Mechanisms of repressive interactions between developmental gene regulatory networks

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Cells acquire their unique identities during development through the progressive emergence of distinct transcriptional programs. These transcriptional programs can be viewed as dynamic networks of interacting genes known as gene regulatory networks (GRNs). GRNs have been well studied for their positive role in activating the expression of genes that endow cells with their specialized properties; however, it is now apparent that an equally important function of these networks is to exclude other, potentially alternative, transcriptional programs. Repressive interactions of this kind play a widespread, fundamental role in determining cellular identities in organisms as diverse as invertebrates and mammals. They are also important in the context of regenerative medicine. The direct reprogramming of somatic cells by lineage-specific transcription factors (TFs) is accompanied by the comprehensive silencing of pre-existing transcriptional programs. Despite the pivotal role that repressive interactions between transcriptional networks play in both embryonic cell fate specification and somatic cell reprogramming, the underlying mechanisms are poorly understood. We will address this important problem using the sea urchin, a prominent experimental model for the analysis of developmental mechanisms and for GRN biology. One of the best characterized sea urchin GRNs underlies the development of cells that form the skeleton. A key component of this network is Alx1, a lineage- specific TF that provides direct, positive inputs into many genes that support skeletogenesis. In parallel with its positive role, Alx1 represses potential, alternative transcriptional programs that are ordinarily restricted to surrounding non-skeletogenic mesoderm (NSM) cells. Perturbation of Alx1 function in skeletogenic cells results in the ectopic deployment of NSM GRNs in these cells and causes them to adopt NSM fates. The repression of NSM GRNs by Alx1 thus provides an outstanding opportunity to uncover mechanisms by which transcriptional networks interact with one another, thereby ensuring the emergence of unique cellular identities. To dissect the mechanisms underlying this GRN interaction, I will begin by defining key spatial and temporal aspects of NSM GRN repression by Alx1, using both quantitative methods and spatial gene expression analysis to characterize changes in gene expression that occur after perturbing Alx1 function (Aim 1). Next, I will explore the hypothesis that Alx1 directly represses NSM genes by using fluorescent reporter constructs and transgenesis to dissect cis-regulatory elements of these genes (Aim 2). Finally, I will test the hypothesis that Alx1 controls a cell-autonomous unresponsiveness to Notch/Delta signaling, a pathway that drives NSM specification during normal development (Aim 3). These studies will shed light on the mechanisms by which Alx1 represses NSM GRNs. More broadly, they will lead to a better understanding of interactions between transcriptional networks that regulate cell identity.