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 low truncates 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.

The zebrafish embryo develops a series of anatomically distinct slow twitch muscle fibres that characteristically express genes encoding lineage-specific isoforms of sarcomeric proteins such as MyHC and troponin. We show here that different subsets of these slow fibres express distinct members of a tandem array of slow MyHC genes. The first slow twitch muscle fibres to differentiate, which are specified by the activity of the transcription factor Prdm1 (also called Ubo or Blimp1) in response to Hedgehog (Hh) signalling, express the smyhc1 gene. Subsequently, secondary slow twitch fibres differentiate in most cases independently of Hh activity. We find that although some of these later-forming fibres also express smyhc1, others express smyhc2 or smyhc3. We show that the smyhc1-positive fibres express the ubo (prdm1) gene and adopt fast twitch fibre characteristics in the absence of Prdm1 activity, whereas those that do not express smyhc1 can differentiate independently of Prdm1 function. Conversely, some smyhc2-expressing fibres, although independent of Prdm1 function, require Hh activity to form. The adult trunk slow fibres express smyhc2 and smyhc3, but lack smyhc1 expression. The different slow fibres in the craniofacial muscles variously express smyhc1, smyhc2 and smyhc3, and all differentiate independently of Prdm1.

All chordates share a basic body plan and many common features of early development. Anteroposterior (AP) regions of the vertebrate neural tube are specified by a combinatorial pattern of Hox gene expression that is conserved in urochordates and cephalochordates. Another primitive feature of Hox gene regulation in all chordates is a sensitivity to retinoic acid during embryogenesis, and recent developmental genetic studies have demonstrated the essential role for retinoid signalling in vertebrates. Two AP regions develop within the chordate neural tube during gastrulation: an anterior ‘forebrain–midbrain’ region specified by Otx genes and a posterior ‘hindbrain–spinal cord’ region specified by Hox genes. A third, intermediate region corresponding to the midbrain or midbrain–hindbrain boundary develops at around the same time in vertebrates, and comparative data suggest that this was also present in the chordate ancestor. Within the anterior part of the Hox –expressing domain, however, vertebrates appear to have evolved unique roles for segmentation genes, such as Krox–20 , in patterning the hindbrain. Genetic approaches in mammals and zebrafish, coupled with molecular phylogenetic studies in ascidians, amphioxus and lampreys, promise to reveal how the complex mechanisms that specify the vertebrate body plan may have arisen from a relatively simple set of ancestral developmental components.

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