Colonization of the murine hindgut by sacral crest-derived neural precursors: experimental support for an evolutionarily conserved model

Enteric ganglia in the hindgut are derived from separate vagal and sacral neural crest populations. Two conflicting models, based primarily on avian data, have been proposed to describe the contribution of sacral neural crest cells. One hypothesizes early colonization of the hindgut shortly after neurulation, and the other states that sacral crest cells reside transiently in the extraenteric ganglion of Remak and colonize the hindgut much later, after vagal crest-derived neural precursors arrive. In this study, I show that Wnt1-lacZ-transgene expression, an "early" marker of murine neural crest cells, is inconsistent with the "early-colonization" model. Although Wnt1-lacZ-positive sacral crest cells populate pelvic ganglia in the mesenchyme surrounding the hindgut, they are not found in the gut prior to the arrival of vagal crest cells. Similarly, segments of murine hindgut harvested prior to the arrival of vagal crest cells and grafted under the renal capsule fail to develop enteric neurons, unless adjacent pelvic mesenchyme is included in the graft. When pelvic mesenchyme from DbetaH-nlacZ transgenic embryos is apposed with nontransgenic hindgut, neural precursors from the mesenchyme colonize the hindgut and form intramural ganglion cells that express the transgenic marker. Contribution of sacral crest-derived cells to the enteric nervous system is not affected by cocolonization of grafts by vagal crest-derived neuroglial precursors. The findings complement recent studies of avian chimeras and support an evolutionarily conserved model in which sacral crest cells first colonize the extramural ganglion and secondarily enter the hindgut mesenchyme.     

Temporal Expression of HNK-1-Reactive Sulfoglucuronyl Glycolipid in Cultured Quail Trunk Neural Crest Cells: Comparison with Other Developmentally Regulated Glycolipids

Monoclonal antibody HNK-1 is an important marker for embryonic neural crest cells and some of their differentiated derivatives. We have identified 3-sulfoglucuronylneolactotetraosylceramide (SGGL-1) as one of the HNK-1 antigens present in cultures of trunk neural crest cells. This lipid was present at 2 days in vitro and increased in amount with time in culture. Other major HNK-1-reactive antigens present in the culture were glycoproteins of apparent molecular masses of 120, 180, and 200 kDa. The 180- and 200-kDa bands were present at 2, 7, and 17 days in vitro, whereas the 120-kDa band was present only at 17 days in vitro. Gangliosides GD3, LD1, and LM1 were also found in the cultures and exhibited distinct temporal patterns of expression. Ganglioside GD3 was present at all stages examined and its expression peaked at 7 days in vitro. In contrast, LD1 was present only at 2 days in vitro and was not detectable at later times. Ganglioside LM1 increased in amount with time in culture in a pattern similar to that seen for SGGL-1. Taken together, these results indicate that several HNK-1-reactive molecules are expressed in neural crest cultures in a temporally regulated manner along with several glycolipids that do not bear this epitope.     

Expression of the HNK-1/NC-1 epitope in early vertebrate neurogenesis

Summary A family of glycoconjugates has recently been shown to share a common carbohydrate epitope recognized by the mouse monoclonal antibody HNK-1. The specificity of HNK-1 was found to be similar to that of another monoclonal antibody, NC-1. These two IgM monoclonal antibodies were raised after immunization of mice with a human T-cell line and avian neural crest-derived ganglia, respectively. The antigens recognized by these antibodies include the myelin-associated glycoprotein, MAG, a glycolipid of defined structure, and a set of molecules involved in cell adhesion. The timing and pattern of appearance of these antigens are distinct. Moreover, the epitope may be absent on an antigen at a given stage or in a given tissue. Therefore, although the molecules able to carry the NC-1/ HNK-1 epitope are numerous and expressed in various tissues, the use of the monoclonal antibodies on tissue sections has proven adequate for following the migration of avian neural crest cells, the major cell lineage recognized by NC-1 and HNK-1 during early embryogenesis. Analogies in several other species have been found on the basis of HNK-1 reactivity. In this study we show that NC-1 and HNK-1 can be used successfully to label migrating neural crest cells in dog, pig and human. On the other hand, the NC-l/HNK-1 epitope was not present on migrating crest cells in amphibians or mice and was found only transiently on the neural crest of rats.     

Characterization of glycoconjugate antigens in mouse embryonic neural precursor cells

Neuronal and glial cells organizing the central nervous system (CNS) are generated from common neural precursor cells (NPCs) during neural development. However, the expression of cell-surface glycoconjugates that are crucial for determining the properties and biological function of these cells at different stages of development has not been clearly defined. In this study, we investigated the expression of several stage-specific glycoconjugate antigens, including several b-series gangliosides GD3, 9-O-acetyl GD3, GT1b and GQ1b, stage-specific embryonic antigen-1 (SSEA-1) and HNK-1, in mouse embryonic NPCs employing immunocytochemistry and flow cytometry. In addition, several of these antigens were positively identified by chemical means for the first time. We further showed that the SSEA-1 immunoreactivity was contributed by both glycoprotein and glycolipid antigens, and that of HNK-1 was contributed only by glycoproteins. Functionally, SSEA-1 may participate in migration of the cells from neurospheres in an NPC cell culture system, and the effect could be repressed by anti-SSEA-1 antibody. Based on this observation, we identified beta1 integrin as one of the SSEA-1 carrier glycoproteins. Our data thus provide insights into the functional role of certain glycoconjugate antigens in NPCs during neural development.     

Sacral neural crest cells colonise aganglionic hindgut in vivo but fail to compensate for lack of enteric ganglia

The vagal neural crest is the origin of majority of neurons and glia that constitute the enteric nervous system, the intrinsic innervation of the gut. We have recently confirmed that a second region of the neuraxis, the sacral neural crest, also contributes to the enteric neuronal and glial populations of both the myenteric and the submucosal plexuses in the chick, caudal to the level of the umbilicus. Results from this previous study showed that sacral neural crest-derived precursors colonised the gut in significant numbers only 4 days after vagal-derived cells had completed their migration along the entire length of the gut. This observation suggested that in order to migrate into the hindgut and differentiate into enteric neurons and glia, sacral neural crest cells may require an interaction with vagal-derived cells or with factors or signalling molecules released by them or their progeny. This interdependence may also explain the inability of sacral neural crest cells to compensate for the lack of ganglia in the terminal hindgut of Hirschsprung's disease in humans or aganglionic megacolon in animals. To investigate the possible interrelationship between sacral and vagal-derived neural crest cells within the hindgut, we mapped the contribution of various vagal neural crest regions to the gut and then ablated appropriate sections of chick vagal neural crest to interrupt the migration of enteric nervous system precursor cells and thus create an aganglionic hindgut model in vivo. In these same ablated animals, the sacral level neural axis was removed and replaced with the equivalent tissue from quail embryos, thus enabling us to document, using cell-specific antibodies, the migration and differentiation of sacral crest-derived cells. Results showed that the vagal neural crest contributed precursors to the enteric nervous system in a regionalised manner. When quail-chick grafts of the neural tube adjacent to somites 1-2 were performed, neural crest cells were found in enteric ganglia throughout the preumbilical gut. These cells were most numerous in the esophagus, sparse in the preumbilical intestine, and absent in the postumbilical gut. When similar grafts adjacent to somites 3-5 or 3-6 were carried out, crest cells were found within enteric ganglia along the entire gut, from the proximal esophagus to the distal colon. Vagal neural crest grafts adjacent to somites 6-7 showed that crest cells from this region were distributed along a caudal-rostral gradient, being most numerous in the hindgut, less so in the intestine, and absent in the proximal foregut. In order to generate aneural hindgut in vivo, it was necessary to ablate the vagal neural crest adjacent to somites 3-6, prior to the 13-somite stage of development. When such ablations were performed, the hindgut, and in some cases also the cecal region, lacked enteric ganglionated plexuses. Sacral neural crest grafting in these vagal neural crest ablated chicks showed that sacral cells migrated along normal, previously described hindgut pathways and formed isolated ganglia containing neurons and glia at the levels of the presumptive myenteric and submucosal plexuses. Comparison between vagal neural crest-ablated and nonablated control animals demonstrated that sacral-derived cells migrated into the gut and differentiated into neurons in higher numbers in the ablated animals than in controls. However, the increase in numbers of sacral neural crest-derived neurons within the hindgut did not appear to be sufficiently high to compensate for the lack of vagal-derived enteric plexuses, as ganglia containing sacral neural crest-derived neurons and glia were small and infrequent. Our findings suggest that the neuronal fate of a relatively fixed subpopulation of sacral neural crest cells may be predetermined as these cells neither require the presence of vagal-derived enteric precursors in order to colonise the hindgut, nor are capable of dramatically altering their proliferation or d     

Perturbation of cranial neural crest migration by the HNK-1 antibody

The HNK-1 antibody recognizes a carbohydrate moiety that is shared by a family of cell adhesion molecules and is also present on the surface of migrating neural crest cells. Here, the effects of the HNK-1 antibody on neural crest cells were examined in vitro and in vivo. When the HNK-1 antibody was added to neural tube explants in tissue culture, neural crest cells detached from laminin substrates but were unaffected on fibronectin substrates. In order to examine the effects of the HNK-1 antibody in vivo, antibody was injected lateral to the mesencephalic neural tube at the onset of cranial neural crest migration. The injected antibody persisted for approximately 16 hr on the injected side of the embryo and appeared to be most prevalent on the surface of neural crest cells. Embryos fixed within the first 24 hr after injection of HNK-1 antibodies (either whole IgMs or small IgM fragments) showed one or more of the following abnormalities: (1) ectopic neural crest cells external to the neural tube, (2) an accumulation of neural crest cell volume on the lumen of the neural tube, (3) some neural tube anomalies, or (4) a reduction in the neural crest cell volume on the injected side. The ectopic cells and neural tube anomalies persisted in embryos fixed 2 days postinjection. Only embryos having 10 or less somites at the time of injection were affected, suggesting a limited period of sensitivity to the HNK-1 antibody. Control embryos injected with a nonspecific antibody or with a nonblocking antibody against the neural cell adhesion molecule (N-CAM) were unaffected. Previous experiments from this laboratory have demonstrated than an antibody against integrin, a fibronectin and laminin receptor caused defects qualitatively similar to those resulting from HNK-1 antibody injection (M. Bronner-Fraser, J. Cell Biol., 101, 610, 1985). Coinjection of the HNK-1 and integrin antibodies resulted in a greater percentage of affected embryos than with either antibody alone. The additive nature of the effects of the two antibodies suggests that they act at different sites. These results demonstrate that the HNK-1 antibody causes abnormalities in cranial neural crest migration, perhaps by perturbing interactions between neural crest cells and laminin substrates.     

Immunostaining to Visualize Murine Enteric Nervous System Development

The enteric nervous system is formed by neural crest cells that proliferate, migrate and colonize the gut. Following colonization, neural crest cells must then differentiate into neurons with markers specific for their neurotransmitter phenotype. Cholinergic neurons, a major neurotransmitter phenotype in the enteric nervous system, are identified by staining for choline acetyltransferase (ChAT), the synthesizing enzyme for acetylcholine. Historical efforts to visualize cholinergic neurons have been hampered by antibodies with differing specificities to central nervous system versus peripheral nervous system ChAT. We and others have overcome this limitation by using an antibody against placental ChAT, which recognizes both central and peripheral ChAT, to successfully visualize embryonic enteric cholinergic neurons. Additionally, we have compared this antibody to genetic reporters for ChAT and shown that the antibody is more reliable during embryogenesis. This protocol describes a technique for dissecting, fixing and immunostaining of the murine embryonic gastrointestinal tract to visualize enteric nervous system neurotransmitter expression.     

Expression and biological functions of sulfoglucuronyl glycolipids (SGGLs) in the nervous system—A review

Sulfoglucuronyl carbohydrate linked to neolactotetraose reacts with HNK-1 antibody. The HNK-1 carbohydrate epitope is found in two major glycolipids, several glycoproteins and in some proteoglycans of the nervous system. Most of the HNK-1 reactive glycoproteins so far identified are neural cell adhesion molecules and/or are involved in cell-cell interactions. HNK-1 carbohydrate is highly immunogenic. Several HNK-1-like antibodies, including IgM of some patients with plasma cell abnormalities and having peripheral neuropathy, have been described. This article summarizes published work mainly on sulfoglucuronyl glycolipids, SGGLs and covers: structural requirements of the carbohydrate epitope for binding to HNK-1 and human antibodies, expression of the lipids in various neural areas, stage and region specific developmental expression in CNS and PNS, immunocytochemical localization, loss of expression in Purkinje cell abnormality murine mutations, biosynthetic regulation of expression by a single enzyme N-acetylglucosaminyl transferase, identification of receptor-like carbohydrate binding neural proteins (lectins), and perceived role of the carbohydrate in physiological functions. The latter includes role in: pathogenesis of certain peripheral neuropathies, in migration of neural crest cells, as a ligand in cell-cell adhesion/interaction and as a promoter of neurite outgrowth for motor neurons. Multiple expression of HNK-1 carbohydrate in several molecules and in various neural cell types at specific stages of nervous system development has puzzled investigators as to its specific biological function, but this may also suggest its importance in multiple systems during cell differentiation and migration processes.Special issue dedicated to Dr. Marjorie B. Lees.     

Activin receptor-like kinase 2 can mediate atrioventricular cushion transformation

The development of enteric and sympathetic neurons from neural crest precursor cells is regulated by signals produced by the embryonic environments to which the cells migrate. Bone morphogenetic proteins (BMPs) are present in the developing embryo and act to induce neuronal differentiation and noradrenergic properties of neural crest cells. We have investigated the role of BMP2 in regulating the appearance of distinct populations of autonomic neurons from postmigratory, HNK-1-positive neural crest precursor cells. BMP2 promotes neuronal differentiation of sympathetic and enteric precursor cells isolated from E14.5 rat. The effects of BMP2 change over time, resulting in a decrease in neuron number that can be attributed to apoptotic cell death. BMP2-dependent neuron death is rescued by gut-derived factors that provide trophic support to maturing neurons, indicating that BMP2 regulates the acquisition of trophic dependence of developing peripheral neurons. In addition to regulating neuron number, BMP2 promotes both panneuronal maturation and the acquisition of an enteric phenotype, as measured by lineage-specific changes in the expression of tyrosine hydroxylase and MASH-1. While BMP2 is sufficient to induce neuronal differentiation and panneuronal development, these results suggest that additional factors in the environment must collaborate with BMP2 to promote the final noradrenergic phenotype of sympathetic neurons.     

Development of the mammalian enteric nervous system.

The mammalian enteric nervous system is derived from neural crest cells which invade the foregut and hindgut mesenchyme. It has been established that signalling molecules produced by the mesenchyme of the gut wall play a critical role in the development of the mammalian enteric nervous system. Recent studies have characterised further the role of such molecules and have identified novel extracellular and intracellular signals that are critical for enteric ganglia formation.     

Analysis of the development of cellular subsets present in the neural crest using cell sorting and cell culture

We have tested the hypothesis that developmentally significant cellular subsets are present in the early stages of neural crest ontogenesis. Cultured quail trunk neural crest cells probed with the monoclonal antibodies HNK-1 and R24 exhibited heterogeneous staining patterns. Fluorescence-activated cell sorting was used to isolate the HNK-1+ and HNK-1- cell populations at 2 days in vitro. When these cell populations were cultured, the HNK-1+ sorted cells differentiated into melanocytes, unpigmented cells, and numerous catecholamine-positive (CA+) cells. In contrast, the HNK-1- sorted cells gave rise to melanocytes and unpigmented cells, but few, if any, CA+ cells. When neural crest cells at 2 days in vitro were labeled with R24 and sorted, both the R24+ the R24- sorted cell populations produced numerous CA+ cell, melanocytes, and unpigmented cells. These results provide evidence for the existence of developmental preferences in some subsets of neural crest cells early in embryogenesis.     

Avian neural crest cell attachment to laminin: involvement of divalent cation dependent and independent integrins.

The mechanisms of neural crest cell interaction with laminin were explored using a quantitative cell attachment assay. With increasing substratum concentrations, an increasing percentage of neural crest cells adhere to laminin. Cell adhesion at all substratum concentrations was inhibited by the CSAT antibody, which recognizes the chick beta 1 subunit of integrin, suggesting that beta 1-integrins mediate neural crest cell interactions with laminin. The HNK-1 antibody, which recognizes a carbohydrate epitope, inhibited neural crest cell attachment to laminin at low coating concentrations (greater than 1 microgram ml-1; Low-LM), but not at high coating concentration of laminin (10 micrograms ml-1; High-LM). Attachment to Low-LM occurred in the absence of divalent cations, whereas attachment to High-LM required greater than 0.1 mM Ca2+ or Mn2+. Neural crest cell adherence to the E8 fragment of laminin, derived from its long arm, was similar to that on intact laminin at high and low coating concentrations, suggesting that this fragment contains the neural crest cell binding site(s). The HNK-1 antibody recognizes a protein of 165,000 Mr which is also found in immunoprecipitates using antibodies against the beta 1 subunit of integrin and is likely to be an integrin alpha subunit or an integrin-associated protein. Our results suggest that the HNK-1 epitope on neural crest cells is present on or associated with a novel or differentially glycosylated form of beta 1-integrin, which recognizes laminin in the apparent absence of divalent cations. We conclude that neural crest cells have at least two functionally independent means of attachment to laminin which are revealed at different substratum concentrations and/or conformations of laminin.     

Phenotypic characterization of Ewing sarcoma cell lines with monoclonal antibodies

The histogenesis of Ewing sarcoma, the second most frequent bone tumor in humans, remains controversial. Four Ewing cell lines were analyzed by immunological methods. A panel of antibodies directed to T, B, and myelomonocytic markers gave negative results. Surface antigens recognized on Ewing cells were found to be related to the neuroectoderm lineage. Ganglioside GD2, a marker of neuroectodermal tissues and tumors, was present on all lines. These were also stained by the mouse monoclonal antibody HNK-1, which detects a carbohydrate epitope present on several glycoconjugates of the nervous system, including two glycoproteins, the myelin-associated glycoprotein and the neural cell-adhesion molecule (N-CAM), and an acidic glycolipid of the peripheral nervous system. The P61 monoclonal antibody, which reacts with a peptide moiety of N-CAM, and a rabbit antiserum, raised to purified mouse N-CAM and not recognizing the HNK-1-defined epitope, were also reactive. By contrast, all antibodies specific for hematopoietic cell surface antigens were totally negative. Besides these antigenic features, Ewing sarcoma cells are characterized by a specific t(11;22)(q24;q12) translocation also observed in neuroepithelioma, a neuroectodermal tumor, suggesting a possible evolutionary related origin. The recent finding that the human N-CAM gene is located at the vicinity of the breakpoint on chromosome 11 indicates that it might be involved in genetic rearrangements occurring in this region.     

Spectrum of in vitro differentiation of quail trunk neural crest cells isolated by cell sorting using the HNK-1 antibody and analysis of the adrenergic development of HNK-1+ sorted subpopulations.

Previous work has demonstrated that catecholamine-containing cells differentiate preferentially from populations of quail trunk neural crest cells isolated by cell sorting using the HNK-1 antibody (Maxwell, Forbes, and Christie, 1988). In the present work, we examine several additional features of the differentiation of these sorted cell populations. As one part of this study, the development of subpopulations of the HNK-(1+)-sorted neural crest cells has been investigated. Twice as many catecholamine-positive and total cells developed from the brightest third of the HNK-1+ cells compared to the remaining HNK-1+ cells, but the proportion of catecholamine-containing cells was similar in both populations. When either of these HNK-1+ subpopulations were grown together with HNK-1- cells, no reduction in the number of adrenergic cells was observed. These results indicate that subpopulations of HNK-1+ cells are qualitatively similar and that their adrenergic development is not affected by HNK-1- cells. In the second part of this study, we investigate the specificity of differentiation of HNK-(1+)- and HNK-(1-)-sorted cells by examining several additional phenotypic markers of development. We found that tyrosine hydroxylase and somatostatin immunoreactive cells developed from the HNK-(1+)-sorted population, while few, if any, cells bearing these phenotypic markers appeared in the HNK-(1-)-sorted population. In marked contrast, substantial numbers of cells immunoreactive for A2B5, E/C8, and NF-160 differentiated from both the HNK-(1+)- and the HNK-(1-)-sorted cell populations. The A2B5, E/C8, and NF-160 immunoreactive cells exhibited a variety of morphologies ranging from nonneuronal to neuronal in both sorted populations. Taken together, these results indicate that the presence of the HNK-1 antigen(s) on the trunk neural crest cell surface at 2 days in vitro is rather tightly correlated with the differentiation of adrenergic and some peptidergic cells, but much less so with other classes of neural cells including A2B5, E/C8, and NF-160 immunoreactive cells. Thus, these findings support the view that cell surface differences are correlated with and may contribute to the generation of the phenotypic diversity of neural crest cell derivatives.     

Identification of Sox8 as a modifier gene in a mouse model of Hirschsprung disease reveals underlying molecular defect

Mice carrying heterozygous mutations in the Sox10 gene display aganglionosis of the colon and represent a model for human Hirschsprung disease. Here, we show that the closely related Sox8 functions as a modifier gene for Sox10-dependent enteric nervous system defects as it increases both penetrance and severity of the defect in Sox10 heterozygous mice despite having no detectable influence on enteric nervous system development on its own. Sox8 exhibits an expression pattern very similar to Sox10 with occurrence in vagal and enteric neural crest cells and later confinement to enteric glia. Loss of Sox8 alleles in Sox10 heterozygous mice impaired colonization of the gut by enteric neural crest cells already at early times. Whereas proliferation, apoptosis, and neuronal differentiation were normal for enteric neural crest cells in the gut of mutant mice, apoptosis was dramatically increased in vagal neural crest cells outside the gut. The defects in enteric nervous system development of mice with Sox10 and Sox8 mutations are therefore likely caused by a reduction of the pool of undifferentiated vagal neural crest cells. Our study suggests that Sox8 and Sox10 are jointly required for the maintenance of these vagal neural crest stem cells.     

Requirement of signalling by receptor tyrosine kinase RET for the directed migration of enteric nervous system progenitor cells during mammalian embryogenesis

The majority of neurones and glia of the enteric nervous system (ENS) are derived from the vagal neural crest. Shortly after emigration from the neural tube, ENS progenitors invade the anterior foregut and, migrating in a rostrocaudal direction, colonise in an orderly fashion the rest of the foregut, the midgut and the hindgut. We provide evidence that activation of the receptor tyrosine kinase RET by glial cell line-derived neurotrophic factor (GDNF) is required for the directional migration of ENS progenitors towards and within the gut wall. We find that neural crest-derived cells present within foetal small intestine explants migrate towards an exogenous source of GDNF in a RET-dependent fashion. Consistent with an in vivo role of GDNF in the migration of ENS progenitors, we demonstrate that Gdnf is expressed at high levels in the gut of mouse embryos in a spatially and temporally regulated manner. Thus, during invasion of the foregut by vagal-derived neural crest cells, expression of Gdnf was restricted to the mesenchyme of the stomach, ahead of the invading NC cells. Twenty-four hours later and as the ENS progenitors were colonising the midgut, Gdnf expression was upregulated in a more posterior region - the caecum anlage. In further support of a role of endogenous GDNF in enteric neural crest cell migration, we find that in explant cultures GDNF produced by caecum is sufficient to attract NC cells residing in more anterior gut segments. In addition, two independently generated loss-of-function alleles of murine Ret, Ret.k- and miRet51, result in characteristic defects of neural crest cell migration within the developing gut. Finally, we identify phosphatidylinositol-3 kinase and the mitogen-activated protein kinase signalling pathways as playing crucial roles in the migratory response of enteric neural crest cells to GDNF.     

P0 is an early marker of the Schwann cell lineage in chickens.

We have generated a monoclonal antibody, termed 1E8, that is specific for myelinating and nonmyelinating Schwann cells in mature chickens. 1E8 first stains cells at the edge of the neural crest; later, cells located between the neural tube and somites and in the sclerotome are immunopositive. Double labeling with HNK-1 indicates that these 1E8-positive cells represent a subset of neural crest cells in the ventral migratory pathways. 1E8-positive cells are later associated with the dorsal and ventral roots and with extending nerve trunks. In Western blots, 1E8 reacts with proteins comigrating with P0. Immunodepletion experiments establish that all P0 molecules carry the 1E8 determinant. The developmental distribution of P0, as determined by 1E8 immunoreactivity, differs from that reported for P0 in mammals and suggests that, in chicken, P0 is an early marker for the Schwann cell lineage.     

Epithelia-mesenchyme interaction plays an essential role in transdifferentiation of retinal pigment epithelium of silver mutant quail: localization of FGF and related molecules and aberrant migration pattern of neural crest cells during eye rudiment formation

Homozygotes of the quail silver mutation, which have plumage color changes, also display a unique phenotype in the eye: during early embryonic development, the retinal pigment epithelium (RPE) spontaneously transdifferentiates into neural retinal tissue. Mitf is considered to be the responsible gene and to function similarly to the mouse microphthalmia mutation, and tissue interaction between RPE and surrounding mesenchymal tissue in organ culture has been shown to be essential for the initiation of the transdifferentiation process in which fibroblast growth factor (FGF) signaling is involved. The immunohistochemical results of the present study show that laminin and heparan sulfate proteoglycan, both acting as cofactors for FGF binding, are localized in the area of transdifferentiation of silver embryos much more abundantly than in wild-type embryos. More intense immunohistochemical staining with FGF-1 antibody, but not with FGF-2 antibody, is also found in the neural retina, RPE, and choroidal tissue of silver embryos than in wild-type embryos. HNK-1 immunohistochemistry revealed that clusters of HNK-1-positive cells (presumptive migrating neural crest cells) are frequently located around the developing eyes and in the posterior region of the silver embryonic eye. Finally, chick-quail chimerical eyes were made by grafting silver quail optic vesicles to chicken host embryos: in most cases, no transdifferentiation occurs in the silver RPE, but in a few cases, transdifferentiation occurs where silver quail cells predominate in the choroid tissue. These observations together with our previous in vitro study indicate that the silver mutation affects not only RPE cells but also cephalic neural crest cells, which migrate to the eye rudiment, and that these crest cells play an essential role in the transdifferentiation of RPE, possibly by modifying the FGF signaling pathway. The precise molecular mechanism involved in RPE-neural crest cell interaction is still unknown, and the quail silver mutation is considered to be a good experimental model for studying the role of neural crest cells in vertebrate eye development.     

Trunk lateral cells are neural crest-like cells in the ascidian Ciona intestinalis: insights into the ancestry and evolution of the neural crest

Neural crest-like cells (NCLC) that express the HNK-1 antigen and form body pigment cells were previously identified in diverse ascidian species. Here we investigate the embryonic origin, migratory activity, and neural crest related gene expression patterns of NCLC in the ascidian Ciona intestinalis. HNK-1 expression first appeared at about the time of larval hatching in dorsal cells of the posterior trunk. In swimming tadpoles, HNK-1 positive cells began to migrate, and after metamorphosis they were localized in the oral and atrial siphons, branchial gill slits, endostyle, and gut. Cleavage arrest experiments showed that NCLC are derived from the A7.6 cells, the precursors of trunk lateral cells (TLC), one of the three types of migratory mesenchymal cells in ascidian embryos. In cleavage arrested embryos, HNK-1 positive TLC were present on the lateral margins of the neural plate and later became localized adjacent to the posterior sensory vesicle, a staging zone for their migration after larval hatching. The Ciona orthologues of seven of sixteen genes that function in the vertebrate neural crest gene regulatory network are expressed in the A7.6/TLC lineage. The vertebrate counterparts of these genes function downstream of neural plate border specification in the regulatory network leading to neural crest development. The results suggest that NCLC and neural crest cells may be homologous cell types originating in the common ancestor of tunicates and vertebrates and support the possibility that a putative regulatory network governing NCLC development was co-opted to produce neural crest cells during vertebrate evolution.