Variation In Phenotypes From A Bmp-Gata3 Genetic Pathway Is Modulated By Shh Signaling

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.Author summary Human birth defects vary widely in their presentation. This is true even in cases where the underlying genetic mutation is the same. In humans, mutation of the gene GATA3 associates with two highly variable birth defects that can disrupt development of the face, microsomia and Hypoparathyroidism, Deafness and Renal dysplasia (HDR) syndrome. We used the zebrafish to identify the causes of variation in facial defects associated with gata3. 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. Instead, we identify a separate signaling pathway, the Sonic Hedgehog (Shh), pathway that is responsible for the variability in gata3 mutant defects. In a genetic background that promotes mild gata3 mutant phenotypes, Shh signaling is elevated relative to mutants in a genetic background sensitized for severe defects. Reduction or elevation of Shh signaling in these two mutants, exacerbates and lessens the phenotypic severity, respectively. Thus, our finding provides important insight into how interactions between signaling pathways cause variation in human birth defects.