Neural crest cells that form the vertebrate head skeleton migrate and interact with surrounding tissues to shape the skull, and defects in these processes underlie many human craniofacial syndromes. Signals at the midline play a crucial role in the development of the anterior neurocranium, which forms the ventral braincase and palate, and here we explore the role of Hedgehog (Hh) signaling in this process. Using sox10:egfp transgenics to follow neural crest cell movements in the living embryo, and vital dye labeling to generate a fate map, we show that distinct populations of neural crest form the two main cartilage elements of the larval anterior neurocranium: the paired trabeculae and the midline ethmoid. By analyzing zebrafish mutants that disrupt sonic hedgehog (shh)expression, we demonstrate that shh is required to specify the movements of progenitors of these elements at the midline, and to induce them to form cartilage. Treatments with cyclopamine, to block Hh signaling at different stages, suggest that although requirements in morphogenesis occur during neural crest migration beneath the brain, requirements in chondrogenesis occur later, as cells form separate trabecular and ethmoid condensations. Cell transplantations indicate that these also reflect different sources of Shh, one from the ventral neural tube that controls trabecular morphogenesis and one from the oral ectoderm that promotes chondrogenesis. Our results suggest a novel role for Shh in the movements of neural crest cells at the midline, as well as in their differentiation into cartilage, and help to explain why both skeletal fusions and palatal clefting are associated with the loss of Hh signaling in holoprosencephalic humans.

The neural crest is a uniquely vertebrate cell type that gives rise to much of the craniofacial skeleton, pigment cells and peripheral nervous system, yet its specification and diversification during embryogenesis are poorly understood. Zebrafish homozygous for the lockjaw (low)mutation show defects in all of these derivatives and we show that low (allelic with montblanc) encodes a zebrafish tfap2a, one of a small family of transcription factors implicated in epidermal and neural crest development. A point mutation in lowtruncates the DNA binding and dimerization domains of tfap2a, causing a loss of function. Consistent with this, injection of antisense morpholino oligonucleotides directed against splice sites in tfap2a into wild-type embryos produces a phenotype identical to low. Analysis of early ectodermal markers revealed that neural crest specification and migration are disrupted in low mutant embryos. TUNEL labeling of dying cells in mutants revealed a transient period of apoptosis in crest cells prior to and during their migration. In the cranial neural crest, gene expression in the mandibular arch is unaffected in low mutants, in contrast to the hyoid arch, which shows severe reductions in dlx2 and hoxa2 expression. Mosaic analysis, using cell transplantation,demonstrated that neural crest defects in low are cell autonomous and secondarily cause disruptions in surrounding mesoderm. These studies demonstrate that low is required for early steps in neural crest development and suggest that tfap2a is essential for the survival of a subset of neural crest derivatives.

SUMMARYBone morphogenetic proteins (BMPs) play crucial roles in craniofacial development but little is known about their interactions with other signals, such as Endothelin 1 (Edn1) and Jagged/Notch, which pattern the dorsal-ventral (DV) axis of the pharyngeal arches. Here, we use transgenic zebrafish to monitor and perturb BMP signaling during arch formation. With a BMP-responsive transgene, Tg(Bre:GFP), we show active BMP signaling in neural crest (NC)-derived skeletal precursors of the ventral arches, and in surrounding epithelia. Loss-of-function studies using a heat shock-inducible, dominant-negative BMP receptor 1a [Tg(hs70I:dnBmpr1a-GFP)] to bypass early roles show that BMP signaling is required for ventral arch development just after NC migration, the same stages at which we detect Tg(Bre:GFP). Inhibition of BMP signaling at these stages reduces expression of the ventral signal Edn1, as well as ventral-specific genes such as hand2 and dlx6a in the arches, and expands expression of the dorsal signal jag1b. This results in a loss or reduction of ventral and intermediate skeletal elements and a mis-shapen dorsal arch skeleton. Conversely, ectopic BMP causes dorsal expansion of ventral-specific gene expression and corresponding reductions/transformations of dorsal cartilages. Soon after NC migration, BMP is required to induce Edn1 and overexpression of either signal partially rescues ventral skeletal defects in embryos deficient for the other. However, once arch primordia are established the effects of BMPs become restricted to more ventral and anterior (palate) domains, which do not depend on Edn1. This suggests that BMPs act upstream and in parallel to Edn1 to promote ventral fates in the arches during early DV patterning, but later acquire distinct roles that further subdivide the identities of NC cells to pattern the craniofacial skeleton.

AP2 transcription factors regulate many aspects of embryonic development. Studies of AP2a (Tfap2a) function in mice and zebrafish have demonstrated a role in patterning mesenchymal cells of neural crest origin that form the craniofacial skeleton, while the mammalian Tfap2b is required in both the facial skeleton and kidney. Here, we show essential functions for zebrafish tfap2a and tfap2b in development of the facial ectoderm, and for signals from this epithelium that induce skeletogenesis in neural crest cells (NCCs). Zebrafish embryos deficient for both tfap2a and tfap2b show defects in epidermal cell survival and lack NCC-derived cartilages. We show that cartilage defects arise after NCC migration during skeletal differentiation, and that they can be rescued by transplantation of wild-type ectoderm. We propose a model in which AP2 proteins play two distinct roles in cranial NCCs: an early cell-autonomous function in cell specification and survival, and a later non-autonomous function regulating ectodermal signals that induce skeletogenesis

Members of the AP-2 transcription factor family have critical roles in many aspects of embryonic development. The zebrafish tfap2a mutant lockjaw (low) displays defects in skeletal and pigment cell derivatives of the neural crest. Here we show essential roles for tfap2a in subsets of embryonic cartilages and pigment cells. Defects in cartilage of the hyoid arch in low correlate with a loss of Hox group 2 gene expression and are suggestive of a transformation to a mandibular fate. In contrast, loss of joints in the mandibular arch and defects in certain types of pigment cells suggest a requirement for tfap2a independent of Hox regulation. Early melanophores do not develop in low mutants, and we propose that this results in part from a loss of kit function, leading to defects in migration, as well as kit-independent defects in melanophore specification. Iridophores are also reduced in low, in contrast to xanthophores, revealing a role for tfap2a in the development of pigment subpopulations. We propose a model of tfap2a function in the neural crest in which there are independent functions for tfap2a in specification of subpopulations of pigment cells and segmental patterning of the pharyngeal skeleton through the regulation of Hox genes. Developmental Dynamics 229:87-98, 2004.