The diverse neural crest: from embryology to human pathology.

We review here some of the historical highlights in exploratory studies of the vertebrate embryonic structure known as the neural crest. The study of the molecular properties of the cells that it produces, their migratory capacities and plasticity, and the still-growing list of tissues that depend on their presence for form and function, continue to enrich our understanding of congenital malformations, paediatric cancers and evolutionary biology. Developmental biology has been key to our understanding of the neural crest, starting with the early days of experimental embryology and through to today, when increasingly powerful technologies contribute to further insight into this fascinating vertebrate cell population.

The "beginnings" of the neural crest.

FOREWORD: The neural crest has been the main object of my investigations during my career in science, up to now. It is a fascinating topic for an embryologist because of its two unique characteristics: its large degree of multipotency and the fact that its development involves a phase during which its component cells migrate all over the embryo and settle in elected sites where they differentiate into a large variety of cell types. Thus, neural crest development raises several specific questions that are at the same time, of general interest: what are the mechanisms controlling the migratory behavior of the cells that detach from the neural plate borders? What are the migration routes taken by the neural crest cells and the environmental factors that make these cells stop in elected sites where they differentiate into a definite series of cell types? When I started to be interested in the neural crest, in the late 1960s, this embryonic structure was the subject of investigations of only a small number of developmental biologists. Fifty years later, it has become the center of interest of many laboratories over the world. The 150th anniversary of its discovery is a relevant opportunity to consider the progress that has been accomplished in our knowledge on the development of this ubiquitous structure, the roles it plays in the physiology of the organism through its numerous and widespread derivatives and its relationships with its environment, as well as the evolutionary advantages it has conferred to the vertebrate phylum. I wish to thank Pr Marianne Bronner, Chief Editor of Developmental Biology and Special Issue Guest Editor, for dedicating a special issue of this journal to this particular structure of the vertebrate embryo. In the following pages, Elisabeth Dupin and I will report some of the highlights of our own acquaintance with the neural crest of the avian embryo, after retracing the main trends of the discoveries of the historical pioneers.

A life in Science with the avian embryo.

My career in research was a second thought. I first (during 8 years) worked as a secondary school teacher and after 4-5 years, during which my two daughters were born, I found a way to escape from what was to be a lifetime job. For two years, my initiation to research was limited to the free time left by my teaching duties. This period of time was a bit "complicated" but not enough to prevent me to realize that research was really what I wanted to do for the rest of my life… And this was when I became acquainted with the chick embryo. This companionship later became extended to another representative of the avian world: the quail (Coturnix coturnix japonica). I recall in the following lines a survey of scientific stories that came out from my association with these precious animals, ... not without a feeling of gratitude.

The issue of the multipotency of the neural crest cells.

In the neural primordium of vertebrate embryos, the neural crest (NC) displays a unique character: the capacity of its component cells to leave the neural primordium, migrate along definite (and, for long, not identified) routes in the developing embryo and invade virtually all tissues and organs, while producing a large array of differentiated cell types. The most striking diversity of the NC derivatives is found in its cephalic domain that produces, not only melanocytes and peripheral nerves and ganglia, but also various mesenchymal derivatives (connective tissues, bones, cartilages…) which, in other parts of the body, are mesoderm-derived. The aim of this article was to review the large amount of work that has been devoted to solving the problem of the differentiation capacities of individual NC cells (NCC) arising from both the cephalic and trunk levels of the neural axis. A variety of experimental designs applied to NCC either in vivo or in vitro are evaluated, including the possibility to culture them in crestospheres, a technique previously designed for cells of the CNS, and which reinforces the notion, previously put forward, of the existence of NC stem cells. At the trunk level, the developmental potentialities of the NCC are more restricted than in their cephalic counterparts, but, in addition to the neural-melanocytic fate that they exclusively express in vivo, it was clearly shown that they harbor mesenchymal capacities that can be revealed in vitro. Finally, a large amount of evidence has been obtained that, during the migration process, most of the NCC are multipotent with a variable array of potentialities among the cells considered. Investigations carried out in adults have shown that multipotent NC stem cells persist in the various sites of the body occupied by NCC. Enlightening new developments concerning the invasive capacity of NCC, the growing peripheral nerves were revealed as migration routes for NCC travelling to distant ventrolateral regions of the body. Designated "Schwann cell precursors" in the mouse embryo, these NCC can leave the nerves and are able to convert to a novel fate. The convertibility of the NC-derived cells, particularly evident in the Schwann cell-melanocyte lineage transition, has also been demonstrated for neuroendocrine cells of the adult carotid body and for the differentiation of parasympathetic neurons of ganglia distant from their origin, the NC. All these new developments attest the vitality of the research on the NC, a field that characterizes vertebrate development and for which the interest has constantly increased during the last decades.

The Pluripotency of Neural Crest Cells and Their Role in Brain Development.

The neural crest (NC) is, in the Chordate phylum, an innovation of vertebrates, which exhibits several original characteristics: its component cells are pluripotent and give rise to both ectodermal and mesodermal cell types. Moreover, during the early stages of neurogenesis, the NC cells exert a paracrine stimulating effect on the development of the preotic brain.

[Ethical problems raised by new reproductive biotechnologies and stem cells]. / Problèmes éthiques posés par les nouvelles biotechnologies de la reproduction et des cellules souches.

Research about the hormonal mechanisms controlling reproduction in mammals has soared during the first half of the 20th century. It has produced a series of discoveries with important outcomes, not only scientific, but also impacting the ways of life. Besides the advent of the contraceptive pill, it has permitted to isolate and cultivate in vitro the female gamete, to fertilize it, thus obtaining a zygote that continues to develop until the blastocyst stage outside the maternal organism. The embryo, transferred into a foster-mother, develops normally until term: the first "test-tube baby" was born in this way in 1978. But the only fact of being able to cultivate the human egg in vitro was to open other possibilities and allow further biological advances: embryonic stem cells (ES cells) obtained from blastocysts and, more recently, from induced Pluripotent Stem cells (iPS), which can potentially be derived from all types of differentiated cell types obtained from adult individuals. From then on, the advent of a new medicine could be anticipated, regenerative because able to replace deficient or absent cells within the organism. As each of these steps was reached, scientists have encountered vigorous opposition from the people: the new potentials disturbed the conceptions that man had of his relationship to nature, in particular in two sensitive domains: sexuality and reproduction. The progress of science has however been accepted by most as soon as it was understood that humanity could anticipate advantages from these advances.

The neural crest, a multifaceted structure of the vertebrates.

In this review, several features of the cells originating from the lateral borders of the primitive neural anlagen, the neural crest (NC) are considered. Among them, their multipotentiality, which together with their migratory properties, leads them to colonize the developing body and to participate in the development of many tissues and organs. The in vitro analysis of the developmental capacities of single NC cells (NCC) showed that they present several analogies with the hematopoietic cells whose differentiation involves the activity of stem cells endowed with different arrays of developmental potentialities. The permanence of such NC stem cells in the adult organism raises the problem of their role at that stage of life. The NC has appeared during evolution in the vertebrate phylum and is absent in their Protocordates ancestors. The major role of the NCC in the development of the vertebrate head points to a critical role for this structure in the remarkable diversification and radiation of this group of animals.

Combinatorial activity of Six1-2-4 genes in cephalic neural crest cells controls craniofacial and brain development.

The combinatorial expression of Hox genes is an evolutionarily ancient program underlying body axis patterning in all Bilateria. In the head, the neural crest (NC)--a vertebrate innovation that contributes to evolutionarily novel skeletal and neural features--develops as a structure free of Hox-gene expression. The activation of Hoxa2 in the Hox-free facial NC (FNC) leads to severe craniofacial and brain defects. Here, we show that this condition unveils the requirement of three Six genes, Six1, Six2, and Six4, for brain development and morphogenesis of the maxillo-mandibular and nasofrontal skeleton. Inactivation of each of these Six genes in FNC generates diverse brain defects, ranging from plexus agenesis to mild or severe holoprosencephaly, and entails facial hypoplasia or truncation of the craniofacial skeleton. The triple silencing of these genes reveals their complementary role in face and brain morphogenesis. Furthermore, we show that the perturbation of the intrinsic genetic FNC program, by either Hoxa2 expression or Six gene inactivation, affects Bmp signaling through the downregulation of Bmp antagonists in the FNC cells. When upregulated in the FNC, Bmp antagonists suppress the adverse skeletal and cerebral effects of Hoxa2 expression. These results demonstrate that the combinatorial expression of Six1, Six2, and Six4 is required for the molecular programs governing craniofacial and cerebral development. These genes are crucial for the signaling system of FNC origin, which regulates normal growth and patterning of the cephalic neuroepithelium. Our results strongly suggest that several congenital craniofacial and cerebral malformations could be attributed to Six genes' misregulation.

Environmental factors unveil dormant developmental capacities in multipotent progenitors of the trunk neural crest.

The neural crest (NC), an ectoderm-derived structure of the vertebrate embryo, gives rise to the melanocytes, most of the peripheral nervous system and the craniofacial mesenchymal tissues (i.e., connective, bone, cartilage and fat cells). In the trunk of Amniotes, no mesenchymal tissues are derived from the NC. In certain in vitro conditions however, avian and murine trunk NC cells (TNCCs) displayed a limited mesenchymal differentiation capacity. Whether this capacity originates from committed precursors or from multipotent TNCCs was unknown. Here, we further investigated the potential of TNCCs to develop into mesenchymal cell types in vitro. We found that, in fact, quail TNCCs exhibit a high ability to differentiate into myofibroblasts, chondrocytes, lipid-laden adipocytes and mineralizing osteoblasts. In single cell cultures, both mesenchymal and neural cell types coexisted in TNCC clonal progeny: 78% of single cells yielded osteoblasts together with glial cells and neurons; moreover, TNCCs generated heterogenous clones with adipocytes, myofibroblasts, melanocytes and/or glial cells. Therefore, alike cephalic NCCs, early migratory TNCCs comprised multipotent progenitors able to generate both mesenchymal and melanocytic/neural derivatives, suggesting a continuum in NC developmental potentials along the neural axis. The skeletogenic capacity of the TNC, which was present in the exoskeletal armor of the extinct basal forms of Vertebrates and which persisted in the distal fin rays of extant teleost fish, thus did not totally disappear during vertebrate evolution. Mesenchymal potentials of the TNC, although not fulfilled during development, are still present in a dormant state in Amniotes and can be disclosed in in vitro culture. Whether these potentials are not expressed in vivo due to the presence of inhibitory cues or to the lack of permissive factors in the trunk environment remains to be understood.

How studies on the avian embryo have opened new avenues in the understanding of development: a view about the neural and hematopoietic systems.

The chick embryo is as ancient a source of knowledge on animal development as the very beginning of embryology. Already, at the time of Caspar Friedrich Wolff, contemplating the strikingly beautiful scenario of the germ deploying on the yellow background of the yolk inspired and supported the tenants of epigenesis at the expense of the preformation theory. In this article, we shall mention some of the many problems of developmental biology that were successfully clarified by research on chick embryos. Two topics, the development of the neural system and that of blood and blood vessels, familiar to the authors, will be discussed in more detail.

Piecing together the vertebrate skull.

In a 1993 Development paper, the quail-chick chimera system was applied to decipher the embryonic origin of the bones of the head skeleton of the avian embryo. The data reported in this article, together with those from previous works, allowed us to assign a precise embryonic origin to all the bones forming the avian skull. It turned out that their major source is the neural crest, with additional contributions from the head paraxial mesoderm and the first five somites, laying to rest a long-standing debate about the origin of the skull.

The neural crest in vertebrate evolution.

Vertebrates belong to the group of chordates characterized by a dorsal neural tube and an anteroposterior axis, the notochord. They are the only chordates to possess an embryonic and pluripotent structure associated with their neural primordium, the neural crest (NC). The NC is at the origin of multiple cell types and plays a major role in the construction of the head, which has been an important asset in the evolutionary success of vertebrates. We discuss here the contribution of the rostral domain of the NC to craniofacial skeletogenesis. Moreover, recent data show that cephalic NC cells regulate the activity of secondary brain organizers, hence being critical for preotic brain development, a role that had not been suspected before.

The neural crest is a powerful regulator of pre-otic brain development.

The role of the neural crest (NC) in the construction of the vertebrate head was demonstrated when cell tracing techniques became available to follow the cells exiting from the cephalic neural folds in embryos of various vertebrate species. Experiments carried out in the avian embryo, using the quail/chick chimera system, were critical in showing that the entire facial skeleton and most of the skull (except for he occipital region) were derived from the NC domain of the posterior diencephalon, mesencephalon and rhombomeres 1 and 2 (r1, r2). This region of the NC was designated FSNC (for Facial Skeletogenic NC). One characteristic of this part of the head including the neural anlage is that it remains free of expression of the homeotic genes of the Hox-clusters. In an attempt to see whether this rostral Hox-negative domain of the NC has a specific role in the development of the skeleton, we have surgically removed it in chick embryos at 5-6 somite stages (5-6 ss). The operated embryos showed a complete absence of facial and skull cartilages and bones showing that the Hox expressing domain of the NC caudally located to the excision did not regenerate to replace the anterior NC. In addition to the deficit in skeletal structures, the operated embryos exhibited severe brain defects resulting in anencephaly. Experiments described here have shown that the neural crest cells regulate the amount of Fgf8 produced by the two brain organizers, the Anterior Neural Ridge (ANR) and the isthmus. This regulation is exerted via the secretion of anti-BMP signaling molecules (e.g. Gremlin and Noggin), which decrease BMP production hence enhancing the amount of Fgf8 synthesized in the ANR (also called "Prosencephalic organizer") and the isthmus. In addition to its role in building up the face and skull, the NC is therefore an important signaling center for brain development.

Modulation of Bmp4 signalling in the epithelial-mesenchymal interactions that take place in early thymus and parathyroid development in avian embryos.

Epithelial-mesenchymal interactions are crucial for the development of the endoderm of the pharyngeal pouches into the epithelia of thymus and parathyroid glands. Here we investigated the dynamics of epithelial-mesenchymal interactions that take place at the earliest stages of thymic and parathyroid organogenesis using the quail-chick model together with a co-culture system capable of reproducing these early events in vitro. The presumptive territories of thymus and parathyroid epithelia were identified in three-dimensionally preserved pharyngeal endoderm of embryonic day 4.5 chick embryos on the basis of the expression of Foxn1 and Gcm2, respectively: the thymic rudiment is located in the dorsal domain of the third and fourth pouches, while the parathyroid rudiment occupies a more medial/anterior pouch domain. Using in vitro quail-chick tissue associations combined with in ovo transplantations, we show that the somatopleural but not the limb bud mesenchyme, can mimic the role of neural crest-derived pharyngeal mesenchyme to sustain development of these glands up to terminal differentiation. Furthermore, mesenchymal-derived Bmp4 appears to be essential to promote early stages of endoderm development during a short window of time, irrespective of the mesenchymal source. In vivo studies using the quail-chick system and implantation of growth factor soaked-beads further showed that expression of Bmp4 by the mesenchyme is necessary during a 24 h-period of time. After this period however, Bmp4 is no longer required and another signalling factor produced by the mesenchyme, Fgf10, influences later differentiation of the pouch endoderm. These results show that morphological development and cell differentiation of thymus and parathyroid epithelia require a succession of signals emanating from the associated mesenchyme, among which Bmp4 plays a pivotal role for triggering thymic epithelium specification.

[Neural crest and vertebrate evolution]. / Crête neurale et évolution des vertébrés.

The neural crest (NC) is a remarkable structure of the Vertebrate embryo, which forms from the lateral borders of the neural plate (designated as neural folds) during neural tube closure. As soon as the NC is formed, its constitutive cells detach and migrate away from the neural primordium along definite pathways and at precise periods of time according to a rostro-caudal progression. The NC cells aggregate in definite places in the developing embryo, where they differentiate into a large variety of cell types including the neurons and glial cells of the peripheral nervous system, the pigment cells dispersed throughout the body and endocrine cells such as the adrenal medulla and the calcitonin producing cells. At the cephalic level only, in higher Vertebrates (but along the whole neural axis in Fishes and Amphibians), the NC is also at the origin of mesenchymal cells differentiating into connective tissue chondrogenic and osteogenic cells. Vertebrates belong to the larger group of Cordates which includes also the Protocordates (Cephalocordates and the Urocordates). All Cordates are characterized by the same body plan with a dorsal neural tube and a notochord which, in Vertebrates, exists only at embryonic stages. The main difference between Protocordates and Vertebrates is the very rudimentary development of cephalic structures in the former. As a result, the process of cephalization is one of the most obvious characteristics of Vertebrates. It was accompanied by the apparition of the NC which can therefore be considered as an innovation of Vertebrates during evolution. The application of a cell marking technique which consists in constructing chimeric embryos between two species of birds, the quail and the chicken, has led to show that the vertebrate head is mainly formed by cells originating from the NC, meaning that this structure was an important asset in Vertebrate evolution. Recent studies, described in this article, have strengthened this view by showing that the NC does not only provide the cells that build up the facial skeleton and most of the skull but plays a major role in early brain neurogenesis. It was shown that the cephalic NC cells produce signaling molecules able to regulate the activity of the two secondary organizing centers previously identified in the developing brain: the anterior neural ridge and the midbrain-hindbrain junction, which secrete Fgf8, a potent stimulator of early brain neurogenesis.

The cephalic neural crest of amniote vertebrates is composed of a large majority of precursors endowed with neural, melanocytic, chondrogenic and osteogenic potentialities.

In the amniote embryo, the neural crest (NC) has the unique capacity to give rise to neuronal and glial cells in the peripheral nervous system (PNS), melanocytes and mesenchymal cells including those forming the head skeleton and connective tissues. In the trunk, mesenchymal cells are derived from the mesoderm. The question was raised whether the NC-derived head mesenchyme arises from a lineage separate from the neural-melanocytic one, or if both skeletogenic and neural-melanocytic derivatives originate from a common putative stem cell in the early cephalic NC. We discuss here these issues and present experimental data that provide evidence for the multipotency of NC cells (NCC), focusing on those at the origin of the craniofacial skeleton. Recent work of in vitro clonal culture revealed that the vast majority (92% of clonogenic cells) of the cephalic quail NCC are capable to yield osteoblasts together with neurones, glial cells and melanocytes. A common pluripotent progenitor for chondrocytes, osteocytes, neurones, glial cells, melanocytes and myofibroblasts has been identified and is present in the early cephalic NC at the frequency of 7 to 13% of clonogenic cells depending on the environmental conditions. Together with recent reports that multipotent NC-related progenitors persist in adult tissues in rodents and humans, these results reinforce a stem cell model for the generation and maintenance of NC-derived lineages during embryogenesis and in adult tissue homeostasis.

High frequency of cephalic neural crest cells shows coexistence of neurogenic, melanogenic, and osteogenic differentiation capacities.

The neural crest (NC) is a vertebrate innovation that distinguishes vertebrates from other chordates and was critical for the development and evolution of a "New Head and Brain." In early vertebrates, the NC was the source of dermal armor of fossil jawless fish. In extant vertebrates, including mammals, the NC forms the peripheral nervous system, melanocytes, and the cartilage and bone of the face. Here, we show that in avian embryos, a large majority of cephalic NC cells (CNCCs) have the ability to differentiate into cell types as diverse as neurons, melanocytes, osteocytes, and chondrocytes. Moreover, we find that the morphogen Sonic hedgehog (Shh) acts on CNCCs to increase endochondral osteogenesis while having no effect on osteoblasts prone to membranous ossification. We have developed culture conditions that demonstrate that "neural-mesenchymal" differentiation abilities are present in more than 90% of CNCCs. A highly multipotent progenitor (able to yield neurons, glia, melanocytes, myofibroblasts, chondrocytes, and osteocytes) comprises 7-13% of the clonogenic cells in the absence and presence of Shh, respectively. This progenitor is a good candidate for a cephalic NC stem cell.

Induction of mirror-image supernumerary jaws in chicken mandibular mesenchyme by Sonic Hedgehog-producing cells.

Previous studies have shown that Sonic Hedgehog (Shh) signaling is crucial for the development of the first branchial arch (BA1) into a lower-jaw in avian and mammalian embryos. We have already shown that if Shh expression is precociously inhibited in pharyngeal endoderm, neural crest cells migrate to BA1 but fail to survive, and Meckel's cartilage and associated structures do not develop. This phenotype can be rescued by addition of an exogenous source of Shh. To decipher the role of Shh, we explored the consequences of providing an extra source of Shh to the presumptive BA1 territory. Grafting quail fibroblasts engineered to produce Shh (QT6-Shh), at the 5- to 8-somite stage, resulted in the induction of mirror-image extra lower jaws, caudolateral to the normal one. It turns out that the oral opening epithelium, in which Shh, Fgf8 and Bmp4 are expressed in a definite pattern, functions as an organizing center for lower-jaw development. In our experimental design, the extra source of Shh activates Fgf8, Bmp4 and Shh genes in caudal BA1 ectoderm in a spatial pattern similar to that of the oral epithelium, and regularly leads to the formation of two extra lower-jaw-organizing centers with opposite rostrocaudal polarities. These results emphasize the similarities between the developmental processes of the limb and mandibular buds, and show that in both cases Shh-producing cells create a zone of polarizing activity for the structures deriving from them.

Developmental patterning deciphered in avian chimeras.

I started my scientific carer by investigating the development of the digestive tract in the laboratory of a well-known embryologist, Etienne Wolff, then professor at the Collège de France. My animal model was the chick embryo. The investigations that I pursued on liver development together with serendipity, led me to devise a cell-marking technique based on the construction of chimeric embryos between two closely related species of birds, the Japanese quail (Coturnix coturnix japonica) and the chick (Gallus gallus). The possibility to follow the migration and fate of the cells throughout development from early embryonic stages up to hatching and even after birth, was a breakthrough in developmental biology of higher vertebrates. This article describes some of scientific achievements based on the use of this technique in my laboratory during the last 38 years.