Thesis (Ph. D. in Biological Sciences)--Vanderbilt University, Dec. 2007. / Title from title screen. Includes bibliographical references.

Thesis (Ph. D.)--University of Washington, 2000. / Vita. Includes bibliographical references (leaves 53-58).

published_or_final_version / abstract / toc / Anatomy / Master / Master of Philosophy

published_or_final_version / abstract / toc / Anatomy / Master / Master of Philosophy

Thesis (Ph. D.)--University of Hong Kong, 2007. / Title proper from title frame. Also available in printed format.

Thesis (Ph. D.)--Duke University, 2007. / Includes bibliographical references.

Thesis (Ph. D.) -- University of Texas Southwestern Medical Center at Dallas, 2005. / Vita. Bibliography: 163-187.

Organisms are comprised of many cells with multiple distinct cell types, each of which must be decided precisely to ensure proper formation of a functional organism. In C. elegans, the basic helix-loop helix transcription factor HLH-2 is required for the specification of the anchor cell, or AC. The AC arises from a group of four somatic gonad cells, all of which initially express HLH-2. Two of the four cells, which we call β cells, lose AC competence early and instead become ventral uterine precursor cells, or VUs. We call the remaining two cells α cells. One α cell becomes the AC, while the other becomes a VU. Which α cell becomes the AC is random—50% of the time one α cell becomes the AC, while the other 50% of the time the other α cell becomes the AC. The choice of which cell becomes the AC and which becomes the VU is called the AC/VU decision, and occurs through reciprocal signaling by LIN-12/Notch and its ligand LAG-2/DSL. At first, both α cells express similar levels of lin-12 and lag-2. As the AC/VU decision progresses, the AC expresses higher levels of lag-2, and the VU expresses higher levels of lin-12. By this time, HLH-2 is only present in the specified AC, while it is post-translationally degraded in VUs. The mechanism by which HLH-2 is degraded and the consequences of disrupting its degradation on AC specification are unknown. In this work, we studied the function and regulation of HLH-2 during two stages of somatic gonad development. First, we used long-term fluorescence microscopy to visualize HLH-2 over the course of somatic gonad development. We found that HLH-2 expression begins in the parents of the α and β cells a consistent amount of time after their birth, and that the parent cell that first expresses HLH-2 almost always gives rise to the α cell that becomes the VU, while the second cell to express HLH-2 gives rise to the AC. This led us to study the effect of a loss of hlh-2 activity in the α and β cells. We generated an α and β cell-specific hlh-2(0) allele using genome editing tools and found that LIN-12 protein is not present in the absence of hlh-2 activity. Based on this discovery, we conceived a model where HLH-2 expression biases the first-expressing cell towards the VU fate by endowing it with an edge in lin-12 activity. Next, we focused on restriction of HLH-2 to the AC. Typically, HLH-2 protein is degraded in VUs, which we hypothesized was a crucial step in restriction of the AC fate to a single cell. We found that in a lin-12(0) background, HLH-2 is stabilized in VUs even when the resulting cell does not become an AC, indicating that lin-12 directly promotes HLH-2 degradation. This led us to search for a lin-12-regulated factor that targets HLH-2 for degradation in VUs. We identified seven ubiquitin-related genes whose depletion resulted in stabilized HLH-2 in VUs, but surprisingly did not cause an AC/VU defect. We suspect that HLH-2 degradation in VUs is one of multiple negative regulatory mechanisms that ensure the robustness of the AC/VU decision. The following research contributes new insights into how stochastic cell fate decisions amplify noise to ensure a consistent and reproducible outcome.

Indiana University-Purdue University Indianapolis (IUPUI) / The basic Helix-Loop-Helix (bHLH) transcription factors Hand1 and Hand2 play critical roles in the development of multiple organ systems during embryogenesis. The dynamic expression patterns of these two factors within developing tissues obfuscates their respective unique and redundant organogenic functions. To define cell lineages potentially dependent upon Hand gene expression, we generated a mutant allele in which the coding region of Hand1 is replaced by Cre recombinase. Subsequent Cre-mediated activation of β-galactosidase or eYFP reporter alleles enabled lineage trace analyses that clearly define the fate of Hand1-expressing cells. Comparisons between Hand1 expression and Hand1-lineage greatly refine our understanding of its dynamic spatio-temporal expression domains and raise the possibility of novel Hand1 functions in structures not thought to be Hand1-dependent. To genetically examine functional overlap between Hand1 and Hand2, we conditionally deleted Hand2 from Hand1-expressing cells. Hand2 conditional knockout mice die midgestation and exhibit cardiovascular and limb defects. Moreover, Hand2 lineage-restricted deletion from the proepicardial organ results in defective epicardialization and failure to form coronary arteries. Together, these novel data demonstrate a hierarchal relationship whereby transient Hand1 expression within the septum transversum defines epicardial precursors that depend upon subsequent Hand2 function.

published_or_final_version / abstract / Anatomy / Doctoral / Doctor of Philosophy