Sophie Creuzet

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.

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.

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. Developmental Dynamics 228:239–246, 2003.

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.

Regulation of pre-otic brain development by the cephalic neural crest

Emergence of the neural crest (NC) is considered an essential asset in the evolution of the chordate phylum, as specific vertebrate traits such as peripheral nervous system, cephalic skeletal tissues, and head development are linked to the NC and its derivatives. It has been proposed that the emergence of the NC was responsible for the formation of a “new head” characterized by the spectacular development of the forebrain and associated sense organs. It was previously shown that removal of the cephalic NC (CNC) prevents the formation of the facial structures but also results in anencephaly. This article reports on the molecular mechanisms whereby the CNC controls cephalic neurulation and brain morphogenesis. This study demonstrates that molecular variations of Gremlin and Noggin level in CNC account for morphological changes in brain size and development. CNC cells act in these processes through a multi-step control and exert cumulative effects counteracting bone morphogenetic protein signaling produced by the neighboring tissues (e.g., adjacent neuroepithelium, ventro-medial mesoderm, superficial ectoderm). These data provide an explanation for the fact that acquisition of the NC during the protochordate-to-vertebrate transition has coincided with a large increase of brain vesicles.

Inhibitory effect of hydroxyflavones on the exogenous nadh dehydrogenase of plant mitochondrial inner membranes

Abstract The inhibitory properties of a series of 21 flavones were investigated on the uncoupled electron transfer of purified potato tuber mitochondria, with different substrates. Thirteen compounds were shown to be selective inhibitors of the external NADH dehydrogenase of the inner membrane. Among mono- and dihydroxyflavones, some good inhibitors were found, but their efficiency depended greatly on the position of hydroxylation (1 50 between 20 and 150 μM). For example, 8-hydroxyflavone was six-fold more effective than 5-hydroxyflavone. Except in the case of norwogonin, the aglycones tested were ineffective when they were substituted by more than two hydroxyls, without other substituents. This was the case for quercetin, for example. The best inhibitor of this series was platanetin (I 50 = 2 μM). This very lipophilic flavonol (-log P = 5.10) was characterized by the presence of a dimethylallyl chain at the 6-position. When this substituent was replaced by a methyl, (platanin), the effectiveness of the product remained high, but its selectivity was lowered. These results suggest that the value of the lipophilicity parameter (-log P), for the molecules able to attain this target must be between 3.7 and 5.1. However, the lipophilicity parameter on its own does not explain why some flavones act selectively on the external NADH dehydrogenase, probably at the level where the electrons are donated to the quinone pool.

Neural crest contribution to forebrain development.

The neural crest (NC), a defining feature of vertebrate embryo, generates most of the skeletal tissues encasing the developing forebrain and provides the prosencephalon with functional vasculature and meninges. Recent findings show that early in development, the cephalic NC is also essential for the pre-otic neural tube closure and promotes the development of the prosencephalic alar plate by regulating the morphogenetic activities of forebrain organizers.

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.

The facial neural crest controls fore- and midbrain patterning by regulating Foxg1 expression through Smad1 activity.

The facial neural crest (FNC), a pluripotent embryonic structure forming craniofacial structures, controls the activity of brain organisers and stimulates cerebrum growth. To understand how the FNC conveys its trophic effect, we have studied the role of Smad1, which encodes an intracellular transducer, to which multiple signalling pathways converge, in the regulation of Foxg1. Foxg1 is a transcription factor essential for telencephalic specification, the mutation of which leads to microcephaly and mental retardation. Smad1 silencing, based on RNA interference (RNAi), was performed in pre-migratory FNC cells. Soon after electroporation of RNAi molecules, Smad1 inactivation abolished the expression of Foxg1 in the chick telencephalon, resulting in dramatic microcephaly and partial holoprosencephaly. In addition, the depletion of Foxg1 activity altered the expression Otx2 and Foxa2 in di/mesencephalic neuroepithelium. However, when mutated forms of Smad1 mediating Fgf and Wnt signalling were transfected into FNC cells, these defects were overcome. We also show that, downstream of Smad1 activity, Dkk1, a Wnt antagonist produced by the FNC, initiated the specification of the telencephalon by regulating Foxg1 activity. Additionally, the activity of Cerberus in FNC-derived mesenchyme synergised with Dkk1 to control Foxg1 expression and maintain the balance between Otx2 and Foxa2.

A conserved role for non-neural ectoderm cells in early neural development

During the early steps of head development, ectodermal patterning leads to the emergence of distinct non-neural and neural progenitor cells. The induction of the preplacodal ectoderm and the neural crest depends on well-studied signalling interactions between the non-neural ectoderm fated to become epidermis and the prospective neural plate. By contrast, the involvement of the non-neural ectoderm in the morphogenetic events leading to the development and patterning of the central nervous system has been studied less extensively. Here, we show that the removal of the rostral non-neural ectoderm abutting the prospective neural plate at late gastrulation stage leads, in mouse and chick embryos, to morphological defects in forebrain and craniofacial tissues. In particular, this ablation compromises the development of the telencephalon without affecting that of the diencephalon. Further investigations of ablated mouse embryos established that signalling centres crucial for forebrain regionalization, namely the axial mesendoderm and the anterior neural ridge, form normally. Moreover, changes in cell death or cell proliferation could not explain the specific loss of telencephalic tissue. Finally, we provide evidence that the removal of rostral tissues triggers misregulation of the BMP, WNT and FGF signalling pathways that may affect telencephalon development. This study opens new perspectives on the role of the neural/non-neural interface and reveals its functional relevance across higher vertebrates.