<p>Regeneration is a remarkable feat that can only be accomplished by a small number of animals. The regeneration of vertebrate limbs is one such case as certain salamanders and fish regenerate robustly while mammalian ability to regenerate is extremely limited. Successful regeneration requires not just cell proliferation after injury but also the patterning of the new tissue into a suitable replacement structure. The process by which this patterning happens is referred to as positional memory. Identification of factors responsible for positional memory in vertebrate appendage regeneration has remained elusive. This dissertation establishes zebrafish pectoral fins as a model system for studying and defining positional memory factors. This has been accomplished through careful morphological measurements, gene expression profiling, construction of transgenic zebrafish strains, and the use of various chemical reagents. Two stunning examples of positional information in the pectoral fin have been discovered. First is the region-specific defect in male pectoral fin regeneration governed by an androgen's influence on GSK3 activity. The second is the role for hand2 in maintaining restricted vitamin D signaling and thus small bones in the posterior region of the pectoral fin. hand2 is the first defined positional memory factor in a zebrafish fin. However, in spite of this success the tools required for further dissection of positional memory are not available and thus the potential for meaningful future work is slight.</p> / Dissertation

The intestinal epithelium is a continuously renewing tissue. This rapid renewal is fueled by stem cells that reside in the base of the crypts of Lieberkühn, the functional unit of the intestinal epithelium. The identity of intestinal stem cells has been the subject of over fifty years of ongoing investigation. Leucine-rich repeats and immunoglobulin-like domains 1 (Lrig1) marks a population of intestinal stem cells, but the identity of Lrig1+ cells has remained controversial due to discrepant reports of Lrig1 expression in the crypt base. We characterized two Lrig1+ populations in the colonic epithelium using two anti-Lrig1 antibodies and a new Lrig1 reporter mouse. We determined that these two populations could be distinguished by reporter expression, Lgr5 expression, and likely the glycosylation status of Lrig1. Since stem cells are widely considered to be tumor-initiating cells in colorectal cancer, we developed new mouse models of colonic neoplasia: Lrig1CreERT2/+;Apcfl/fl and Lrig1CreERT2/+;Apcfl/fl;KrasLSL-G12D/+. We found that Apc loss resulted in high-grade tumors; expression of mutant Kras expression did not significantly affect histological grade of tumors, but may contribute to increased invasion.

Heritable Pulmonary Arterial Hypertension (HPAH) is a rare, fatal disease of the pulmonary vasculature for which there is no cure. The majority of HPAH patients inherit mutations in the BMP type 2-receptor gene, BMPR2, but how these promote pulmonary vascular disease is unclear. In this work, we show BMPR2 mutations promote Src-kinase activation pulmonary endothelial cells (PECs) isolated from Bmpr2 mutant mice. We show increased Src activation leads to endothelial barrier defects due in part to enhanced Src-mediated caveolar endocytosis and that these defects can be rescued using Src kinase inhibitors. We go on to show that pulmonary endothelial cells and late outgrowth endothelial progenitor cells isolated from idiopathic PAH patients show similar increases in Src kinase activation suggesting that Src kinase activation may be a common disease mechanism in PAH. Therefor, these studies provide evidence for the use of Src kinase inhibitors in the treatment of pulmonary arterial hypertension.

Proteins that regulate cell polarity are fundamental for metazoan biology and are necessary for proper development of tissues and organs. In light of polarity genes fundamental role in tissue organization, disruptions in polarity networks have been suspected to promote neoplasia. Studies in Drosophila melanogaster models and correlative data from human tumor samples have supported this hypothesis, but direct experimental support for polarity genes as mammalian tumor suppressors has only recently been reported. The polarity regulator Par3 has emerged as a suppressor of growth and metastasis in mammary and skin tumors. The mechanisms by which Par3 restrains tumor progression, however, remain obscure. In the studies reported here, I show that loss of Par3 can promote activation of an oncogenic signaling pathway in mouse mammary cells by permitting aPKCι/λ to activate NF-κB signaling. These studies demonstrate that preventing aberrant aPKC activity is a key tumor suppressor function of Par3. This mechanism may be relevant to human tumors.

CELL AND DEVELOPMENTAL BIOLOGY Identification and characterization of MAFA coregulators: MLL3/4 and its role in mouse and human islet β-cells David William Scoville Dissertation under the direction of Professor Roland Stein Pancreatic islet β-cells are critical regulators of glycemic control, and their dysfunction or absence leads to Diabetes Mellitus. Several transcription factors play crucial roles in the development of functional β-cells, including the transcription factor MafA. MafA expression is restricted to islet β-cells in the pancreas where it regulates postnatal β-cell maturation and function in the mouse. However, little is known about the mechanisms through which MafA regulates its target genes. The goal of this dissertation work was to identify coregulators which interact with and contribute to the function of the MafA transcription factor. Utilizing an unbiased proteomics approach in a mouse β-cell line, I identified several potential coregulators of MafA that could control the many activities associated with this protein, including β-cell proliferation and glucose-stimulated insulin secretion. Among them were the mixed-lineage leukemia 3 and 4 (Mll3/4) complexes, known for their role in histone 3 lysine 4 (H3K4) methylation and gene activation. MafA was bound to the Mll3/4 complexes in experiments performed with size fractionated β-cell extracts and by immunoprecipitation analysis. Likewise, the MLL3 and MLL4 complexes bound human MAFB, which is closely related to MAFA, important to mouse β-cell development, and co-produced with MAFA in adult human islet β-cells. Knockdown of NCOA6, a core subunit of these methyltransferases, reduced expression of only a small subset of MAFA and MAFB target genes in mouse and human β-cell lines. In contrast, a more profound effect on MafA/MafB gene activation was observed upon NCoA6 deletion specifically in embryonic β-cells using rat insulin promoter-driven Cre. We propose that this broader impact is due to the coordinated recruitment of the Mll3/4 complexes by MafB during development and MafA postnatally. Future studies may elucidate the context-dependent mechanisms involved in regulating MAFA and MAFB coregulator binding. This may allow for development of therapeutic strategies for enhancing β-cell function and mass in the treatment of diabetes.

Retinoic acid (RA) has been used therapeutically to reduce injury and fibrosis in models of acute kidney injury (AKI), but little is known about whether and how this pathway is normally regulated, and what role it plays in regulating injury and repair after AKI. In these studies we show that RA signaling is activated in mouse and zebrafish models of AKI, and that these responses limit the extent of injury and promote normal repair. These effects are mediated through a novel mechanism by which RA signaling coordinates the dynamic equilibrium of pro-inflammatory M1 spectrum vs. alternatively activated M2 spectrum macrophages. According to this model, direct repression of pro-inflammatory macrophages by locally synthesized RA reduces macrophage-dependent injury post-AKI, while locally synthesized RA activates RA signaling in injured tubular epithelium, which in turn promotes alternatively activated M2 spectrum macrophages. Since RA signaling plays an essential role in kidney development but is repressed in the adult, these findings provide evidence of an embryonic signaling pathway that is reactivated after injury and plays an important role in reducing injury and enhancing repair after AKI.

CELL AND DEVELOPMENTAL BIOLOGY Dominant and Context-Specific Control of Endodermal Organ Allocation by Ptf1a Spencer Gaffney Willet Dissertation under the direction of Professor Christopher V.E. Wright The timing and gene-regulatory logic of organ-fate commitment from within the posterior foregut of the mammalian endoderm is largely unexplored. Transient misexpression of a presumed pancreatic-commitment transcription factor, Ptf1a, in embryonic mouse endoderm (Ptf1aEDD) dramatically expanded the pancreatic gene regulatory network within the foregut. Early-stage Ptf1aEDD rapidly expanded the endogenous endodermal Pdx1-positive domain, and recruited other pancreas-fate-instructive genes, thereby spatially enlarging the potential for pancreatic multipotency. Early Ptf1aEDD converted essentially the entire glandular stomach, rostral duodenum, and extrahepatic biliary system to pancreas. Sliding the Ptf1aEDD expression window through embryogenesis revealed differential temporal competencies for stomach-pancreas respecification. The response to later-stage Ptf1aEDD changed radically towards unipotent, acinar-restricted conversion.

The TGF-â ligand Nodal is a key regulator of body axis formation and patterning in the developing vertebrate embryo. How the Nodal signaling pathway is activated and regulated, and how precise spatiotemporal control of the pathway is translated into morphogenetic cues for asymmetric morphogenesis is unclear. Attempts to gain insight into the dynamics of active Nodal signaling have been hindered by the inability to detect the endogenous Nodal ligand. Antibodies against the signal transducer phospho-Smad2 (pSmad2) have been used to detect active TGF-â signaling, but are of poor quality and cannot accurately depict Nodal signaling in situ. We generated a new pSmad2 antibody, with the goal of using it to create a spatiotemporal map of Nodal signaling during development. We planned to integrate such maps with our data showing increased F-actin labeling in the R LPM, and possible L vs. R cell shape differences, just prior to organ looping to aid in understanding how Nodal signaling directly translates into morphogenetic cues. Fully understanding the role of Nodal signaling in morphogenesis fundamentally includes understanding its regulation at the transcriptional level. Current models have the winged-helix transcription factor Foxh1 bound with pSmad2 as a central transcriptional activator of Nodal. However, a conserved Engrailed-homology-1 (EH1) motif, which is recognized by Groucho co-repressors, in Foxh1 suggests that Foxh1 functions as a transcriptional switch, toggling between transcriptional on and off states via pSmad2-Grg switching, to ensure properly timed initiation and suppression, and/or amplitude, of Nodal. We minimally mutated the Foxh1 EH1 motif, creating a novel Foxh1mEH1 allele to test the contribution of Foxh1-Grgmediated repression on the transient, dynamic pattern of Nodal signaling in mice. We find that Foxh1-Grgmediated repression is not essential for Nodal expression during mouse embryogenesis. This suggests that other regulators compensate for the loss of Foxh1-Grgmediated repression, and that Nodal signaling exists within the context of a strongly buffered regulatory system that contributes to resilience and accuracy of its dynamic expression pattern.

Mitotic kinesins represent the new age targets of spindle-poisoning chemotherapies. The kinesin-5 Eg5 is one such example, as kinesin-5 inhibitors (K5Is) induce a lethal mitotic arrest from failed spindle assembly. Documented here is the discovery of a novel spindle assembly pathway that confers K5I-resistance to human tumor cells in culture. This reverse-jackknifing pathway is mechanically distinct from the canonical Eg5-driven mechanism, relying instead on the activity of the kinesin-12 Kif15. The work shown here details the molecular function and biochemical properties of Kif15, while also providing insight into the larger question of why anti-mitotic pharmacological agents fail as chemotherapies. Future efforts will evaluate the combination of K5Is with Kif15-inhibitors as a novel strategy for the treatment of neoplasias. Ultimately, this study bridges mitotic mechanisms with clinically relevant problems, being of interest to cell biologists, cancer biologists, and clinicians.

Colorectal carcinoma is the third leading cause of cancer related mortality within the United States. Over 90% of these deaths can be attributed to distant metastasis. Therefore, understanding the signaling perturbations that underlie the development of carcinoma and process of metastasis are paramount in combatting the progression of this disease. Many signaling pathways are dysregulated in the pathogenesis of colorectal carcinoma, but over 80% of sporadic colorectal cancer cases have alterations within the WNT pathway, and over 50% within the TGF-β signaling pathway. Both the WNT and TGF-β pathways are critical in development and intestinal homeostasis. The central mediator of TGF-β signaling pathway is Smad4, a transcription factor that is mutated in approximately 15-30% of colorectal cancer cases. Loss of Smad4 is associated with distant metastasis and overall poor patient prognosis. This body of work uses both in vitro and in vivo models to describe a role for Smad4 in supporting normal epithelial homeostasis within the intestinal tract. These models also describe a new tumor suppressor role of Smad4 wherein Smad4 signaling transcriptionally suppresses WNT pathway activation within intestinal epithelial cells. Overall, these results aid in understanding the biological function of Smad4 signaling in the context of intestinal homeostasis and tumorigenesis and permit further insight into potential therapeutic targets in colorectal carcinoma.