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

['Mary E. Swartz', 'Department Of Molecular Biosciences', 'University Of Texas At Austin', 'Austin', 'Texas', 'United States Of America', 'C. Ben Lovely', 'Johann K. Eberhart']
Date: None

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.

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.

Funding: This work was supported by (NIDCR https://www.nidcr.nih.gov ) RO1 DE020884 and R35 DE029086 to JKE, (NIH/NIAAA https://www.niaaa.nih.gov ) F32AA021320 and K99AA023560 to CBL The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Copyright: © 2021 Swartz et al. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

We previously demonstrated that phenotypes in zebrafish gata3 mutants were highly variable, similar to human disorders associated with GATA3, and that this variability associated with genetic background [ 13 ]. Here, we determine the role of gata3 in development of the zebrafish palate and characterize the signaling pathways that regulate the variability in gata3 mutant phenotypes. We show that neural crest cells require the function of Gata3 shortly after their migration into the pharyngeal arches. Bmp signaling is necessary for the expression of gata3 in the maxillary neural crest and loss of Bmp signaling recapitulates the craniofacial defects in gata3 mutants. Transgenic overexpression of GATA3 restores facial development in Bmp deficient zebrafish, demonstrating that Gata3 functions downstream of Bmp. We demonstrate that the variability in gata3 mutant phenotypes is due to the actions of a second pathway, Shh. Elevating and attenuating Shh signaling ameliorates and exacerbates the phenotypes of gata3 mutants, respectively. Importantly, in a sensitized gata3 mutant genetic background, reduction of Shh signaling is sufficient to cause gata3 haploinsufficiency, similar to the human condition. Our results demonstrate that the coordination of two pathways, a Bmp-Gata3 pathway and Shh, regulate trabeculae phenotypes. These findings provide important insights into the causes of variability in craniofacial disease phenotypes.

Our understanding of the roles of GATA3 in craniofacial development is limited due to the early lethality of mouse Gata3 mutants caused by parathyroid defects [ 27 , 28 ]. Gata3 mutant mice pharmacologically rescued display severe craniofacial defects and neural crest patterning defects [ 28 , 29 ], consistent with a critical role in craniofacial development. Work in the mouse mandible has demonstrated that Smad1/5 binds regions adjacent to Gata3, suggesting that it is a target of Bmp signaling [ 30 ] furthermore Gata3 is a BMP target in limb mesenchyme [ 31 ]. However, the precise roles of Gata3, its regulation during palate development and the modulation of resulting phenotypes remain unknown.

The zinc finger transcription factor, GATA3, associates with craniofacial syndromes. Haploinsufficiency of GATA3 causes Hypoparathyroidism, Deafness and Renal dysplasia (HDR) syndrome [ 19 ]. HDR is an extremely variable birth defect, even among individuals sharing the same mutation within a family [ 20 ]. Palatal defects and choanal atresia (defects of the nasal bones) are craniofacial defects that can co-occur with the HDR triad [ 21 , 22 ]. Furthermore, GATA3 associates with craniofacial microsomia [ 23 – 25 ], another highly variable disease. Humans with microsomia can have unilateral shortening and clefts of the palate as well as defects to other craniofacial bones, ears and cranial ganglia [ 24 , 26 ]. While HDR is a relatively rare disease microsomia is very common affecting 1 in 5600 conceptuses. Collectively, these findings in human patients suggest that phenotypes associated with loss of GATA3 function are inherently variable.

Defining the causes of phenotypic variation is important for our understanding of development, disease and evolution. However, there are a limited number of studies defining the cause of phenotypic variation. Such variation can conceptually be caused by three general mechanisms: 1) genetic background, 2) gene-environment interactions and 3) stochastic developmental events [ 10 ]. Our understanding of all of these mechanisms is limited. Recent work is beginning to shed light on the mechanisms of gene-environment interactions and how such interactions can synergistically effect phenotypes [ 11 , 12 ]. Similarly, mutant analyses in mouse and zebrafish have pointed to the importance of genetic background, with phenotypes differing depending upon the strain carrying the mutation [ 13 – 16 ]. Recent studies have demonstrated that selective breeding for heritable variation in phenotypic penetrance of mef2ca mutants results in altered methylation in a transposon at the mef2ca promoter and a compensatory downregulation of the opposing Notch pathway [ 17 , 18 ], providing some insights into these mechanisms. However, much remains to be understood regarding the nature of these genetic background effects.

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. Cranial neural crest cells (CNCC) that generate the palatal skeleton are generated in the dorsal neural tube from which they must migrate into the periphery to differentiate. Palatal precursors occupy the maxillary region of the first pharyngeal arch and the frontonasal prominence in human, mouse and zebrafish [ 1 – 4 ]. The zebrafish palate (also referred to as the anterior neurocranium) is comprised of an anterior, midline, ethmoid plate and the posterior bilateral trabeculae. Fate mapping shows that the medial ethmoid palate is formed from frontonasal CNCC and the remaining palate forms from maxillary CNCC [ 2 , 3 ]. The trabeculae fuse to the posterior neurocranium which is primarily composed of mesodermally-derived cells [ 5 ]. 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 – 9 ]. Yet we still have an incomplete knowledge of the genes involved in craniofacial development and a poor understanding of how they interact to generate variability.

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. The causes of most birth defects are thought to be complex and include genetic and environmental risk factors. Furthermore, the precise phenotypes observed within a specific birth defect can be highly variable and this variability is also thought to arise from genetic and environmental modifiers. Craniofacial defects are among the most common birth defects and offer an excellent model of variability. For instance, orofacial clefts affect 1 in 700 live births and appear to be caused by an interplay of genetic and environmental factors [ 1 ].

Results

[1] Url: https://journals.plos.org/plosgenetics/article?id=10.1371/journal.pgen.1009579

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