Cephalic Neural Crest Cells Research Articles

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.

Irreversible effects of retinoic acid pulse onXenopusjaw morphogenesis: new insight into cranial neural crest specificationb

Jaws are formed by cephalic neural crest (CNCCs) and mesodermal cells migrating to the first pharyngeal arch (PA1). A complex signaling network involving different PA1 components then establishes the jaw morphogenetic program. To gather insight on this developmental process, in this study, we analyze the teratogenic effects of brief (1-15 min) pulses of low doses of retinoic acid (RA: 0.25-2 µM) or RA agonists administered to early Xenopus laevis (X.l.) embryos. We show that these brief pulses of RA cause permanent craniofacial defects specifically when treatments are performed during a 6-hr window (developmental stages NF15-NF23) that covers the period of CNCCs maintenance, migration, and specification. Earlier or later treatments have no effect. Similar treatments performed at slightly different developmental stages within this temporal window give rise to different spectra of malformations. The RA-dependent teratogenic effects observed in Xenopus can be partially rescued by folinic acid. We provide evidence suggesting that in Xenopus, as in the mouse, RA causes craniofacial malformations by perturbing signaling to CNCCs. Differently from the mouse, where RA affects CNCCs only at the end of their migration, in Xenopus, RA has an effect on CNCCs during all the period ranging from their exit from the neural tube until their arrival in the PA1. Our findings provide a conceptual framework to understand the origin of individual facial features and the evolution of different craniofacial morphotypes.

Spatio‐temporal dynamics of gene expression of the Edn1‐Dlx5/6 pathway during development of the lower jaw

The morphogenesis of the vertebrate skull results from highly dynamic integrated processes involving the exchange of signals between the ectoderm, the endoderm, and cephalic neural crest cells (CNCCs). Before migration CNCCs are not committed to form any specific skull element, molecular signals exchanged in restricted regions of tissue interaction are crucial in providing positional identity to the CNCCs mesenchyme and activate the specific morphogenetic process of different skeletal components of the head. In particular, the endothelin-1 (Edn1)-dependent activation of Dlx5 and Dlx6 in CNCCs that colonize the first pharyngeal arch (PA1) is necessary and sufficient to specify maxillo-mandibular identity. Here, to better analyze the spatio-temporal dynamics of this process, we associate quantitative gene expression analysis with detailed examination of skeletal phenotypes resulting from combined allelic reduction of Edn1, Dlx5, and Dlx6. We show that Edn1-dependent and -independent regulatory pathways act at different developmental times in distinct regions of PA1. The Edn1-->Dlx5/6-->Hand2 pathway is already active at E9.5 during early stages of CNCCs colonization. At later stages (E10.5) the scenario is more complex: we propose a model in which PA1 is subdivided into four adjacent territories in which distinct regulations are taking place. This new developmental model may provide a conceptual framework to interpret the craniofacial malformations present in several mouse mutants and in human first arch syndromes. More in general, our findings emphasize the importance of quantitative gene expression in the fine control of morphogenetic events.

Expression of chick Coactosin in cells in morphogenetic movement

Coactosin is a 17 kDa actin binding protein that belongs to the actin depolymerizing factor/cofilin homology family. Coactosin inhibits barbed-end capping of actin filament, and is involved in actin polymerization. Coactosin is expressed in cephalic and trunk neural crest cells, cranial ganglia and dorsal root ganglia. Coactosin is also expressed in the cells that are forming mesonephric duct, and endodermal cells. Immunocytochemistry with anti-Coactosin antibody shows that Coactosin is localized in the cytoplasm, and associated with actin stress fibers in cultured neural crest cells. Coactosin is also expressed in the axon of oculomotor nerve and trigeminal nerve. In the growth cone of the oculomotor nerve axons, both Coactosin mRNA and protein were localized, which is indicative of the role of Coactosin in pathfinding of the growth cone. Coactosin is expressed in those that require dynamic and highly coordinated regulation of actin cytoskeleton, that is, neural crest cells, cells in the tip of the mesonephros, endodermal cells and axons.

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.

Heterochronic shift between early organogenesis and migration of cephalic neural crest cells in two divergent evolutionary phenotypes of archosaurs: crocodile and ostrich

Living archosaurs (crocodiles and birds) represent an intriguing evo-devo model system. Although close in phylogenetic relationship, the two lineages show considerable divergence in trends of phenotypic evolution. The head anatomy of recent crocodilians has changed little in comparison with that of their crocodylomorph ancestors. The head phenotype of the avians (birds), as well as some non-avian theropods, shows numerous evolutionary innovations that differ considerably from the crocodylomorph pattern. Most of the novel head structures, such as features of the craniofacial skeleton, cranial nerves, head muscles, and integument are derived from the same cellular source common to all archosaurs, the cephalic neural crest (CNC). Therefore, other factors must be involved in the developmental disparity of homologous structures in the aforementioned lineages. The present study analyzes the earliest developmental events that are associated with the appearance of the neural crest cells in the two archosaur models: Crocodylus niloticus and Struthio camelus. I found that both models share unique developmental features, the presence of an unpaired, rostrally migrating population of CNC cells, showing that the two are closely related to each other. On the other hand, the crocodile and the ostrich differ substantially in (1) timing, (2) duration, and (3) expression patterns of the CNC. Compared with the crocodile, the CNC cells in the ostrich (1) migrate much later into the embryonic head, (2) but relocate to their terminal positions faster, and (3) take specifically directed migratory routes in the mandibular/oral region and head/trunk-interface regions. I suggest that accelerated relocation of CNC cells combined with delayed head organogenesis may represent important innovative conditions in the developmental evolution of a new archosaur head phenotype.

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.

Elucidating timing and function of endothelin-A receptor signaling during craniofacial development using neural crest cell-specific gene deletion and receptor antagonism

Cranial neural crest cells (NCCs) play an intimate role in craniofacial development. Multiple signaling cascades participate in patterning cranial NCCs, some of which are regulated by endothelin-A receptor (Ednra) signaling. Ednra −/− embryos die at birth from severe craniofacial defects resulting from disruption of neural crest cell patterning and differentiation. These defects include homeotic transformation of lower jaw structures into upper jaw-like structures, suggesting that some cephalic NCCs alter their “identity” in the absence of Ednra signaling. To elucidate the temporal necessity for Ednra signaling in vivo, we undertook two strategies. We first used a conditional knockout strategy in which mice containing a conditionally targeted Ednra allele ( Ednra fl ) were bred with mice from the Hand2-Cre and Wnt1-Cre transgenic mouse strains, two strains in which Cre expression occurs at different time periods within cranial NCCs. In our second approach, we used an Ednra-specific antagonist to treat wild type pregnant mice between embryonic days E8.0 and E10.0, a time frame encompassing the early migration and proliferation of cranial NCCs. The combined results suggest that Ednra function is crucial for NCC development between E8.25 and E9.0, a time period encompassing the arrival of NCCs in the arches and/or early post-migratory patterning. After this time period, Ednra signaling is dispensable. Interestingly, middle ear structures are enlarged and malformed in a majority of Ednra fl/fl;Wnt1-Cre embryos, instead resembling structures found in extinct predecessors of mammals. These observations suggest that the advent of Ednra signaling in cranial NCCs may have been a crucial event in the evolution of the mammalian middle ear ossicles.

An endothelin-1 switch specifies maxillomandibular identity

Articulated jaws are highly conserved structures characteristic of gnathostome evolution. Epithelial-mesenchymal interactions within the first pharyngeal arch (PA1) instruct cephalic neural crest cells (CNCCs) to form the different skeletal elements of the jaws. The endothelin-1 (Edn1)/endothelin receptor type-A (Ednra)-->Dlx5/6-->Hand2 signaling pathway is necessary for lower jaw formation. Here, we show that the Edn1 signaling is sufficient for the conversion of the maxillary arch to mandibular identity. Constitutive activation of Ednra induced the transformation of upper jaw, maxillary, structures into lower jaw, mandibular, structures with duplicated Meckel's cartilage and dermatocranial jaws constituted by 4 dentary bones. Misexpression of Hand2 in the Ednra domain caused a similar transformation. Skeletal transformations are accompanied by neuromuscular remodeling. Ednra is expressed by most CNCCs, but its constitutive activation affects predominantly PA1. We conclude that after migration CNCCs are not all equivalent, suggesting that their specification occurs in sequential steps. Also, we show that, within PA1, CNCCs are competent to form both mandibular and maxillary structures and that an Edn1 switch is responsible for the choice of either morphogenetic program.

Sonic hedgehog signalling from foregut endoderm patterns the avian nasal capsule

Morphogenesis of the facial skeleton depends on inductive interactions between cephalic neural crest cells and cephalic epithelia, including the foregut endoderm. We show that Shh expression in the most rostral zone of the endoderm, endoderm zone I (EZ-I), is necessary to induce the formation of the ventral component of the avian nasal capsule: the mesethmoid cartilage. Surgical removal of EZ-I specifically prevented mesethmoid formation, whereas grafting a supernumerary EZ-I resulted in an ectopic mesethmoid. EZ-I ablation was rescued by Shh-loaded beads, whereas inhibition of Shh signalling suppressed mesethmoid formation. This interaction between the endoderm and cephalic neural crest cells was reproduced in vitro, as evidenced by Gli1 induction. Our work bolsters the hypothesis that early endodermal regionalisation provides the blueprint for facial morphogenesis and that its disruption might cause foetal craniofacial defects, including those of the nasal region.

Sonic Hedgehog promotes the development of multipotent neural crest progenitors endowed with both mesenchymal and neural potentials

In the vertebrate embryo, the cephalic neural crest cells (CNCCs) produce cells belonging to two main lineages: the neural [including neurons, glial cells of the peripheral nervous system (PNS), and melanocytes] and the mesenchymal (chondrocytes, osteoblasts, smooth muscle cells, and connective tissue cells), whereas the trunk NCCs (TNCCs) in amniotes yield only neural derivatives. Although multipotent cells have previously been evidenced by in vitro clonal analysis, the issue as to whether all of the mesenchymal and neural phenotypes can be derived from a unique NC stem cell has remained elusive. In the present work, we devised culture conditions that led us to identify a highly multipotent NCC endowed with both neural and mesenchymal potentials, which lies upstream of all the other NC progenitors known so far. We found that addition of recombinant Sonic Hedgehog (Shh) increased the number of CNCC progenitors yielding both mesenchymal and neural lineages and promoted the development of such precursors from the TNCC. Shh decreased the neural-restricted precursors without affecting the overall CNCC survival and proliferation. By showing a differential positive effect of Shh on the expression of mesenchymal phenotypes (i.e., chondrocytes and smooth muscle cells) by multipotent CNCCs, these results shed insights on the in vivo requirement of Shh for craniofacial morphogenesis. Together with evolutionary considerations, these data also suggest that the mesenchymal-neural precursor represents the ancestral form of the NC stem cell, which in extinct forms of vertebrates (the ostracoderms) was able to yield both the PNS and superficial skeleton.

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.

Cephalic neural crest cells and the evolution of craniofacial structures in vertebrates: morphological and embryological significance of the premandibular–mandibular boundary

The vertebrate head characteristically has two types of mesenchyme: the neural crest-derived ectomesenchyme and the mesoderm derived mesenchyme. Conserved patterns of development in various animal taxa imply the presence of shared inductive events for cephalic mesenchyme. These developmental programs can serve as developmental constraints that emerge as morphological homology of embryonic patterns. To understand the evolutionary changes in the developmental programs that shape the skull, we need to separate ancestral and derived patterns of vertebrate craniogenesis. This review deals with the terminology for neural crest cell subpopulations at each developmental stage, based on the topographical relationships and possible mechanisms for specification. The aim is to identify the changes that could have occurred in the evolutionary history of vertebrates. From comparisons of a lamprey species, Lethenteron japonicum, with gnathostomes it is clear that the initial distribution of cephalic crest cells is identical in the two animal lineages. In all vertebrate embryos, the trigeminal crest (TC) cells of an early pharyngula are subdivided into three subpopulations. At this stage, only the posterior subpopulation of the TC cells is specified as the mandibular arch, as compared to the more rostral components, the ‘premandibular crest cells’. Later in development, the local specification patterns of the lamprey and the gnathostomes differ, so that homology cannot be established in the craniofacial primordia, including the oral apparatus. Therefore, embryological terminology should reflect these hierarchical patterns in developmental stages and phylogeny.

The instability of the neural crest phenotypes: Schwann cells can differentiate into myofibroblasts

In the vertebrate embryo, the neural crest cells (NCCs) that migrate out from the neural primordium yield multiple phenotypes, including melanocytes, peripheral neurones and glia and, in the head, cartilage, bone, connective cells and myofibroblasts / vascular smooth muscle cells (SMCs). The differentiation of pluripotent NCCs is mainly directed by local growth factors. Even at postmigratory stages, NC-derived cells exhibit some fate plasticity. Thus, we reported earlier that pigment cells and Schwann cells are able in vitro to interconvert in the presence of endothelin 3 (ET3). Here, we further investigated the capacity of Schwann cells to reprogram their phenotype. We show that purified quail Schwann cells in dissociated cultures produce alpha smooth muscle actin ((alpha)SMA)-expressing myofibroblasts through the generation of a pluripotent progeny. This transdifferentiation took place independently of ET3, but was promoted by transforming growth factor beta1 (TGF(beta)1). Moreover, when implanted into chick embryos, the Schwann cells were found to contribute with host cephalic NCCs to perivascular SMCs. These data provided the first evidence for the acquisition of an NC-derived mesenchymal fate by Schwann cells and further demonstrate that the differentiation state of NC-derived cells is unstable and capable of reprogramming. The high plasticity of Schwann cells evidenced here also suggests that, as in the CNS, glial cells of the PNS may function as NC stem cells in particular circumstances such as repair.

Identification of a novel type II classical cadherin: Rat cadherin19 is expressed in the cranial ganglia and Schwann cell precursors during development

To identify a novel type II classical cadherin, we searched the genome database and found rat cadherin19 (cad19) with high similarity to human cadherin19. We also found nucleotide sequences corresponding to rat cad19 in mouse and chicken genomes. In situ hybridization of rat cad19 revealed that rat cad19 mRNA was initially expressed in cephalic neural crest cells, and then in the cranial ganglia, migrating trunk neural crest cells, the nascent dorsal root ganglia, and the sympathetic ganglia. Expression of cad19 overlapped with that of neural crest markers, including Sox10 and AP-2, but cad19 expression was confined to subpopulations of the neural crest-derived cells, those typically observed in the satellite glia at the periphery of the ganglia and Schwann cell precursors along the peripheral nerves. cad19 mRNA was not detected in cells expressing Phox2b, an epibranchial placode-derived neurons, nor in those expressing neuronal markers such as Hu protein. These observations suggest that cad19 is expressed in neural crest-derived, non-neuronal cells. Although the expression of cad19 mRNA persisted in Schwann cell precursors at E14.5, it was no longer detected in maturing Schwann cells at later stages. These results suggest that cad19 is an evolutionarily conserved cadherin and may be involved in the early development of Schwann cells in the peripheral nervous system.

Evolution of the vertebrate jaw: comparative embryology and molecular developmental biology reveal the factors behind evolutionary novelty

It is generally believed that the jaw arose through the simple transformation of an ancestral rostral gill arch. The gnathostome jaw differentiates from Hox-free crest cells in the mandibular arch, and this is also apparent in the lamprey. The basic Hox code, including the Hox-free default state in the mandibular arch, may have been present in the common ancestor, and jaw patterning appears to have been secondarily constructed in the gnathostomes. The distribution of the cephalic neural crest cells is similar in the early pharyngula of gnathostomes and lampreys, but different cell subsets form the oral apparatus in each group through epithelial-mesenchymal interactions: and this heterotopy is likely to have been an important evolutionary change that permitted jaw differentiation. This theory implies that the premandibular crest cells differentiate into the upper lip, or the dorsal subdivision of the oral apparatus in the lamprey, whereas the equivalent cell population forms the trabecula of the skull base in gnathostomes. Because the gnathostome oral apparatus is derived exclusively from the mandibular arch, the concepts 'oral' and 'mandibular' must be dissociated. The 'lamprey trabecula' develops from mandibular mesoderm, and is not homologous with the gnathostome trabecula, which develops from premandibular crest cells. Thus the jaw evolved as an evolutionary novelty through tissue rearrangements and topographical changes in tissue interactions.

Endothelin-A receptor-dependent and -independent signaling pathways in establishing mandibular identity.

The lower jaw skeleton is derived from cephalic neural crest (CNC) cells that reside in the mandibular region of the first pharyngeal arch. Endothelin-A receptor (Ednra) signaling in crest cells is crucial for their development, as Ednra(-/-) mice are born with severe craniofacial defects resulting in neonatal lethality. In this study, we undertook a more detailed analysis of mandibular arch development in Ednra(-/-) embryos to better understand the cellular and molecular basis for these defects. We show that most lower jaw structures in Ednra(-/-) embryos undergo a homeotic transformation into maxillary-like structures similar to those observed in Dlx5/Dlx6(-/-) embryos, though lower incisors are still present in both mutant embryos. These structural changes are preceded by aberrant expansion of proximal first arch gene expression into the distal arch, in addition to the previously described loss of a Dlx6/Hand2 expression network. However, a small distal Hand2 expression domain remains. Although this distal expression is not dependent on either Ednra or Dlx5/Dlx6 function, it may require one or more GATA factors. Using fate analysis, we show that these distal Hand2-positive cells probably contribute to lower incisor formation. Together, our results suggest that the establishment of a 'mandibular identity' during lower jaw development requires both Ednra-dependent and -independent signaling pathways.

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.

Differential expression of beta3 integrin gene in chick and mouse cranial neural crest cells.

RNA in situ hybridization on early chicken embryos revealed that the beta3 integrin gene started to be expressed after Hamburger and Hamilton (HH) stage 6 in the presumptive epidermis adjacent to the neural plate, before closure of the neural tube. The beta3 integrin gene was also strongly expressed in cephalic neural crest cells at the same stage in which they begin their migration but disappeared progressively in these cells along the route they take to the branchial arches. The gene was weakly expressed in the differentiating cranial neural crest cells. The alphaVbeta3 integrin protein complex was also mainly detected in the migratory cephalic neural crest cells. However, during early mouse embryogenesis and in contrast to the chick, the beta3 integrin gene was expressed in the foregut diverticulum and in the heart and not in the cephalic neural crest cells. Therefore, the difference in the beta3 integrin expression suggests that mouse and chicken cranial neural crest cells may have distinct integrin requirements during their ontogenesis.

Dhand-cre transgenic mice reveal specific potential functions of dHAND during craniofacial development

Most of the bone, cartilage, and connective tissue of the craniofacial region arise from cephalic neural crest cells. Presumably, patterning differences in crest cells are a result of regional action of transcription factors within the developing pharyngeal arches. The basic helix–loop–helix transcription factor dHAND/HAND2 is expressed throughout much of the neural crest-derived mesenchyme of the pharyngeal arches, suggesting that it plays a crucial role in craniofacial development. However, targeted inactivation of the dHAND gene results in embryonic lethality by E10.5 due to vascular defects, preventing further analysis of the role of dHAND in cephalic neural crest cell development. In order to examine putative roles of dHAND during later stages of embryogenesis, we have used a transgenic lineage marker approach, in which a portion of the dHAND upstream region containing an enhancer that directs dHAND expression to the pharyngeal arches is used to drive Cre recombinase expression. By crossing these dHAND-Cre transgenic mice with R26R mice, we can follow the fate of cells that expressed dHAND at any time during development by examining β-galactosidase activity. We show that dHAND is first expressed in postmigratory cephalic neural crest cells within the pharyngeal arches. In older embryos, β-galactosidase-labeled cells are observed in most of the neural crest-derived lower jaw skeleton and surrounding connective tissues. However, labeled cells only contribute to substructures within the middle ear, indicating that our transgene is not globally expressed in cephalic neural crest cells within the pharyngeal arches. Moreover, dHAND-Cre mice will provide a valuable tool for tissue-specific inactivation of gene expression in multiple tissue types of neural crest origin.