Specification of both neural crest cells and Rohon-Beard (RB) sensory neurons involves a complex series of interactions between the neural and non-neural ectoderm. The molecular mechanisms directing this process are not well understood. The zebrafish narrowminded (nrd) mutation is unique, since it is one of two mutations in which defects are observed in both cell populations: it leads to a complete absence of RB neurons and a reduction in neural crest cells and their derivatives. Here, we show that nrd is a mutation in prdm1, a SET/zinc-finger domain transcription factor. A Morpholino-mediated depletion of prdm1 phenocopies the nrd mutation, and conversely overexpression of prdm1 mRNA rescues the nrd RB sensory neuron and neural crest phenotype. prdm1 is expressed at the border of the neural plate within the domain where neural crest cells and RB sensory neurons form. Analysis of prdm1 function by overexpression indicates that prdm1 functions to promote the cell fate specification of both neural crest cells and RB sensory neurons, most likely as a downstream effector of the BMP signaling pathway.

B1-type SOXs (SOXs 1, 2, and 3) are the most evolutionarily conserved subgroup of the SOX transcription factor family. To study their maternal functions, we used the affinity-purified antibody antiSOX3c, which inhibits the binding of Xenopus SOX3 to target DNA sequences [Development. 130(2003)5609]. The antibody also cross-reacts with zebrafish embryos. When injected into fertilized Xenopus or zebrafish eggs, antiSOX3c caused a profound gastrulation defect; this defect could be rescued by the injection of RNA encoding SOX3DeltaC-EnR, a SOX3-engrailed repression domain chimera. In antiSOX3c-injected Xenopus embryos, normal animal-vegetal patterning of mesodermal and endodermal markers was disrupted, expression domains were shifted toward the animal pole, and the levels of the endodermal markers SOX17 and endodermin increased. In Xenopus, SOX3 acts as a negative regulator of Xnr5, which encodes a nodal-related TGFbeta-family protein. Two nodal-related proteins are expressed in the early zebrafish embryo, squint and cyclops; antiSOX3c-injection leads to an increase in the level of cyclops expression. In both Xenopus and zebrafish, the antiSOX3c phenotype was rescued by the injection of RNA encoding the nodal inhibitor Cerberus-short (CerS). In Xenopus, antiSOX3c's effects on endodermin expression were suppressed by injection of RNA encoding a dominant negative version of Mixer or a morpholino against SOX17alpha2, both of which act downstream of nodal signaling in the endoderm specification pathway. Based on these data, it appears that maternal B1-type SOX functions together with the VegT/beta-catenin system to regulate nodal expression and to establish the normal pattern of germ layer formation in Xenopus. A mechanistically conserved system appears to act in a similar manner in the zebrafish.

BACKGROUND The regulatory mechanisms underpinning facial development are conserved between diverse species. Therefore, results from model systems provide insight into the genetic causes of human craniofacial defects. Previously, we generated a comprehensive dataset examining gene expression during development and fusion of the mouse facial prominences. Here, we used this resource to identify genes that have dynamic expression patterns in the facial prominences, but for which only limited information exists concerning developmental function. RESULTS This set of ~80 genes was used for a high throughput functional analysis in the zebrafish system using Morpholino gene knockdown technology. This screen revealed three classes of cranial cartilage phenotypes depending upon whether knockdown of the gene affected the neurocranium, viscerocranium, or both. The targeted genes that produced consistent phenotypes encoded proteins linked to transcription (meis1, meis2a, tshz2, vgll4l), signaling (pkdcc, vlk, macc1, wu:fb16h09), and extracellular matrix function (smoc2). The majority of these phenotypes were not altered by reduction of p53 levels, demonstrating that both p53 dependent and independent mechanisms were involved in the craniofacial abnormalities. CONCLUSIONS This Morpholino-based screen highlights new genes involved in development of the zebrafish craniofacial skeleton with wider relevance to formation of the face in other species, particularly mouse and human.

Invertebrate and vertebrate vestigial (vg) and vestigial-like (vgl) genes are involved in embryonic patterning and cell fate determination. These genes encode cofactors that interact with members of the TEAD/Scalloped family of transcription factors and modulate their activity. We have previously shown that, in mice, Vgll2 is differentially expressed in the developing facial prominences. In this study, we show that the zebrafish ortholog vgll2a is expressed in the pharyngeal endoderm and ectoderm surrounding the neural crest derived mesenchyme of the pharyngeal arches. Moreover, both the FGF and retinoic acid (RA) signaling pathways, which are critical components of the hierarchy controlling craniofacial patterning, regulate this domain of vgll2a expression. Consistent with these observations, vgll2a is required within the pharyngeal endoderm for NCC survival and pharyngeal cartilage development. Specifically, knockdown of Vgll2a in zebrafish embryos using Morpholino injection results in increased cell death within the pharyngeal arches, aberrant endodermal pouch morphogenesis, and hypoplastic cranial cartilages. Overall, our data reveal a novel non-cell autonomous role for Vgll2a in development of the NCC-derived vertebrate craniofacial skeleton.

SUMMARYThe neural crest comprises multipotent precursor cells that are induced at the neural plate border by a series of complex signaling and genetic interactions. Several transcription factors, termed neural crest specifiers, are necessary for early neural crest development; however, the nature of their interactions and regulation is not well understood. Here, we have established that the PR/SET domaincontaining transcription factor Prdm1a is co-expressed with two essential neural crest specifiers, foxd3 and tfap2a, at the neural plate border. Through rescue experiments, chromatin immunoprecipitation and reporter assays, we have determined that Prdm1a directly binds to and transcriptionally activates enhancers for foxd3 and tfap2a and that they are functional, direct targets of Prdm1a at the neural plate border. Additionally, analysis of dominant activator and dominant repressor Prdm1a constructs suggests that Prdm1a is required both as a transcriptional activator and transcriptional repressor for neural crest development in zebrafish embryos.