Grafts of supplementary thymuses injected with allogeneic pancreatic islets protect nonobese diabetic mice against diabetes.

In nonobese diabetic (NOD) mice, the autoimmune attack of the beta-cells in pancreatic islets is now believed to result from abnormal thymic selection. Accordingly, grafts of thymic epithelium from NOD donors to athymic recipients promote autoimmune islet inflammation in normal strains, and intrathymic islet grafts decrease the incidence of disease in NOD animals. Two competing hypotheses of abnormal thymic selection in diabetic mice have been proposed: deficient negative selection with poor elimination of aggressive organ-specific T cells vs. deficient positive selection of protective T regulatory cells. We have now addressed these alternatives by grafting, into young NOD mice whose own thymus was left intact, newborn NOD thymuses containing allogeneic pancreatic islets. If the NOD defect represented poor negative selection, these animals would develop disease at control rates, as the generation of autoreactive T cells proceeds undisturbed in the autologous thymus. In contrast, if NOD thymuses are defective in the production of T regulatory cells, lower disease incidence is expected in the chimeras, as more protective cells can be produced in the grafted thymus. The results show a reduced incidence of diabetes in the chimeras (24%) as compared with control (72%) NOD mice, throughout adult life. We conclude that amelioration of NOD mice by intrathymic islet grafts is not caused by enhanced negative selection and suggest that autoimmune diabetes in this system is the result of inefficient generation of T regulatory cells in the thymus.

Anti-apoptotic role of Sonic hedgehog protein at the early stages of nervous system organogenesis.

In vertebrates the neural tube, like most of the embryonic organs, shows discreet areas of programmed cell death at several stages during development. In the chick embryo, cell death is dramatically increased in the developing nervous system and other tissues when the midline cells, notochord and floor plate, are prevented from forming by excision of the axial-paraxial hinge (APH), i.e. caudal Hensen's node and rostral primitive streak, at the 6-somite stage ( Charrier, J. B., Teillet, M.-A., Lapointe, F. and Le Douarin, N. M. (1999). Development 126, 4771-4783). In this paper we demonstrate that one day after APH excision, when dramatic apoptosis is already present in the neural tube, the latter can be rescued from death by grafting a notochord or a floor plate fragment in its vicinity. The neural tube can also be recovered by transplanting it into a stage-matched chick embryo having one of these structures. In addition, cells engineered to produce Sonic hedgehog protein (SHH) can mimic the effect of the notochord and floor plate cells in in situ grafts and transplantation experiments. SHH can thus counteract a built-in cell death program and thereby contribute to organ morphogenesis, in particular in the central nervous system.

BMP4 plays a key role in left-right patterning in chick embryos by maintaining Sonic Hedgehog asymmetry.

In chick embryos, the first signs of left-right asymmetry are detected in Hensen's node, essentially by left-sided Sonic Hedgehog (Shh) expression. After a gap of several hours, SHH induces polarized gene activities in the left paraxial mesoderm. We show that during this time period, BMP4 signaling is necessary and sufficient to maintain Shh asymmetry within the node. SHH and BMP4 proteins negatively regulate each other's transcription, resulting in a strict complementarity between these two gene patterns on each side of the node. Noggin, present in the midline at this stage, limits BMP4 spreading. Moreover, BMP4 is downstream to Activin signals and controls Fgf8. Thus, early BMP4 signaling coordinates left and right pathways in Hensen's node.

Distribution of HOX genes in the chicken genome reveals a new segment of conservation between human and chicken.

Homeobox genes play an important role in the regulation of early embryonic development. They represent a family of evolutionarily highly conserved transcription factors. In this work, several genes that belong to the four HOX gene clusters are assigned by in situ hybridization to four distinct chicken chromosomes. The four gene clusters are mapped to 2p2.1 (HOXA), 3q3.1 (HOXB), 1q3.1 (HOXC) and 7q1.3--> q1.4 (HOXD). We confirm partial homologies already detected by genetic mapping between chicken chromosomes 1, 2 and 7 and human chromosomes 12, 7 and 2 and we describe a new conserved segment between chicken chromosome 3 and human chromosome 17. These results represent the first data that confirm the physical linkage between chicken HOX genes and may improve our understanding of phylogenetic relationships and genome evolution.

Early neurogenesis in Amniote vertebrates.

Labelling of Hensen's node in a 6-somite stage chick embryo by the quail/chick chimera method has revealed that, while moving caudalwards as the embryo elongates, the node leaves in its wake not only the notochord but also the floor plate and a longitudinal strand of dorsal endoderm. The node itself contains cells endowed with the capacity to yield midline cells (i.e. notochord and floor plate) along the whole length of the neural axis. Caudal node cells function as stem cells. They are responsible for the apical growth of the cord of cells that are at the origin of the midline structures since, if removed, neither the notochord nor the floor plate, are formed caudally to the ablation. The embryo extends however in the absence of midline cells and a neural tube develops posterior to the excision. Only dorsal molecular markers are detectable on this neural tube (e.g. Pax3 and Slug). The posterior region of the embryo in which the structures secreting Shh are missing undergo cell death within the 24 to 48 hours following its formation. Unpublished results indicate that rescue of the posterior region of the embryo can be obtained by implantation of Shh secreting cells. One of the critical roles of floor plate and notochord is therefore to inhibit the cell death programme in the axial and paraxial structures of the embryo at gastrulation and neurulation stages.

The cephalic neural crest provides pericytes and smooth muscle cells to all blood vessels of the face and forebrain.

Most connective tissues in the head develop from neural crest cells (NCCs), an embryonic cell population present only in vertebrates. We show that NCC-derived pericytes and smooth muscle cells are distributed in a sharply circumscribed sector of the vasculature of the avian embryo. As NCCs detach from the neural folds that correspond to the future posterior diencephalon, mesencephalon and rhombencephalon, they migrate between the ectoderm and the neuroepithelium into the anterior/ventral head, encountering mesoderm-derived endothelial precursors. Together, these two cell populations build a vascular tree rooted at the departure of the aorta from the heart and ramified into the capillary plexi that irrigate the forebrain meninges, retinal choroids and all facial structures, before returning to the heart. NCCs ensheath each aortic arch-derived vessel, providing every component except the endothelial cells. Within the meninges, capillaries with pericytes of diencephalic and mesencephalic neural fold origin supply the forebrain, while capillaries with pericytes of mesodermal origin supply the rest of the central nervous system, in a mutually exclusive manner. The two types of head vasculature contact at a few defined points, including the anastomotic vessels of the circle of Willis, immediately ventral to the forebrain/midbrain boundary. Over the course of evolution, the vertebrate subphylum may have exploited the exceptionally broad range of developmental potentialities and the plasticity of NCCs in head remodelling that resulted in the growth of the forebrain.

Enteric nervous system development: analysis of the selective developmental potentialities of vagal and sacral neural crest cells using quail-chick chimeras.

The majority of the enteric nervous system (ENS) is derived from vagal neural crest cells (NCC). For many years, the contribution from a second region of the neuraxis (the sacral neural crest) to the ENS has been less clear, with conflicting reports appearing in the literature. To resolve this longstanding issue, we documented the spatiotemporal migration and differentiation of vagal and sacral-derived NCC within the developing chick embryo using quail-chick grafting and antibody labelling. Results showed that vagal NCC colonised the entire length of the gut in a rostrocaudal direction. The hindgut, the region of the gastrointestinal tract most frequently affected in developmental disorders, was found to be colonised in a complex manner. Vagal NCC initially migrated within the submucosa, internal to the circular muscle layer, before colonising the myenteric plexus region. In contrast, sacral NCC, which colonised the hindgut in a caudorostral direction, were primarily located in the myenteric plexus region from where they subsequently migrated to the submucosa. We also observed that sacral NCC migrated into the hindgut in significant numbers only after vagal-derived cells had colonised the entire length of the gut. This suggested that to participate in ENS formation, sacral cells may require an interaction with vagal-derived cells, or with factors or signalling molecules released by them or their progeny. To investigate this possible inter-relationship, we ablated sections of vagal neural crest (NC) to prevent the rostrocaudal migration of ENS precursors and, thus, create an aganglionic hindgut model. In the same NC ablated animals, quail-chick sacral NC grafts were performed. In the absence of vagal-derived ganglia, sacral NCC migrated and differentiated in an apparently normal manner. Although the numbers of sacral cells within the hindgut was slightly higher in the absence of vagal-derived cells, the increase was not sufficient to compensate for the lack of enteric ganglia. As vagal NCC appear to be more invasive than sacral NCC, since they colonise the entire length of the gut, we investigated the ability of transplanted vagal cells to colonise the hindgut by grafting the vagal NC into the sacral region. We found that when transplanted, vagal cells retained their invasive capacity and migrated into the hindgut in large numbers. Although sacral-derived cells normally contribute a relatively small number of precursors to the post-umbilical gut, many heterotopic vagal cells were found within the hindgut enteric plexuses at much earlier stages of development than normal. Heterotopic grafting of invasive vagal NCC into the sacral neuraxis may, therefore, be a means of rescuing an aganglionic hindgut phenotype.

Induction of melanogenesis by tetradecanoylphorbol-13 acetate and endothelin 3 in embryonic avian peripheral nerve cultures.

In this study, we have analyzed the melanogenic potential of Schwann cells using in vitro cell cultures of embryonic quail peripheral nerves. It is shown that in Schwann cells, two factors, 12-O-tetradecanoylphorbol-13 acetate (TPA) and endothelin 3, trigger a differentiation pathway toward melanocytes, and that Steel factor has no effect on these cells unless treated simultaneously with TPA. In these cultures, TPA induces the expression of c-kit, whereas Steel factor enhances the development of melanocytes. In the assay system we employed, neither neuronal nor catecholaminergic phenotypes were obtained, regardless of various combinations of related factors added to the culture medium. These data support our previous observations indicating the existence of bipotent progenitors that are capable of differentiating into Schwann cells or into melanocytes, and the regulatory role of endothelin 3 on those precursors, as revealed by the clonal culture of neural crest cells.

The developmental potentials of the caudalmost part of the neural crest are restricted to melanocytes and glia.

The avian spinal cord is characterized by an absence of motor nerves and sensory nerves and ganglia at its caudalmost part. Since peripheral sensory neurons derive from neural crest cells, three basic mechanisms could account for this feature: (i) the caudalmost neural tube does not generate any neural crest cells; (ii) neural crest cells originating from the caudal part of the neural tube cannot give rise to dorsal root ganglia or (iii) the caudal environment is not permissive for the formation of dorsal root ganglia. To solve this problem, we have first studied the pattern of expression of ventral (HNF3beta) and dorsal (slug) marker genes in the caudal region of the neural tube; in a second approach, we have recorded the emergence of neural crest cells using the HNK1 monoclonal antibody; and finally, we have analyzed the developmental potentials of neural crest cells arising from the caudalmost part of the neural tube in avian embryo in in vitro culture and by means of heterotopic transplantations in vivo. We show here that neural crest cells arising from the neural tube located at the level of somites 47-53 can differentiate both in vitro and in vivo into melanocytes and Schwann cells but not into neurons. Furthermore, the neural tube located caudally to the last pair of somites (i.e. the 53rd pair) does not give rise to neural crest cells in any of the situations tested. The specific anatomical aspect of the avian spinal cord can thus be accounted for by limited developmental potentials of neural crest cells arising from the most caudal part of the neural tube.

Endothelin 3 induces the reversion of melanocytes to glia through a neural crest-derived glial-melanocytic progenitor.

Functional signaling of endothelin 3 (ET3) and its receptor B (ETRB) has been shown to be required for the development of neural crest (NC)-derived pigment cells in mouse, but the precise role of ET3 is not completely understood. Using the avian embryo as a model, we previously reported that ET3 promotes the survival and proliferation of unipotent melanocyte and bipotent glia-melanocyte precursors in trunk NC cultures. Here we investigated whether, at later stages, embryonic pigment cells respond to ET3. Such a possibility is supported by the previous finding that, in vivo, avian melanocytes express endothelin receptor B2 (ETRB2) during migration and after their differentiation in the skin. We found that in vitro ET3 exerts a dose-dependent stimulation of proliferation and melanogenesis in NC cells that had homed to the epidermis of embryonic quail dorsal skin. Moreover, in clonal cultures of skin-derived pigment cells, ET3 induces rapid cell divisions of clonogenic melanocytes that generate a mixed progeny of melanocytes and cells devoid of pigment granules and expressing glial markers in more than 40% of the colonies. It can therefore be concluded that ET3 is strongly mitogenic to embryonic pigment cells and able to alter their differentiation program, leading them to recapitulate the glial-melanocyte bipotentiality of their NC ancestors.

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

Defining subregions of Hensen's node essential for caudalward movement, midline development and cell survival.

Hensen's node, also called the chordoneural hinge in the tail bud, is a group of cells that constitutes the organizer of the avian embryo and that expresses the gene HNF-3(&bgr;). During gastrulation and neurulation, it undergoes a rostral-to-caudal movement as the embryo elongates. Labeling of Hensen's node by the quail-chick chimera system has shown that, while moving caudally, Hensen's node leaves in its wake not only the notochord but also the floor plate and a longitudinal strand of dorsal endodermal cells. In this work, we demonstrate that the node can be divided into functionally distinct subregions. Caudalward migration of the node depends on the presence of the most posterior region, which is closely apposed to the anterior portion of the primitive streak as defined by expression of the T-box gene Ch-Tbx6L. We call this region the axial-paraxial hinge because it corresponds to the junction of the presumptive midline axial structures (notochord and floor plate) and the paraxial mesoderm. We propose that the axial-paraxial hinge is the equivalent of the neuroenteric canal of other vertebrates such as Xenopus. Blocking the caudal movement of Hensen's node at the 5- to 6-somite stage by removing the axial-paraxial hinge deprives the embryo of midline structures caudal to the brachial level, but does not prevent formation of the neural tube and mesoderm located posteriorly. However, the whole embryonic region generated posterior to the level of Hensen's node arrest undergoes widespread apoptosis within the next 24 hours. Hensen's node-derived structures (notochord and floor plate) thus appear to produce maintenance factor(s) that ensures the survival and further development of adjacent tissues.

Patterning signals acting in the spinal cord override the organizing activity of the isthmus.

The regionalization of the neural tube along the anteroposterior axis is established through the action of patterning signals from the endomesoderm including the organizer. These signals set up a pre-pattern which is subsequently refined through local patterning events. The midbrain-hindbrain junction, or isthmus, is endowed with such an organizing activity. It is able to induce graded expression of the Engrailed protein in the adjacent mesencephalon and rhombencephalon, and subsequently elicits the development of tectal and cerebellar structures. Ectopically grafted isthmus was also shown to induce Engrailed expression in diencephalon and otic and pre-otic rhombencephalon. Fgf8 is a signalling protein which is produced by the isthmus and which is able to mimic most isthmic properties. We show here that the isthmus, when transposed to the level of either rhombomere 8 or the spinal cord, loses its ability to induce Engrailed and cerebellar development in adjacent tissues. This is accompanied by the down-regulation of fgf8 expression in the grafted isthmus and by the up-regulation of a marker of the recipient site, Hoxb-4. Moreover, these changes in gene activity in the transplant are followed by a transformation of the fate of the grafted cells which adjust to their novel environment. These results show that the fate of the isthmus is not determined at 10-somite stage and that the molecular loop of isthmic maintenance can be disrupted by exogenous signals.

Anterior cephalic neural crest is required for forebrain viability.

The prosencephalon, or embryonic forebrain, grows within a mesenchymal matrix of local paraxial mesoderm and of neural crest cells (NCC) derived from the posterior diencephalon and mesencephalon. Part of this NCC population forms the outer wall of capillaries within the prosencephalic leptomeninges and neuroepithelium itself. The surgical removal of NCC from the anterior head of chick embryos leads to massive cell death within the forebrain neuroepithelium during an interval that precedes its vascularization by at least 36 hours. During this critical period, a mesenchymal layer made up of intermingled mesodermal cells and NCC surround the neuroepithelium. This layer is not formed after anterior cephalic NCC ablation. The neuroepithelium then undergoes massive apoptosis. Cyclopia ensues after forebrain deterioration and absence of intervening frontonasal bud derivatives. The deleterious effect of ablation of the anterior NC cannot be interpreted as a deficit in vascularization because it takes place well before the time when blood vessels start to invade the neuroepithelium. Thus the mesenchymal layer itself exerts a trophic effect on the prosencephalic neuroepithelium. In an assay to rescue the operated phenotype, we found that the rhombencephalic but not the truncal NC can successfully replace the diencephalic and mesencephalic NC. Moreover, any region of the paraxial cephalic mesoderm can replace NCC in their dual function: in their early trophic effect and in providing pericytes to the forebrain meningeal blood vessels. The assumption of these roles by the cephalic neural crest may have been instrumental in the rostral expansion of the vertebrate forebrain over the course of evolution.

BEN/SC1/DM-GRASP expression during neuromuscular development: a cell adhesion molecule regulated by innervation.

BEN/SC1/DM-GRASP is a cell adhesion molecule belonging to the Ig superfamily that is transiently expressed during avian embryogenesis in a variety of cell types, including the motoneurons of the spinal cord. We have investigated the pattern of BEN expression during neuromuscular development of the chick. We show that both motoneurons and their target myoblasts express BEN during early embryonic development and that the protein becomes restricted at neuromuscular contacts as soon as postsynaptic acetylcholine receptor clusters are observed in muscle fibers. Muscle cells grown in vitro express and maintain BEN expression even when they fuse and give rise to mature myotubes. When embryos are deprived of innervation by neural tube ablation, BEN expression is observed in muscle fibers, whereas, in control, the protein is already restricted at neuromuscular synaptic sites. These results demonstrate that all myogenic cells intrinsically express BEN and maintain the protein in the absence of innervation. Conversely, when neurons are added to myogenic cultures, BEN is rapidly downregulated in muscle cells, demonstrating that innervation controls the restricted pattern of BEN expression seen in innervated muscles. After nerve section in postnatal muscles, BEN protein becomes again widely spread over muscle fibers. When denervated muscles are allowed to be reinnervated, the protein is reexpressed in regenerating motor axons, and reinnervation of synaptic sites leads to the concentration of BEN at neuromuscular junctions. Our results suggest that BEN cell adhesion molecule acts both in the formation of neuromuscular contacts during development and in the events leading to muscle reinnervation.

Neurulation in amniote vertebrates: a novel view deduced from the use of quail-chick chimeras.

Two apparently different mechanisms successively contribute to the formation of the neural tube in the avian embryo: bending of the neural plate during the primary neurulation in the cephalo-cervico-thoracic region and cavitation of the medullary cord during the secondary neurulation in the lumbo-sacral region. During both these processes, gastrulation continues by the caudal regression of Hensen's node--also called cordoneural hinge in the secondary neurulation. Labeling of Hensen's node or cordoneural hinge by the quail chick marker system revealed that this structure, which is the equivalent of the dorsal blastoporal lip of the Amphibian embryo, i.e., of the Spemann's organizer, gives rise to the midline cells of the three germ layers: the floor plate of the neural tube, the notocord and the dorsal cells of the intestinal endoderm. Caudally to the organizer, both in primary and secondary neurulation, the presumptive territory of the alar plates of the future neural tube overlies the precursors of the paraxial mesoderm. Regression of Hensen's node bisects the ectoderm in two bilateral neural plates leaving in its wake the floor plate, the notocord and the dorsal endoderm.

Endothelin 3 selectively promotes survival and proliferation of neural crest-derived glial and melanocytic precursors in vitro.

Genetic data in the mouse have shown that endothelin 3 (ET3) and its receptor B (ETRB) are essential for the development of two neural crest (NC) derivatives, the melanocytes and the enteric nervous system. We report here the effects of ET3 in vitro on the differentiation of quail trunk NC cells (NCC) in mass and clonal cultures. Treatment with ET3 is highly mitogenic to the undifferentiated NCC population, which leads to expansion of the population of cells in the melanocytic, and to a lesser extent, the glial lineages. The effect of ET3 on these two NC derivatives was confirmed by the quantitative analysis of clones derived from individual NCC subjected to ET3: we found a large increase in the survival and proliferation of unipotent and bipotent precursors for glial cells and melanocytes, with no significant effect on multipotent cells generating neurons. ET3 first stimulates expression of both ETRB and ETRB2 by cultured NCC. Then, under prolonged exposure to ET3, ETRB expression decreases and switches toward an ETRB2-positive melanogenic cell population. We therefore propose that the present in vitro experiments (long-lasting exposure to a high concentration of ET3) mimic the environment encountered by NCC in vivo when they migrate to the skin under the ectoderm that expresses ET3.