Author's School

Graduate School of Arts & Sciences

Author's Department/Program

Biology and Biomedical Sciences: Molecular Genetics and Genomics

Language

English (en)

Date of Award

Spring 4-30-2013

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Chair and Committee

Paul H Taghert

Abstract

Neuroendocrine cells feature a large capacity for the processing, accumulation and regulated release of bioactive peptides and peptide hormones. The ultrastructural correlate of this regulated secretory pathway is a specialized organelle, called a dense core vesicle: DCV). DCVs are typically larger than conventional, small synaptic vesicles. Past work has identified intrinsic DCV proteins: non-cargo proteins, like the processing enzyme, carboxypeptidase) or ancillary ones that play a role in DCV trafficking and exocytosis: like CAPS, the Ca2+-dependent activator protein for secretion). Currently, there is a lack of understanding of the developmental and physiological mechanisms that permit neurosecretory cells to coordinate and scale the regulated secretory pathway. In this context, the basic helix-loop-helix transcription factor dimmed: dimm) is especially important in the fruit fly Drosophila, but it is not involved in neuroendocrine cell fate determination.

Neuroendocrine cells require DIMM to accumulate, and process large amounts of secretory peptides, but DIMM does not target individual neuropeptide-encoding genes. Instead, we show that DIMM supports the complete resolution of NE-specific cellular properties by organizing the cellular machinery required to support a highly active RSP. The mouse orthologue Mist1 likewise plays a role in supporting the RSP of serous exocrine cells. This thesis has three goals. First, I evaluated a set of putative DIMM targets obtained by another scientist in the lab, and ask whether or not these are direct targets of this transcription factor. To accomplish this, I use in vivo chromatin immunoprecipitation: ChIP) followed by measuring DIMM binding to putative DIMM enhancers by quantitative Polymerase Chain Reaction: qPCR). This work is described in Chapter 2. Secondly, I extend DIMM ChIP analysis to identify direct DIMM transcriptional targets on a genome-wide level in vivo in adult neurons. This was done by DIMM chromatin immunoprecipitation coupled to tiling microarrays: ChIP-chip), and also applying Fluorescence Activated Cell Sorting: FACS) and deep sequencing: RNA-seq) to define the transcriptome of DIMM neuroendocrine cells, as described in Chapter 3.

I then integrate the ChIP-chip and RNA-seq datasets to provide new viewpoints on how DIMM is used to coordinate and appropriately scale the RSP in NE cells. The intersection of the RNA-Seq and ChIP-chip data presents a list of genes that is likely to mediate the bulk of the transcriptional output of DIMM - i.e., its molecular "mechanism". In order to conduct a functional assay and thus validate the list of intersected genes, I conducted a behavioral genetic screen. DIMM-expressing cells have previously been shown to regulate sleep amount in flies. I conducted an RNA interference-based screen, in which expression of individual DIMM target genes was knocked down in DIMM neurons and the effects of this manipulation on sleep were quantified. This in vivo validation provides an important filter with which to ascribe single gene functions and gives further insights into the general mechanisms by which DIMM operates.

Comments

This work is not available online per the author’s request. For access information, please contact digital@wumail.wustl.edu or visit http://digital.wustl.edu/publish/etd-search.html.

Permanent URL: http://dx.doi.org/10.7936/K7HH6H3W

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