Precise specification of tissues and organs within the developing embryo requires the translation of cell position into cell fate. Both positional and functional information is provided by gradients of signaling molecules called morphogens, which specify several cell fates in a concentration-dependent manner. The Bone Morphogenetic Proteins (BMPs) act as a morphogen to pattern the dorsoventral (DV) axis in all vertebrates. In zebrafish, a gradient of BMP signaling activity across the embryo is formed during gastrulation, where the highest level of BMP signaling is found ventrally and the lowest levels dorsally. BMP signaling specifies multiple ventral cell fates, whereas dorsal tissue specification requires suppression of BMP signaling. Proper positioning of gene expression is essential during development because these domains specify the relative abundance of distinct tissue types. However, it is unknown how cells along the DV axis interpret and translate distinct levels of BMP signaling into differential gene activation to specify cell fate. Morphogen gradients have been shown to induce differential gene activation by multiple distinct mechanisms, including the steady-state amount of signaling, distinct temporal signaling duration, and the steepness of the graded signal across cells. I will test the hypothesis that distinct threshold levels of BMP signaling activity establish discrete gene expression domains. Using a quantitative immunofluorescence assay of nuclear phosphorylated Smad5, the transcriptional effector of BMP signaling, the Mullins lab has determined the shape of the BMP signaling gradient within the zebrafish gastrula. I will identify the ventral genes that directly respond to BMP signaling and the threshold level of BMP activity required to induce their expression within their endogenous domain. I will test the ability of the threshold level of BMP signaling activity to position target gene expression within a distinct domain. BMPs are a member of the TGF- ? signaling family, which signal through highly conserved Smad transcription factors. Association of Smads with additional sequence specific transcription factors increases affinity and selectivity for specific target genes in response to different signals. Discovery of additional Smad5- cofactor interactions is critical for understanding BMP induced transcriptional regulation and cell fate specification. I will identify the DNA-binding cofactors that direct the specificity of nuclear pSmad5 to target genes essential for patterning the DV axis of the zebrafish embryo. I will identify Smad5-cofactor interactions through screening and analysis of transcription cofactor motifs captured by pSmad5 chromatin immunoprecipitation and sequencing. I will test if identified cofactors are required for BMP-dependent expression patterns and cell fate specification by using CRISPR-Cas9 to knock out cofactor expression. Together, these experiments will elucidate the mechanism for interpreting the BMP activity gradient into distinct expression domains and cell fates.
The Bone Morphogenetic Protein (BMP) morphogen gradient patterns many tissues during development including the neural tube, developing limbs, stem cell niches, and the dorsal-ventral (DV) body axis. Misregulation of BMP signaling is associated with cancers, pulmonary hypertension, osteoporosis, and ectopic bone disease. Investigating BMP-mediated gene regulation is critical for understanding developmental patterning and cell fate specification, but also for understanding its role in the progression and treatment of human diseases.
