Elena S. Casey - US grants

DESCRIPTION (provided by applicant): The study of neural induction addresses a fundamental question, in development: How is the fate of ectodermal cells directed toward the formation of neural tissue and away from the formation of epidermis? Answering this question has led to much interest in elucidating the signal transduction pathways involved in the formation and patterning off the vertebrate nervous system. A number of molecules have been defined that are involved in neural formation, but many steps in the pathways remain unknown. Two proteins expressed early in response to neural induction are Sox2 and Sox3, members of the Sox family of HMG box transcription factors. These proteins have been implicated in maintaining a neural progenitor population; both genes are expressed in proliferating neural cells and down-regulated when these cells differentiate into specific neural cell types. By studying their regulation and function we will identify steps necessary for the induction, proliferation and differentiation of neural tissue. Our Specific Aims are to: (1) identify cis-regulatory modules of Sox2 and -3 using 'promoter bashing' and transgenesis (2) identify regulatory factors that interact with the cis sequences of these genes through the candidate gene approach and (3) use gain and loss of function studies to characterize their roles in neural induction and proliferation. These studies will be done in Xenopus laevis and X. tropicalis as much of our knowledge of neural formation comes from embryological studies on the frog and improved transgenic capabilities allow for the rapid identification of regulatory elements and networks. Combined with molecular assays and comparative computational analysis we will study the regulation of Xenopus Sox2 and 3 and define their role in neural development.

DESCRIPTION (Provided by Applicant): A change in the regulatory protein profile of a cell drives fate specification. This change is in part controlled at the transcriptional level, but as cells progress to their final fate during embryonic development, regulatory proteins must also be inactivated. One mechanism is removal by ubiquitin-mediated protein degradation which involves the attachment of a multi-ubiquitin chain to target the selected proteins to the 26S proteasome for destruction. These proteins are selected by ubiquitin ligases (E3), and while there are numerous E3 ligases, the best understood are the Cullin based ligases (SCF) which contain Skp1, the scaffold protein Cullin, and the target-binding F-box proteins. There is a rich history demonstrating a role for ubiquitin-mediated protein destruction in cell-cycle regulation, and defects in this process underlie a number of cancers. Furthermore, E3 ligase deficiencies have been linked to neural crest disorders and neurodegenerative diseases, indicating that protein degradation plays a key role throughout embryonic development. While the multistep process of ubiquitination is well understood and targets have been identified, we know less about its function in the regulation of cell fate specification in vertebrate development. To gain a better understanding of the role of protein degradation in neural development, the investigators propose to identify all F-box proteins in Xenopus and use gain- and loss-of-function assays to characterize those expressed in the nervous system throughout development and in the epidermis during neural plate specification. With the completion of these aims, they will then be able to identify F-box binding partners and therefore potential targets whose degradation is required for ectodermal fate specification as well as for progression from neural precursor to differentiated neuron. F-box proteins are the largest class of E3 substrate-interacting proteins and since many bind multiple substrates, they are likely to facilitate the ubiquitination of hundreds of proteins in a variety of cellular and developmental processes. Using bioinformatic tools, thus far the investigators have identified 58 F-box proteins in Xenopus and classified them into three groups based on their protein interaction domain. Furthermore, with in silico genome scanning for REST binding sites, they have identified three F-box proteins that are expressed in the nervous system. The investigators will confirm that they play a role in ubisquitination and that they have a role in neural development by demonstrating interaction with Skp1 of the modular Cullin-based E3 ligase, and with loss-of-function analysis, respectively. The long term goal of this research program, to determine the role of these specific degradation events in the specification and differentiation of the nervous system, is fundamental to understanding the regulation of normal and disordered neural development. NARRATIVE: To transform the fertilized egg into a perfectly formed individual, specific combinations of proteins must be expressed at the right time and place. Loss of expression or misexpression of a single protein can have catastrophic consequences including cyclopia, dwarfism, connective tissue disorders, and limb as well as craniofacial defects. This application will address how the levels of proteins are controlled by degradation to ensure ordered progression through neurogenesis and therefore the proper development of the central nervous system.