The Sp 2H / Sp 2H mesenchyme also remains competent for tubulogenesis. We conclude that the NC cells fate mapped with the aid of Wnt-1 promoter migrate to the close to the metanephros and form later derivatives integrating with the kidney, but they may not be essential to the development of the stromal cells nor they may provide critical morphogenetic signals to regulate early kidney development in vivo.

Abstract 3937: Generation and Analysis of Neural Crest- and Endothelial-specific Mutant Mice for Foxc1

The forkhead transcription factor Foxc1 has been implicated in craniofacial, ocular and cardiovascular development. Foxc1 is expressed in mesoderm- and neural crest (NC)-derived cells, including endothelial cells (ECs) of the heart and blood vessels and vascular smooth muscle cells. However, the precise role of Foxc1 in specific cell types still remains unclear. Therefore, to define the distinct function of Foxc1 in NC and EC, we have generated conditional mutant mice for Foxc1 crossed with either Tie2-Cre or Wnt1-Cre mice. EC-specific Foxc1 mutants survive until adulthood with no apparent embryonic defects, although they show vascular abnormalities in the adult. By contrast, NC-specific Foxc1 mutants die perinately with haemorrhagic hydrocephalus, rudimentary frontal bones, and abnormal patterning of the aortic arch. NC-derived cells also give rise to the stroma and endothelium of the cornea, an avascular organ whose transparency is critical for vision. Importantly, we found that NC-specific Foxc1 mutants show failure of the formation of the anterior chamber and corneal endothelium in the eye. Mutant corneal stroma is much thicker than normal with increased cell proliferation. Most Intriguingly, NC-specific Foxc1 mutants exhibit ectopic neovascularization in the cornea with significant upregulation of Mmp9, sFlt1, Flt1 and Tek at E15.5, while Vegfa and Fgf expression is not changed. By contrast, the cornea of EC-specific Foxc1 mutants is normally formed and avascular. These data suggest that the cell-autonomous function of Foxc1 in the neural crest is essential for craniofacial and cardiovascular development and that Foxc1 plays an important role in inhibition of vascular formation in the cornea.

IN THIS ISSUE

![Figure][1]</img> Contact between neural crest (NC)-derived mesenchymal cells and thymic epithelial cells has been shown to be important in early thymic development, but whether NC-derived cells are relevant to the mature thymus is disputed. Müller et al. (p. [5344][2] ) developed a fate mapping

Neural crest origin of perivascular mesenchyme in the adult thymus.

The endodermal epithelial thymus anlage develops in tight association with neural crest (NC)-derived mesenchyme. This epithelial-NC interaction is crucial for thymus development, but it is not known how NC supports thymus development or whether NC cells or their progeny make any significant contribution to the adult thymus. By nude mouse blastocyst complementation and by cell surface phenotype, we could previously separate thymus stroma into Foxn1-dependent epithelial cells and a Foxn1-independent mesenchymal cell population. These mesenchymal cells expressed vascular endothelial growth factor-A, and contributed to thymus vascularization. These data suggested a physical or functional association with thymic blood vessels, but the origin, location in the thymus, and function of these stromal cells remained unknown. Using a transgenic mouse expressing Cre recombinase in premigratory NC (Sox10-Cre), we have now fate-mapped the majority of these adult mesenchymal cells to a NC origin. NC-derived cells represent tightly vessel-associated pericytes that are sandwiched between endothelium and epithelium along the entire thymus vasculature. The ontogenetic, phenotypic, and positional definition of this distinct perivascular mesenchymal compartment provides a cellular basis for the role of NC in thymus development and possibly maintenance, and might be useful to address properties of the endothelial-epithelial barrier in the adult thymus.