Transcriptional regulation in developing sensory neurones

Genetic studies of simple multicellular organisms have defined a number of transcriptional regulators involved in neuronal specification, and in some cases mammalian homologues have been identified. In this thesis I have focused on two such mammalian transcription factors which may play regulatory roles in sensory neurone development and function. Mammalian Brn-3c is a homologue of the C. elegans POU domain factor, unc-86, which is required for cell fate determination and for the postmitotic development of many neurones. Brn-3c is expressed in a restricted pattern within the nervous system, including a subset of dorsal root ganglion (DRG) neurones and may therefore be involved in the specification or later development of these neurones. To investigate this possibility, I decided to generate a Brn-3c null mutant mouse, using homologous recombination in embryonic stem (ES) cells, followed by injection of genetically altered ES cells into mouse blastulae. Control and knockout genomic DNA constructs were generated. Transfection of ES cells with these constructs, followed by sequential screening techniques, allowed the identification firstly of control cell lines and subsequently of four cell lines containing a null deletion in one allele at the Brn-3c locus. Parallel work by another group has shown, via the generation of a Brn-3c null mutant mouse, that Brn-Sc is essential for hair cell development in the inner ear. A second line of research involved the LIM-homeodomain transcription factor, Islet-1 (Isl1). Isl1 is required at an early stage in the generation of motor neurones, and its later expression in motor neurone subsets suggests roles in neuronal subtype generation. Its Drosophila homologue, Is1, is required for axonal pathfinding and neurotransmitter production and in the vertebrate pancreas Isl1 is involved in the regulation of at least three genes for secreted polypeptides. Isl1 is expressed in developing and adult DRG and may therefore play roles in the development and maintenance of trunk sensory neurones. I have shown that Isl1 is expressed in both SD and L neurones; that all or almost all nociceptors express Isl1, but Isl1 is not restricted to nociceptive neurones; and that the neurotransmitter CGRP is expressed exclusively by a subset of Isl1 positive neurones. I have also demonstrated that Isl1 is first expressed at around the time of the last mitotic division in sensory neurones, with some cells initiating Isl1 expression prior to their final mitosis. Using Isl1 null mutant mice I have shown that neural crest cells are formed in the absence of Isl1 and are able to migrate to their correct target areas, including the prospective DRG, and begin to coalesce into ganglia. I have putatively identified SCGIO expression in the location of cervical DRG in embryos lacking Isl1, which suggests that the initial stages of sensory neurone formation do not require Isl1. However, expression of other early neuronal markers could not be detected and there is evidence for increased cell death in the region of prospective DRG. Analysis of later stages of sensory neurone development have been hindered by the early death of the null mutant embryos. In addition to these studies, I attempted to use sequence homology to define conserved cis-acting elements within the regulatory regions of a number of genes expressed only, or selectively in sensory neurones. Putative cis-acting regions were analysed functionally using EMSAs to determine whether these regions were bound by DRG specific nuclear factors. On the basis of these experiments, three cis-acting sequences have been identified which bind to proteins present within DRG. These elements could therefore be involved in neuronal specific expression of adjacent genes