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Date of Award

9-2010

Access Type

Campus Access

Document type

dissertation

Degree Name

Doctor of Philosophy (PhD)

Degree Program

Molecular and Cellular Biology

First Advisor

Rolf O. Karlstrom

Second Advisor

Gerald B. Downes

Third Advisor

John Nambu

Subject Categories

Molecular Biology

Abstract

The secreted Hedgehog (Hh) molecule acts as a morphogen, mitogen, and cell survival factor during embryogenesis and during later growth and tissue homeostasis periods in vertebrates. Hh plays these diverse roles by regulating the transcription of a number of genes that promote cellular processes including differentiation, proliferation, and resistance to programmed cell death. At the cellular level, the response to Hh signaling appears to change depending on the period during development that the cells are exposed to Hh, potentially allowing the switch, particularly in the brain, from early morphogen to a late mitogen. Mutations that reduce Hh signaling lead to congenital disorders in humans (e.g. holoprosencephaly), while mutations that inappropriately activate Hh signaling can lead to cancer (e.g. medulloblastoma and basal cell carcinoma).

One goal of my research was to identify the major transcriptional targets of this important signaling pathway, work that may identify new genes that are targets of mutation in human disease. A collaborative effort using gene profiling techniques on zebrafish embryos with either increased or decreased Hh signaling was undertaken to identify genes regulated by the Hh/Gli signaling cascade. Our microarray approach led us to both known and novel zebrafish genes that are regulated by Hh signaling, including several with putative Gli transcription factor binding sites in their regulatory regions. We have confirmed the regulation of some known and novel genes by Hh in embryos with increased and decreased Hh signaling. This now published work has identified a large number of direct and indirect targets of the Hh signaling pathway.

The major goal of my research was to both phenotypically and genetically characterize the zebrafish umleitung (uml) (meaning "detour" in German) mutation. The umlty54 mutant was identified due to its aberrant retinal tectal axon projections, and here I show that these mutants also have defects in Hh-mediated ventral neural patterning. These defects are accompanied by reduced expression of Hh-regulated genes in the forebrain and spinal cord, similar to known mutations that affect the Hh signaling pathway. Consistent with impaired Hh signaling, uml mutants have fewer endocrine cell types in the developing anterior pituitary, a reduced number of differentiated slow-muscle fibers in the tail, and defects in development of the jaw. To further understand how uml affects Hh signaling, I isolated the gene disrupted in uml by positional cloning. I have now shown that the defects seen in uml are due to a loss of function mutation in the gene encoding an immunoglobulin (Ig) and fibronectin type III (FNIII) domain containing cell-surface Hh-receptor, Brother of Cdo (Boc). Mosaic analysis indicates that Boc exerts its affects on Hh signaling cell-autonomously and may have a very early role in establishing the dose dependent response of cells along the dorsal/ventral axis of the central nervous system (CNS) to Sonic Hedgehog (Shh). This study demonstrates a previously unknown necessity for Boc in multiple Hh-mediated vertebrate developmental processes including, forebrain patterning, endocrine cell type specification, and jaw development. My analysis also reveals intriguing new ways in which Hh signaling is regulated within the vertebrate embryo and further implicates boc as a candidate gene in the treatment of disease caused by aberrant Hh signaling.

DOI

https://doi.org/10.7275/5675266

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