Biomedical subjects

Nicole M Le Douarin

Publications and source records attributed to Nicole M Le Douarin.

At least 19 recordsLinked to original sources

PubMed2006

Clonally cultured differentiated pigment cells can dedifferentiate and generate multipotent progenitors with self-renewing potential.

The differentiation of a given cell should be irreversible in order to ensure cell-type-specific function and stability of resident tissue. However, under stimulation in vitro or during regeneration, differentiated cells may recover properties of immature cells. Yet the mechanisms whereby differentiated cells can change fate or reverse to precursor cells are poorly understood. We show here that neural crest (NC)-derived pigment cells that have differentiated in quail embryo, when isolated from the skin and clonally cultured in vitro, are able to generate glial and myofibroblastic cells. The phenotypic reprogramming involves dedifferentiation of dividing pigment cells into cells that re-express NC early marker genes Sox10, FoxD3, Pax3 and Slug. Single melanocytes generate multipotent progenitors able to self-renew along serial subcloning, thus exhibiting stem cell properties. The presence of endothelin 3 promotes the emergence and maintenance of multipotent progenitors in melanocyte progeny. These multipotent cells are heterogeneous with respect to marker identity, including pigmented cells and dedifferentiated cells that have reacquired expression of the early NC marker HNK1. These data provide evidence that, when removed from their niche and subjected to appropriate culture conditions, pigment cells are phenotypically unstable and can reverse to their NC-like ancestors endowed with self-renewal capacity.

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PubMed2006

The cephalic neural crest exerts a critical effect on forebrain and midbrain development.

Encephalisation is the most important characteristic in the evolutionary transition leading from protochordates to vertebrates. This event has coincided with the emergence of a transient and pluripotent structure, the neural crest (NC), which is absent in protochordates. In vertebrates, NC provides the rostral cephalic vesicles with skeletal protection and functional vascularization. The surgical extirpation of the cephalic NC, which is responsible for building up the craniofacial skeleton, results in the absence of facial skeleton together with severe defects of preotic brain development, leading to exencephaly. Here, we have analyzed the role of the NC in forebrain and midbrain development. We show that (i) NC cells (NCC) control Fgf8 expression in the anterior neural ridge, which is considered the prosencephalic organizer; (ii) the cephalic NCC are necessary for the closure of the neural tube; and (iii) NCC contribute to the proper patterning of genes that are expressed in the prosencephalic and mesencephalic alar plate. Along with the development of the roof plate, NCC also concur to the patterning of the pallial and subpallial structures. We show that the NC-dependent production of FGF8 in anterior neural ridge is able to restrict Shh expression to the ventral prosencephalon. All together, these findings support the notion that the cephalic NC controls the formation of craniofacial structures and the development of preotic brain.

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PubMed2006

An early role for sonic hedgehog from foregut endoderm in jaw development: ensuring neural crest cell survival.

We have investigated the role of Sonic hedgehog (Shh) in the development of facial structures by depriving chicken embryos of the most anterior sources of this morphogen, including the prechordal plate and the anterior ventral endoderm of the foregut, before the onset of neural crest cell (NCC) migration to the first branchial arch (BA1). The entire forehead, including the foregut endoderm, was removed at 5- to 10-somite stage (ss), which led to the absence of the lower jaw when the operation was performed before 7-ss. If the embryos were deprived of their forehead at 8- to 10-ss, they were later on endowed with a lower beak. In embryos that were operated on early, the NCCs migrated normally to BA1 but were subjected to massive apoptosis a few hours later. Cell death did not occur when forehead excision was performed at a later stage. In this case, onward expression of Shh in the ventral foregut endoderm extended caudally over the excision limit, and we hypothesized that absence of Shh production by the endoderm in embryos that were operated on early could be responsible for the NCC apoptosis and the failure of BA1 development. We thus provided exogenous Shh to the embryos that were operated on before 7-ss. In this case, the development of the lower jaw was rescued. Therefore, Shh derived from the ventral foregut endoderm ensures the survival of NCCs at a critical stage of BA1 development.

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PubMed2006

The contribution of the neural crest to the vertebrate body.

As a transitory structure providing adult tissues of the vertebrates with very diverse cell types, the neural crest (NC) has attracted for long the interest of developmental biologists and is still the subject of ongoing research in a variety of animal models. Here we review a number of data from in vivo cell tracing and in vitro single cell culture experiments, which gained new insights on the mechanisms of cell migration, proliferation and differentiation during NC ontogeny. We put emphasis on the role of Hox genes, morphogens and interactions with neighbouring tissues in specifying and patterning the skeletogenic NC cells in the head. We also include advances made towards characterizing multipotent stem cells in the early NC as well as in various NC derivatives in embryos and even in adult.

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PubMed2005

Patterning the neural crest derivatives during development of the vertebrate head: insights from avian studies.

Studies carried out in the avian embryo and based on the construction of quail-chick chimeras have shown that most of the skull and all the facial and visceral skeleton are derived from the cephalic neural crest (NC). Contribution of the mesoderm is limited to its occipital and (partly) to its otic domains. NC cells (NCCs) participating in membrane bones and cartilages of the vertebrate head arise from the diencephalon (posterior half only), the mesencephalon and the rhombencephalon. They can be divided into an anterior domain (extending down to r2 included) in which genes of the Hox clusters are not expressed (Hox-negative skeletogenic NC) and a posterior domain including r4 to r8 in which Hox genes of the four first paraloguous groups are expressed. The NCCs that form the facial skeleton belong exclusively to the anterior Hox-negative domain and develop from the first branchial arch (BA1). This rostral domain of the crest is designated as FSNC for facial skeletogenic neural crest. Rhombomere 3 (r3) participates modestly to both BA1 and BA2. Forced expression of Hox genes (Hoxa2, Hoxa3 and Hoxb4) in the neural fold of the anterior domain inhibits facial skeleton development. Similarly, surgical excision of these anterior Hox-negative NCCs results in the absence of facial skeleton, showing that Hox-positive NCCs cannot replace the Hox-negative domain for facial skeletogenesis. We also show that excision of the FSNC results in dramatic down-regulation of Fgf8 expression in the head, namely in ventral forebrain and in BA1 ectoderm. We have further demonstrated that exogenous FGF8 applied to the presumptive BA1 territory at the 5-6-somite stage (5-6ss) restores to a large extent facial skeleton development. The source of the cells responsible for this regeneration was shown to be r3, which is at the limit between the Hox-positive and Hox-negative domain. NCCs that respond to FGF8 by survival and proliferation are in turn necessary for the expression/maintenance of Fgf8 expression in the ectoderm. These results strongly support the emerging picture according to which the processes underlying morphogenesis of the craniofacial skeleton are regulated by epithelial-mesenchymal bidirectional crosstalk.

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PubMed2004

Reciprocal relationships between Fgf8 and neural crest cells in facial and forebrain development.

Fgf8 exerts a strong effect on the mesenchymal cells of neural crest (NC) origin that are fated to form the facial skeleton. Surgical extirpation of facial skeletogenic NC domain (including mid-diencephalon down through rhombomere 2), which does not express Hox genes, results in the failure of facial skeleton development and inhibition of the closure of the forebrain neural tube, while Fgf8 expression in the telencephalon and in the branchial arch (BA) ectoderm is abolished. We demonstrate here that (i) exogenous FGF8 is able to rescue facial skeleton development by promoting the proliferation of NC cells from a single rhombomere, r3, which in normal development contributes only marginally to mesenchyme of BA1, and (ii) expression of Fgf8 in forebrain and in BA ectoderm is subjected to signal(s) arising from NC cells, thus showing that the development of cephalic NC-derived structures depends on FGF8 signaling, which is itself triggered by the NC cells.

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PubMed2004

Self-renewal capacity is a widespread property of various types of neural crest precursor cells.

In vertebrates, trunk neural crest (NC) generates glia, neurons, and melanocytes. In addition, it yields mesectodermal derivatives (connective tissues, chondrocytes, and myofibroblasts lining the blood vessels) in the head. Previous in vitro clonal analyses of avian NC cells unraveled a hierarchical succession of highly pluripotent, followed by various intermediate, progenitors, suggesting a model of progressive restrictions in the multiple potentialities of a totipotent stem cell, as prevails in the hematopoietic system. However, which progenitors are able to self-renew within the hierarchy of the NC lineages is still undetermined. Here, we explored further the stem cell properties of quail NC cells by means of in vitro serial subcloning. We identified types of multipotent and oligopotent NC progenitors that differ in their developmental repertoire, ability to self-maintain, and response to exogenous endothelin 3 according to their truncal or cephalic origin. The most striking result is that bipotent progenitors are endowed with self-renewal properties. Thus glia-melanocyte and glia-myofibroblast progenitors behave like stem cells in that they are able both to self-renew and generate a restricted progeny. In our culture conditions, glia-myofibroblast precursors display a modest capacity to self-renew, whereas glia-melanocyte precursors respond to endothelin 3 by extensive self-renewal. These findings may explain the etiology of certain multiphenotypic NC-derived tumors in humans. Moreover, the presence of multiple stem cell phenotypes along the NC-derived lineages may account for the rarity of the "totipotent NC stem cell" and may be related to the large variety and widespread dispersion of NC derivatives throughout the body.

3T3 Cells↗

PubMed2004

The avian embryo as a model to study the development of the neural crest: a long and still ongoing story.

The aim of this review is to evoke briefly the progress that has been made in our knowledge about the contribution of the neural crest to the vertebrate body since it was discovered by Wilhelm His in 1868. Although first studied essentially in amphibian embryos, a large amount of what is known on this very special structure was gained by experimental work carried out on the avian embryo. The making of chimeras between quail and chick has permitted not only to analyse the normal course of neural crest cell migration and differentiation but also to reveal some of the cellular interactions that regulate these events. Looking to the future, we can foresee that the novel methods, which now allow to manipulate gene activities in definite groups of cells and at elected times in the developing embryo, will make the avian model even more instrumental than ever to approach the developmental problems raised by neural crest cell differentiation.

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PubMed2004

From the hemangioblast to self-tolerance: a series of innovations gained from studies on the avian embryo.

During the last decades of the 20th century, studies on the vertebrate hematopoietic and immune systems have largely been performed, on mammalian models. The mouse has been the preferred material for several cogent reasons: (i) numerous well defined genetic strains are available; (ii) this species has been and still is instrumental in the study of gene activity through transgenesis; and (iii) in vitro culture techniques and in vivo assays for blood cells together with a wide array of antibodies and nucleic acid probes have been developed to investigate the cellular interactions occurring during hematopoiesis and immune reactivity. However, important and fundamental notions have emerged from using another higher vertebrate model, the avian embryo. The distinction among small lymphocytes of two populations, the T and B lymphocytes, endowed with different roles in adaptive immunity and dependant on different environments for their specification, has relied on experiments carried out in birds. The avian model has been critical for the analysis of the origin and traffic of hematopoietic precursor cells. It allowed the demonstration that both hematopoietic and angioblastic lineages arise from a common precursor, a cell whose existence had been proposed but never undoubtedly proven, the hemangioblast. Finally a form of thymus-dependant 'dominant' tolerance was demonstrated on the basis of experiments in the avian embryo, which initiated a large current of studies on 'regulatory T-cells'. Work in this model during the last decades has relied strongly on the construction of chimeras between quail and chick embryos that allowed a refined analysis of cell behaviour during embryogenesis. The novel perception of developmental neuropoiesis and immunopoiesis that followed proved to be largely applicable to lower and higher vertebrates, notably mammals.

Allantois↗

PubMed2004

Neural crest cell plasticity and its limits.

The neural crest (NC) yields pluripotent cells endowed with migratory properties. They give rise to neurons, glia, melanocytes and endocrine cells, and to diverse 'mesenchymal' derivatives. Experiments in avian embryos have revealed that the differentiation of the NC 'neural' precursors is strongly influenced by environmental cues. The reversibility of differentiated cells (such as melanocytes or glia) to a pluripotent precursor state can even be induced in vitro by a cytokine, endothelin 3. The fate of 'mesenchymal' NC precursors is strongly restricted by Hox gene expression. In this context, however, facial skeleton morphogenesis is under the control of a multistep crosstalk between the epithelia (endoderm and ectoderm) and NC cells.

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PubMed2003

Inhibition of neuroepithelial patched-induced apoptosis by sonic hedgehog.

During early development in vertebrates, Sonic hedgehog (Shh) is produced by the notochord and the floor plate. A ventrodorsal gradient of Shh directs ventrodorsal patterning of the neural tube. However, Shh is also required for the survival of neuroepithelial cells. We show that Patched (Ptc) induces apoptotic cell death unless its ligand Shh is present to block the signal. Moreover, the blockade of Ptc-induced cell death partly rescues the chick spinal cord defect provoked by Shh deprivation. Thus, the proapoptotic activity of unbound Ptc and the positive effect of Shh-bound Ptc on cell differentiation probably cooperate to achieve the appropriate spinal cord development.

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PubMed2003

Msx genes are expressed in the carapacial ridge of turtle shell: a study of the European pond turtle, Emys orbicularis.

The turtle shell forms by extensive ossification of dermis ventrally and dorsally. The carapacial ridge (CR) controls early dorsal shell formation and is thought to play a similar role in shell growth as the apical ectodermal ridge during limb development. However, the molecular mechanisms underlying carapace development are still unknown. Msx genes are involved in the development of limb mesenchyme and of various skeletal structures. In particular, precocious Msx expression is recorded in skeletal precursors that develop close to the ectoderm, such as vertebral spinous processes or skull. Here, we have studied the embryonic expression of Msx genes in the European pond turtle, Emys orbicularis. The overall Msx expression in head, limb, and trunk is similar to what is observed in other vertebrates. We have focused on the CR area and pre-skeletal shell condensations. The CR expresses Msx genes transiently, in a pattern similar to that of fgf10. In the future carapace domain, the dermis located dorsal to the spinal cord expresses Msx genes, as in other vertebrates, but we did not see expansion of this expression in the dermis located more laterally, on top of the dermomyotomes. In the ventral plastron, although the dermal osseous condensations form in the embryonic Msx-positive somatopleura, we did not observe enhanced Msx expression around these elements. These observations may indicate that common mechanisms participate in limb bud and CR early development, but that pre-differentiation steps differ between shell and other skeletal structures and involve other gene activities than that of Msx genes.

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PubMed2003

Development of melanocyte precursors from the vertebrate neural crest.

Pigment cells that differentiate in the vertebral skin arise from the neural crest (NC), a transitory structure formed at the dorsal borders of the neural plate and which gives rise to migratory cells of multiple fates. How NC cells become committed to the melanocytic lineage and what factors control the survival, proliferation and differentiation of melanocyte precursors remain largely unknown. These issues are of great importance for understanding the mechanisms of several pigment cell pathologies including melanomas. Recent in vivo and in vitro analyses of the fate of single NC cells have indicated that multipotent cells yield melanocyte precursors that become spatially and temporally segregated from other, non melanogenic, NC-derived cell types. The proper development of subsets of NC precursors is governed by environmental local cytokines acting in a paracrine manner. The conjunction of recent studies in mammals and birds reviewed here focuses on the action of endothelin 3 in controlling both the emergence and the maintenance of the NC-derived melanocyte phenotype.

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PubMed2003

Reversal of developmental restrictions in neural crest lineages: transition from Schwann cells to glial-melanocytic precursors in vitro.

In vertebrate embryos, diversification of the lineages arising from the neural crest (NC) is controlled to a large extent by environmental factors. In previous work, we showed that endothelin 3 (ET3) peptide favors the development of glial and melanocytic NC precursors in vitro. This factor is also capable of inducing proliferation of cultured epidermal pigment cells and their conversion to glia. ET3 therefore strongly promotes the emergence of melanocytic and glial phenotypes from precursors and acts on the maintenance of these phenotypes. In the present work, we explored the capacity of ET3 to reprogram glial cells into melanocytes. Schwann cells expressing glial-specific markers [such as the Schwann cell myelin protein (SMP)] were isolated from sciatic nerves of quail embryos and cultured in vitro. We found that ET3 promotes cell growth and sequential expression of melanocyte differentiation markers in cultures of purified SMP-expressing cells, whereas it had no significant effect on SMP-negative cells from the same nerves. Moreover, we provide evidence for the transition of differentiated Schwann cells to melanocytes in clonal cultures. This transition involves the production of a mixed progeny of melanoblasts/melanocytes, glia, and cells bearing differentiation markers of both phenotypes. Therefore, Schwann cells exposed to ET3 transdifferentiate to melanocytes through reversion to the stage of bipotent glial-melanocytic NC precursors. These findings show that NC-derived pigment and glial cells are phenotypically unstable in vitro and may undergo reversal of precursor hierarchy to function as bipotent stem cells.

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PubMed2003

Patterning of the hyoid cartilage depends upon signals arising from the ventral foregut endoderm.

Hyoid bone is a part of the visceral skeleton which arises from both Hox-expressing (Hox+) and Hox-nonexpressing (Hox-) cephalic neural crest cells. In a previous work, we have demonstrated that the Hox- neural crest domain behaves as a naïve entity to which the ventral foregut endoderm confers patterning cues to specify the shape and orientation of the nasal and mandibular skeleton. By using ablation and grafting approaches, we have extended our study to the formation of the hyoid bone and tested the patterning ability of more caudal levels of the lateroventral foregut endoderm in the chick embryo at the early neurula stage. In this study, endodermal stripes have first been delineated according to the projection of mid- and posterior rhombencephalic structures. The extirpation of endodermal transverse stripes along the anteroposterior axis selectively hampers the formation of the ceratobranchials and epibranchials. Thus defined, the patterning ability of the endodermal stripes was further explored in their medial and lateral parts. When homotopically engrafted on the migration pathway of cephalic neural crest cells, ventromedial zones of endoderm lead to the formation of supernumerary basihyal and basibranchial, while lateral zones generate additional cartilaginous pieces recognizable as ceratobranchial and epibranchial. Taken together, our data demonstrate that, early in development, the ventral foregut endoderm exerts a regionalized patterning activity on the cephalic neural crest to build up the primary facial and visceral skeleton in jaws and neck and enable a map of the endodermal skeletogenic areas to be drawn. This map reveals that a cryptic metamerization of the anterior foregut endoderm precedes the formation of the branchial arches.

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PubMed2003

Multipotentiality of the neural crest.

Multiple neural and non-neural cell types arise from the neural crest (NC) in vertebrate embryos. Recent work has provided evidence for multipotent stem cells and intermediate precursors in the early NC cell population as well as in various NC derivatives in embryos and even in adult. Advances have been made towards understanding how cytokines, regulatory genes and cell-cell interactions cooperate to control commitment and differentiation to pigment cells, glia and neurone subtypes. In addition, NC cell fates appeared to be unstable, as differentiated NC cells can reverse to multipotent precursors and transdifferentiate in vitro.

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PubMed2002

Morphogenesis of the branchial vascular sector.

The branchial and dorsal cephalic vascular sectors correspond to the blood vessels contained within evolutionarily recent and ancestral parts of the head, respectively. Recent work demonstrates that neural crest cells (NCCs) provide the pericytes, and connective and smooth muscle cells to the entire branchial sector in an ordered fashion. Initial NCC position is transposed to the vascular distal-to-proximal axis, explaining why circumscribed cephalic vascular anomalies are often associated with reproducible malformations in head tissues derived from the neural crest. Unlike the rest of the central nervous system, the forebrain requires mesenchyme-containing vascular-competent NCCs to survive during embryogenesis and beyond.

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PubMed2002

In ovo transplantation of enteric nervous system precursors from vagal to sacral neural crest results in extensive hindgut colonisation.

The enteric nervous system (ENS) is derived from vagal and sacral neural crest cells (NCC). Within the embryonic avian gut, vagal NCC migrate in a rostrocaudal direction to form the majority of neurons and glia along the entire length of the gastrointestinal tract, whereas sacral NCC migrate in an opposing caudorostral direction, initially forming the nerve of Remak, and contribute a smaller number of ENS cells primarily to the distal hindgut. In this study, we have investigated the ability of vagal NCC, transplanted to the sacral region of the neuraxis, to colonise the chick hindgut and form the ENS in an experimentally generated hypoganglionic hindgut in ovo model. Results showed that when the vagal NC was transplanted into the sacral region of the neuraxis, vagal-derived ENS precursors immediately migrated away from the neural tube along characteristic pathways, with numerous cells colonising the gut mesenchyme by embryonic day (E) 4. By E7, the colorectum was extensively colonised by transplanted vagal NCC and the migration front had advanced caudorostrally to the level of the umbilicus. By E10, the stage at which sacral NCC begin to colonise the hindgut in large numbers, myenteric and submucosal plexuses in the hindgut almost entirely composed of transplanted vagal NCC, while the migration front had progressed into the pre-umbilical intestine, midway between the stomach and umbilicus. Immunohistochemical staining with the pan-neuronal marker, ANNA-1, revealed that the transplanted vagal NCC differentiated into enteric neurons, and whole-mount staining with NADPH-diaphorase showed that myenteric and submucosal ganglia formed interconnecting plexuses, similar to control animals. Furthermore, using an anti-RET antibody, widespread immunostaining was observed throughout the ENS, within a subpopulation of sacral NC-derived ENS precursors, and in the majority of transplanted vagal-to-sacral NCC. Our results demonstrate that: (1) a cell autonomous difference exists between the migration/signalling mechanisms used by sacral and vagal NCC, as transplanted vagal cells migrated along pathways normally followed by sacral cells, but did so in much larger numbers, earlier in development; (2) vagal NCC transplanted into the sacral neuraxis extensively colonised the hindgut, migrated in a caudorostral direction, differentiated into neuronal phenotypes, and formed enteric plexuses; (3) RET immunostaining occurred in vagal crest-derived ENS cells, the nerve of Remak and a subpopulation of sacral NCC within hindgut enteric ganglia.

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