Dataset Information

A zebrafish screen for craniofacial mutants identifies wdr68 as a highly conserved gene required for endothelin-1 expression.

ABSTRACT:

Background

Craniofacial birth defects result from defects in cranial neural crest (NC) patterning and morphogenesis. The vertebrate craniofacial skeleton is derived from cranial NC cells and the patterning of these cells occurs within the pharyngeal arches. Substantial efforts have led to the identification of several genes required for craniofacial skeletal development such as the endothelin-1 (edn1) signaling pathway that is required for lower jaw formation. However, many essential genes required for craniofacial development remain to be identified.

Results

Through screening a collection of insertional zebrafish mutants containing approximately 25% of the genes essential for embryonic development, we present the identification of 15 essential genes that are required for craniofacial development. We identified 3 genes required for hyomandibular development. We also identified zebrafish models for Campomelic Dysplasia and Ehlers-Danlos syndrome. To further demonstrate the utility of this method, we include a characterization of the wdr68 gene. We show that wdr68 acts upstream of the edn1 pathway and is also required for formation of the upper jaw equivalent, the palatoquadrate. We also present evidence that the level of wdr68 activity required for edn1 pathway function differs between the 1st and 2nd arches. Wdr68 interacts with two minibrain-related kinases, Dyrk1a and Dyrk1b, required for embryonic growth and myotube differentiation, respectively. We show that a GFP-Wdr68 fusion protein localizes to the nucleus with Dyrk1a in contrast to an engineered loss of function mutation Wdr68-T284F that no longer accumulated in the cell nucleus and failed to rescue wdr68 mutant animals. Wdr68 homologs appear to exist in all eukaryotic genomes. Notably, we found that the Drosophila wdr68 homolog CG14614 could substitute for the vertebrate wdr68 gene even though insects lack the NC cell lineage.

Conclusion

This work represents a systematic identification of approximately 25% of the essential genes required for craniofacial development. The identification of zebrafish models for two human disease syndromes indicates that homologs to the other genes are likely to also be relevant for human craniofacial development. The initial characterization of wdr68 suggests an important role in craniofacial development for the highly conserved Wdr68-Dyrk1 protein complexes.

SUBMITTER: Nissen RM

PROVIDER: S-EPMC1523201 | biostudies-literature | 2006 Jun

REPOSITORIES: biostudies-literature

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Publications

A zebrafish screen for craniofacial mutants identifies wdr68 as a highly conserved gene required for endothelin-1 expression.

Nissen Robert M RM Amsterdam Adam A Hopkins Nancy N

BMC developmental biology 20060607

<h4>Background</h4>Craniofacial birth defects result from defects in cranial neural crest (NC) patterning and morphogenesis. The vertebrate craniofacial skeleton is derived from cranial NC cells and the patterning of these cells occurs within the pharyngeal arches. Substantial efforts have led to the identification of several genes required for craniofacial skeletal development such as the endothelin-1 (edn1) signaling pathway that is required for lower jaw formation. However, many essential gen ...[more]

PMID: 16759393

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Project description:BackgroundFetal alcohol spectrum disorders (FASD) are a highly variable set of phenotypes caused by fetal alcohol exposure. Numerous factors influence FASD phenotypes, including genetics. The zebrafish is a powerful vertebrate model system with which to identify these genetic factors. Many zebrafish mutants are housed at the Zebrafish International Resource Center (ZIRC). These mutants are readily accessible and an excellent source to screen for ethanol (EtOH)-sensitive developmental structural mutants.MethodsWe screened mutants obtained from ZIRC for sensitivity to EtOH teratogenesis. Embryos were treated with 1% EtOH (41 mM tissue levels) from 6 hours postfertilization onward. Levels of apoptosis were evaluated at 24 hours postfertilization. At 4 days postfertilization, the craniofacial skeleton, peripheral axon projections, and sensory neurons of neuromasts were examined. Fish were genotyped to determine whether there were phenotype/genotype correlations.ResultsFive of 20 loci interacted with EtOH. Notable among these was that vangl2, involved in convergent extension movements of the embryonic axis, interacted strongly with EtOH. Untreated vangl2 mutants had normal craniofacial morphology, while severe midfacial defects including synophthalmia and narrowing of the palatal skeleton were found in all EtOH-treated mutants and a low percentage of heterozygotes. The cell cycle gene, plk1, also interacted strongly with EtOH. Untreated mutants have slightly elevated levels of apoptosis and loss of ventral craniofacial elements. Exposure to EtOH results in extensive apoptosis along with loss of neural tissue and the entire craniofacial skeleton. Phenotypes of hinfp, mars, and foxi1 mutants were also exacerbated by EtOH.ConclusionsOur results provide insight into the gene-EtOH interactions that may underlie EtOH teratogenesis. They support previous findings that EtOH disrupts elongation of the embryonic axis. Importantly, these results show that the zebrafish is an efficient model with which to test for gene-EtOH interactions. Understanding these interactions will be crucial to understanding of the FASD variation.

Project description:Birth defects are among the leading causes of infant mortality and contribute substantially to illness and long-term disability. Defects in Bone Morphogenetic Protein (BMP) signaling are associated with cleft lip/palate. Many craniofacial syndromes are caused by defects in signaling pathways that pattern the cranial neural crest cells (CNCCs) along the dorsal-ventral axis. For example, auriculocondylar syndrome is caused by impaired Endothelin-1 (Edn1) signaling, and Alagille syndrome is caused by defects in Jagged-Notch signaling. The BMP, Edn1, and Jag1b pathways intersect because BMP signaling is required for ventral edn1 expression that, in turn, restricts jag1b to dorsal CNCC territory. In zebrafish, the scaffolding protein Wdr68 is required for edn1 expression and subsequent formation of the ventral Meckel's cartilage as well as the dorsal Palatoquadrate. Here we report that wdr68 activity is required between the 17-somites and prim-5 stages, that edn1 functions downstream of wdr68, and that wdr68 activity restricts jag1b, hey1, and grem2 expression from ventral CNCC territory. Expression of dlx1a and dlx2a was also severely reduced in anterior dorsal and ventral 1st arch CNCC territory in wdr68 mutants. We also found that the BMP agonist isoliquiritigenin (ISL) can partially rescue lower jaw formation and edn1 expression in wdr68 mutants. However, we found no significant defects in BMP reporter induction or pSmad1/5 accumulation in wdr68 mutant cells or zebrafish. The Transforming Growth Factor Beta (TGF-?) signaling pathway is also known to be important for craniofacial development and can interfere with BMP signaling. Here we further report that TGF-? interference with BMP signaling was greater in wdr68 mutant cells relative to control cells. To determine whether interference might also act in vivo, we treated wdr68 mutant zebrafish embryos with the TGF-? signaling inhibitor SB431542 and found partial rescue of edn1 expression and craniofacial development. While ISL treatment failed, SB431542 partially rescued dlx2a expression in wdr68 mutants. Together these findings reveal an indirect role for Wdr68 in the BMP-Edn1-Jag1b signaling hierarchy and dorso-anterior expression of dlx1a/2a.

Dataset Information

A zebrafish screen for craniofacial mutants identifies wdr68 as a highly conserved gene required for endothelin-1 expression.

Background

Results

Conclusion

Publications

A zebrafish screen for craniofacial mutants identifies wdr68 as a highly conserved gene required for endothelin-1 expression.

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