Variation in phenotypes from a Bmp-Gata3 genetic pathway is modulated by Shh signaling.

Mary E Swartz,C Ben Lovely,Johann K Eberhart,Robert A Cornell

doi:10.1371/journal.pgen.1009579

Abstract

We sought to understand how perturbation of signaling pathways and their targets generates variable phenotypes. In humans, GATA3 associates with highly variable defects, such as HDR syndrome, microsomia and choanal atresia. We previously characterized a zebrafish point mutation in gata3 with highly variable craniofacial defects to the posterior palate. This variability could be due to residual Gata3 function, however, we observe the same phenotypic variability in gata3 null mutants. Using hsp:GATA3-GFP transgenics, we demonstrate that Gata3 function is required between 24 and 30 hpf. At this time maxillary neural crest cells fated to generate the palate express gata3. Transplantation experiments show that neural crest cells require Gata3 function for palatal development. Via a candidate approach, we determined if Bmp signaling was upstream of gata3 and if this pathway explained the mutant's phenotypic variation. Using BRE:d2EGFP transgenics, we demonstrate that maxillary neural crest cells are Bmp responsive by 24 hpf. We find that gata3 expression in maxillary neural crest requires Bmp signaling and that blocking Bmp signaling, in hsp:DN-Bmpr1a-GFP embryos, can phenocopy gata3 mutants. Palatal defects are rescued in hsp:DN-Bmpr1a-GFP;hsp:GATA3-GFP double transgenic embryos, collectively demonstrating that gata3 is downstream of Bmp signaling. However, Bmp attenuation does not alter phenotypic variability in gata3 loss-of-function embryos, implicating a different pathway. Due to phenotypes observed in hypomorphic shha mutants, the Sonic Hedgehog (Shh) pathway was a promising candidate for this pathway. Small molecule activators and inhibitors of the Shh pathway lessen and exacerbate, respectively, the phenotypic severity of gata3 mutants. Importantly, inhibition of Shh can cause gata3 haploinsufficiency, as observed in humans. We find that gata3 mutants in a less expressive genetic background have a compensatory upregulation of Shh signaling. These results demonstrate that the level of Shh signaling can modulate the phenotypes observed in gata3 mutants.

Highlights

Congenital birth defects are a leading cause of infant mortality worldwide and the leading cause of mortality in many industrial nations according to the World Health Organization
We show that the cells that generate the palate require the function of Gata3 and that the Bone Morphogenetic Protein (Bmp) pathway is necessary for the expression of gata3 by these cells
While Gata3 functions downstream of Bmp, we find no evidence that alteration of the Bmp pathway causes the variability in skeletal defects in gata3 mutants

Summary

Introduction

The causes of most birth defects are thought to be complex and include genetic and environmental risk factors. Orofacial clefts affect 1 in 700 live births and appear to be caused by an interplay of genetic and environmental factors [1]. The high rate and variable nature of craniofacial defects such as orofacial clefts are largely because proper palatogenesis requires the precise coordination of many events that are subject to genetic and/or environmental perturbations. The zebrafish palate ( referred to as the anterior neurocranium) is comprised of an anterior, midline, ethmoid plate and the posterior bilateral trabeculae. While the evolutionary homologies remain unclear, a growing body of evidence demonstrates that the gene function required for craniofacial development, including palatogenesis, in mammals is conserved in zebrafish [6,7,8,9]. We still have an incomplete knowledge of the genes involved in craniofacial development and a poor understanding of how they interact to generate variability

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