Understanding how the mammalian nervous system is formed during development is a formidable undertaking. We will approach the problem by focusing on one subdivision of the nervous system, the sympathetic division (SNS) of the peripheral nervous system (PNS). Our ultimate goal is to gain a comprehensive understanding of the genetic pathways that direct SNS development. Although the focus of this proposal is SNS development, knowledge gained will further our understanding of the mechanisms underlying nervous system development in general. The regulatory mechanisms directing development of the PNS lineage are among the best characterized of the neural lineages on a molecular and genetic level yet much is still not understood. Regulators of PNS development include members of the basic helix-loop-helix (bHLH) family of transcription factors. Within the SNS, the bHLH factor Mash1, is required for differentiation of SNS neuroblasts into neurons, but little is known about the transcription factors that regulate its expression or the genes regulated by Mash1. We found two other bHLH factors, HAND1 and HAND2, that are also expressed in the SNS during development and our preliminary results suggest that they function in a cascade with Mash1 to regulate SNS development. Of these two factors, HAND1 is expressed exclusively in the sympathetic lineage of the nervous system making it and ideal gene to investigate SNS-specific gene regulation. In this proposal we wish to unravel the transcriptional hierarchy regulating SNS development. Our approach is to identify the cis-acting sequences that regulate HAND1 expression in the SNS. Aim 1) To investigate the genetic hierarchy regulating SNS development. We will dissect the transcriptional regulatory network specifying SNS-specific gene expression through analysis of the cis-acting DNA elements regulating HAND1. HAND1 is expressed exclusively in the SNS lineage of the developing nervous system. Using transgenic mouse and chicken embryos, the DNA sequences flanking the HAND1 gene will be analyzed for their ability to regulate expression in a SNS-specific manner. After the core regulatory elements have been identified, we will identify the transcription factors that bind these sequences and regulate SNS-specific transcription. The research approach proposed here relies on a combination of state-of-the-art molecular and genetic techniques. The proposal will fund two graduate students who will perform most of the experiments. The training provided to students during the course of this work will prepare them to work in any laboratory employing molecular biology or molecular genetic techniques. After our mouse models have been generated, characterization of these mice will provide projects for high school and undergraduate students.
