Zebrafish con/disp1 reveals multiple spatiotemporal requirements for Hedgehog-signaling in craniofacial development

Tyler Schwend,Sara C Ahlgren

doi:10.1186/1471-213x-9-59

Abstract

BackgroundThe vertebrate head skeleton is derived largely from cranial neural crest cells (CNCC). Genetic studies in zebrafish and mice have established that the Hedgehog (Hh)-signaling pathway plays a critical role in craniofacial development, partly due to the pathway's role in CNCC development. Disruption of the Hh-signaling pathway in humans can lead to the spectral disorder of Holoprosencephaly (HPE), which is often characterized by a variety of craniofacial defects including midline facial clefting and cyclopia [1,2]. Previous work has uncovered a role for Hh-signaling in zebrafish dorsal neurocranium patterning and chondrogenesis, however Hh-signaling mutants have not been described with respect to the ventral pharyngeal arch (PA) skeleton. Lipid-modified Hh-ligands require the transmembrane-spanning receptor Dispatched 1 (Disp1) for proper secretion from Hh-synthesizing cells to the extracellular field where they act on target cells. Here we study chameleon mutants, lacking a functional disp1(con/disp1).Resultscon/disp1 mutants display reduced and dysmorphic mandibular and hyoid arch cartilages and lack all ceratobranchial cartilage elements. CNCC specification and migration into the PA primorida occurs normally in con/disp1 mutants, however disp1 is necessary for post-migratory CNCC patterning and differentiation. We show that disp1 is required for post-migratory CNCC to become properly patterned within the first arch, while the gene is dispensable for CNCC condensation and patterning in more posterior arches. Upon residing in well-formed pharyngeal epithelium, neural crest condensations in the posterior PA fail to maintain expression of two transcription factors essential for chondrogenesis, sox9a and dlx2a, yet continue to robustly express other neural crest markers. Histology reveals that posterior arch residing-CNCC differentiate into fibrous-connective tissue, rather than becoming chondrocytes. Treatments with Cyclopamine, to inhibit Hh-signaling at different developmental stages, show that Hh-signaling is required during gastrulation for normal patterning of CNCC in the first PA, and then during the late pharyngula stage, to promote CNCC chondrogenesis within the posterior arches. Further, loss of disp1 disrupted normal expression of bapx1 and gdf5, markers of jaw joint patterning, thus resulting in jaw joint defects in con/disp1 mutant animals.ConclusionThis study reveals novel requirements for Hh-signaling in the zebrafish PA skeleton and highlights the functional diversity and differential sensitivity of craniofacial tissues to Hh-signaling throughout the face, a finding that may help to explain the spectrum of human facial phenotypes characteristic of HPE.

Highlights

The vertebrate head skeleton is derived largely from cranial neural crest cells (CNCC)
To better understand the role of Hh-signaling in this process, we studied cartilage development within two con/ disp1 mutant alleles: the con/disp1tm15a and con/disp1b392. con/ disp1 mutant phenotypes are less severe than slow-muscleomitted mutant phenotypes, making them more amenable for detection of subtle deficits [33]. con/ disp1tm15a and con/disp1b392 alleles displayed similar penetrance in their craniofacial phenotype, and shared similar gene expression reductions in gli1, a Hh-signaling pathway transcription factor that is sensitive to the pathway's attenuation
5 day old con/disp1 mutants were distinguished by an overall reduction in head size and displayed micro- or agnathia due to reduced jaw outgrowth and significant heart edema when compared to wild type larvae (Figure 1A, B)

Summary

Introduction

The vertebrate head skeleton is derived largely from cranial neural crest cells (CNCC). The vertebrate head skeleton is largely derived from cranial neural crest cells (CNCC) which originate in the dorsal neural tube and migrate ventrally to populate the pharyngeal arches (PA) [3]. The CNCC of the first PA (mandibular) forms the jaw, while those in the second PA (hyoid) add support to the jaw structure. Posterior to these two PA, the CNCC populate five posterior PA to form bilateral gill support structures All of these elements derive from CNCC, the neural crest arise from different anterior-posterior regions of the neural axis, travel in different streams to arrive at the final destination, express different Hox genes, and encounter different local environments at the point of condensation [6,12]. The genes that regulate the production of cartilage from CNCC are shared regardless of location [13]

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Conclusion