Identification of multipotent cardiac progenitors has provided fresh insight into the mechanisms of myocardial lineage specification, yet has done little to clarify the origin of the endocardium. Despite its essential role in heart development, the lineage classification of endocardium has remained undefined due to a lack of specific markers of this early vascular subpopulation. To distinguish endocardium from other vasculature, we generated an NFATc1-nuc-LacZ BAC transgenic mouse line that faithfully recapitulated endogenous NFATc1 expression in the heart and other cell populations during development. Utilizing this novel specific marker, the endocardium can be distinguished from other endothelial subpopulations, and tracked as it emerges from the cardiac mesoderm and participates in early cardiac morphogenesis. To further characterize endocardiogenesis, embryonic stem cells (ESCs) derived from NFATc1-nuc-LacZ blastocysts were utilized to demonstrate that endocardial differentiation occurs in a pattern consistent with its mesodermal origin and its close association with myocardium. Endocardium is specified as a cardiac cell lineage, independent from other vascular populations, responding to BMP and Wnt signals that enhance cardiomyocyte differentiation. Furthermore, a population of Flk1+ cardiovascular progenitors, distinct from hemangioblast precursors, represents a mesodermal precursor of the endocardial endothelium, as well as other cardiovascular lineages. Taken together, this work emphasizes the endocardium as a unique cardiac lineage and provides further evidence that endocardium and myocardium are derived from a common precursor. Furthermore, we have established a method to identify the endocardium at the onset of differentiation and throughout subsequent stages of development, providing the framework for future work to delineate mechanisms important in the ontogeny of this unique population.

The trachea and esophagus are respectively respiratory and digestive organs that originate from a common foregut endoderm during development. Perturbed patterning in this endodermal tissue can result in a variety of foregut anomalies, such as tracheal agenesis (TA), esophageal atresia (EA) and tracheoesophageal fistula (TEF); however, their etiologies remain unknown. We found that mice lacking Noggin displayed Type C EA/TEF, the most common form in humans, and notochordal defects strikingly similar to the adriamycin-induced rat EA/TEF model. In accord with esophageal atresia, Noggin-/- embryos displayed reduction in the dorsal foregut endoderm which was associated with reduced adhesion and disrupted basement membrane. However, no significant apoptosis in the Noggin-/- dorsal foregut was observed. Instead, non-notochordal, likely endodermal, cells were found in Noggin-/- notochord suggesting that Noggin function is required in the notochordal plate for its proper delamination from the dorsal foregut. Notably, ablating Bmp7 function in Noggin-/- embryos rescued EA/TEF and notochord branching defects, establishing a critical role of Noggin-mediated Bmp7 antagonism in EA/TEF pathogenesis. In addition to the critical role of Bmp signaling in EA/TEF pathogenesis, it also plays a vital role in tracheal development. We found that conditional ablation of Bmp4 in the primitive foregut by Foxg1Cre resulted in complete loss of trachea. While tracheal specification was not affected in Bmp4-deficient foreguts, tracheal outgrowth was severely impaired. The anterior foregut domain also displayed significantly reduced epithelial and mesenchymal proliferation without apparent alteration in apoptotic cell death. While we did not observe alteration in Wnt/â-catenin signaling in the Bmp4-deficient foregut, we detected consistent reduction in the expression level of Shh, a signaling molecule known to promote cell proliferation. In line with this observation, expression of Cyclin D1, a known downstream target of Shh signaling, was also found to be reduced in Bmp4- deficient foreguts. Taken together, these findings elucidate a critical role of Bmp signaling and cell proliferation in tracheal morphogenesis and implicate potential Bmp-Shh crosstalk in anterior foregut morphogenesis.

The small GTPase Rab11 family proteins have been implicated in the plasma membrane recycling system in such diverse model systems as H/K-ATPase trafficking in parietal cells to GLUT4 trafficking in heart and skeletal muscle. Rab11 family interacting protein 2 (Rab11-FIP2) was previously identified as binding to both Rab11a and the motor protein, myosin Vb. Therefore, we hypothesized that Rab11-FIP2 is a critical regulator of the plasma membrane recycling system. In these studies, we sought to characterize the role of Rab11-FIP2 function in polarized epithelial cells. To address the role of Rab11-FIP2, we uncovered a new role for Rab11-FIP2 in the establishment of polarity. We found that Rab11-FIP2 is phosphorylated by MARK2, and that this phosphorylation is necessary for the proper formation of the adherens junction. Next, we characterized a new dominant mutant Rab11-FIP2 mutant, which has unique effects on the recycling system than all previously characterized mutants. Analysis of the data suggests that FIP2 is involved in multiple stages in passage through the Rab11a associated recycling system. Multiple points of entry into the Rab11a/ FIP2 recycling system may be exploited depending upon the origin of the protein and possibly, its destination. This model supports a dynamic vision of the recycling system trafficking. We also analyzed new Rab11-FIP2 interacting proteins utilizing a novel approach of immunoprecipitation from stable cell lines overexpressing either wild type Rab11-FIP2 or its mutants followed by identification of the associated proteins using mass spectrometry. This proteomics approach revealed novel interactions with proteins known to be involved in trafficking (dynein and Rab10) in early endosomal membrane regulation (Rab5b and EpsinR), and vesicle coat proteins (AP-1 and clathrin heavy chain). Validation of these interactions proves that this methodology reveals robust interactions that are readily confirmed. All together, this body of work significantly advances our understanding of the diverse roles of Rab11-FIP2 in the regulation of epithelial cell function.

The inhibitor of DNA-binding (Id) genes encode a family of helix-loop-helix proteins lacking the basic DNA-binding domain. The Id proteins function as dominant negative factors by dimerizing with other transcription factors, inhibiting DNA binding and transcriptional activation. Four members of the Id family (Id1-4) have been identified in mammals, expressed in spatially and temporally restricted patterns in many developing organs and have been implicated in the regulation of cell proliferation, apoptosis, differentiation and migration. Both Id1 and Id3 are highly expressed in the lung mesenchyme during vascular development, suggesting that they play indispensable roles in endothelial morphogenesis. In agreement, we found that Id1-/-Id3-/- lungs exhibit defects in distal angiogenesis after prolonged culture and implantation in renal capsules. MMP-2 is significantly under-expressed in Id1-/-Id3-/- lung endothelial cells, suggesting its contribution to the phenotype. These findings indicate that Id proteins are key components of embryonic lung vascular development. In this study we also found that, upon bleomycin treatment, Id1 expression was upregulated in a number of lung cell types but predominantly in endothelial cells, as revealed by double immunolabeling and quantitative FACS sorting analysis. As the result of ablated Id1 function, bleomycin-injured Id1-/- lungs showed increased vascular permeability and endothelial apoptosis. Accordingly, bleomycin-treated Id1-/- lung microvascular endothelial cells also showed decreased survival in culture. We detected a decrease in the level of Bcl-2, a primary anti-apoptotic protein, in Id1-/- endothelial cells, suggesting that downregulated Bcl-2 may promote endothelial apoptosis in the lung. Therefore, we propose that Id1 plays an important role in promoting endothelial survival in the adult lung upon injury. In addition, mice lacking Id1 function displayed increased collagen accumulation and fibrogenesis after long-term bleomycin exposure, suggesting that Id1 upregulation in the endothelium and other lung cell types may play a critical role in lung homeostasis.

While localized tumor growth may cause organ dysfunction and even death, metastases cause the vast majority (~90%) of human cancer deaths. Both autocrine and paracrine transforming growth factor-beta (TGF-beta) have been linked to invasive and metastatic tumor growth. The mechanism whereby autocrine TGF-beta elicits tumorigenic effects was investigated. The metastatic breast cancer cells, MDA-MB-231, secrete TGF-beta and express both the urokinase plasminogen activator (uPA) and matrix metalloproteinase-9 (MMP-9) that are important for cancer metastatic growth. Whether autocrine TGF-beta promotes invasive growth of MDA-MB-231 cells through regulation of uPA and/or MMP-9 protein levels and/or activity was studied. Inhibition of autocrine TGF-beta signaling decreased MDA-MB-231 cell invasion and uPA secretion. Inhibition of uPA proteolytic activity decreased cell invasion to the similar extent. The Smad proteins are the intracellular mediators for the canonical TGF-beta signaling pathway. However, TGF-beta receptors may transduce signals through Smad-independent pathways. My study demonstrates that the self-sufficiency of promoting invasive potential of tumor cells is through enhanced uPA secretion by autocrine TGF-beta in a Smad-dependent manner. While autocrine TGF-beta signaling modulates uPA protein secretion, exogenous TGF-beta increased uPA mRNA expression through RNA stabilization, suggesting distinct post-transcriptional mechanisms for regulation of uPA by different magnitudes of TGF-beta stimulation. Smad4 is both a tumor promoter and a suppressor. We previously observed inverse protein expression of Smad4 and claudin-1 in intestinal epithelial and colorectal cancer (CRC) cells and tissues. Claudin-1 is a tight junction protein with potential of enhancing metastatic growth of CRC cells. Whether Smad4 may act as a tumor suppressor by inhibiting claudin-1 expression in CRC was studied. In the Smad4-deficient, claudin-1-positive HT29 and SW480 CRC cells, Smad4 expression specifically downregulated claudin-1 protein expression through possibly transcriptional suppression. Previous findings suggest TGF-beta signaling-independent functions of Smad4. My study demonstrates that the Smad4-mediated suppression of claudin-1 expression is independent of TGF-beta signaling in SW480 and HT29 cells. These findings suggest a novel mechanism underlying the Smad4 tumor suppressive function through regulation of a potential metastatic modulator, claudin-1, in a TGF-beta-independent manner in CRC cells.

Wnt/β‐catenin signaling plays a critical role in metazoan development, stem cell maintenance, and human disease. The multicomponent β‐catenin destruction complex maintains low cellular β‐catenin in the absence of a signal and becomes inhibited in the presence of a Wnt ligand, allowing β‐catenin levels to rise. We have identified an FDA-approved drug, pyrvinium, as a potent inhibitor of Wnt signaling. We show pyrvinium binds CK1α, enhances kinase activity and CK1α knockdown abrogates the effects of pyrvinium on the Wnt pathway. In addition to effects on Axin and β‐catenin levels, pyrvinium promotes degradation of Pygopus, a Wnt transcriptional component. Pyrvinium treatment of colon cancer cells with mutation of Adenomatous Polyposis Coli or β‐catenin inhibits both Wnt signaling and proliferation. These findings reveal allosteric activation of CK1α as an effective mechanism to inhibit Wnt signaling. We also performed biochemical studies that identified positive feedback within the β‐catenin destruction complex between essential components, Axin and GSK3. Theoretical modeling of this positive feedback loop predicts bistability in the activity of the β‐catenin destruction complex. Through single cell studies, we generated experimental evidence for our theoretical predictions. These findings elucidate molecular design features that convert gradients into discrete binary cell fate decisions. In conclusion, this work combines chemical studies and systems-level analysis to uncover novel mechanisms of regulating the Wnt/β‐catenin pathway. Our findings highlight new strategies for targeted therapeutics directed against the Wnt pathway.

<p>Dynein is a minus-end directed microtubule motor complex that is required for a diverse range of biological processes, from intracellular-cargo transport to cell migration. Dynein is regulated at multiple levels and its functions are dictated by its affinity for microtubules, motor action, associations with specific cargos, and subcellular localization.</p> <p>During <i>Drosophila</i> spermatogenesis, dynein localizes to the nuclear surface at entry into meiotic prophase where it plays an essential role in mediating nucleus-centrosome coupling. Dynein also localizes to the nuclear surface of early postmeiotic spermatids, where it maintains associations between the nucleus and basal body and controls the position of the nucleus during elongation. I have identified the conserved gene <i>asunder</i> (<i>asun</i>) as a novel regulator of dyneins localization to the nuclear surface during <i>Drosophila</i> spermatogenesis. In addition, I have found that the dynein regulatory factor, <i>Lis-1</i>, also plays an essential role in promoting the localization of dynein to the nuclear surface, and that it may cooperate with <i>asun</i> in this process.</p>

In humans, the proper growth and homeostasis of endocrine islets in the pancreas is of great medical importance, in that loss and dysfunction of islet cells result in Type 1 and Type 2 Diabetes Mellitus. One of the major goals of diabetic-related researches is to understand how islets are formed and matured during normal development, so as to aid functional islet production in vitro for transplantation-based diabetes therapies. It has been well established that a bHLH transcription factor Neurogenin3 (Neurog3 or Ngn3) plays essential roles in endocrine islet differentiation in mice. However, it remains unclear how Ngn3 levels are regulated in endocrine progenitors and how Ngn3 coordinates islet cell differentiation and function. Previously, a zinc-finger transcription factor Myt1 (Myelin transcription factor 1) was identified from a microarray-based study aiming for factors that are specifically enriched in Ngn3+ pancreatic endocrine progenitors. Here, we report that Myt1 and Ngn3 form a feed-forward expression loop to promote endocrine differentiation. Specifically, loss of Myt1 partially compromises endocrine differentiation and islet function in the mouse pancreas, demonstrating that Myt1 plays a role in the generation of fully functional islet cells. Furthermore, although Myt1 expression largely depends on Ngn3 activity, Myt1 can enhance Ngn3 expression, suggesting that Myt1 contributes to endocrine commitment through Ngn3. To this end, reduced Ngn3 production at per cell level significantly impairs endocrine differentiation and endocrine/exocrine allocation. Finally, we discovered a previously unsuspected role of Ngn3 in islet cells. Sustained Ngn3 expression in hormone expressing endocrine islet cells is required for islet maturation and function. These studies not only provide important information regarding the regulation of endocrine differentiation, but also open up new directions to improve islet function under diabetic conditions.

Coordinated movement depends on specific connections between neurons, yet mechanisms that govern synaptic specificity are poorly understood. The simple, well-defined motor circuit of the nematode, C. elegans provides an optimal model system to study how neurons choose synaptic partners. Here, interneurons AVA, AVD, and AVE synapse with VA and DA motor neurons to establish the backward motor circuit, whereas interneurons AVB and PVC make connections with VB and DB motor neurons in the forward circuit. Mutations in the UNC-4 homeodomain transcription factor miswire VA motor neurons with inputs normally reserved for VBs, thereby disrupting backward locomotion. The Miller lab has shown that UNC-4 functions in post-synaptic VA motor neurons to block expression of VB genes that specify these aberrant connections. One VB gene, the transcription factor, CEH-12/HB9, is required for miswiring posterior VA motor neurons. My dissertation addresses two important questions: (1) What mechanism limits ceh-12/HB9 expression to posterior VAs and (2) What additional unc-4 pathway genes regulate inputs onto anterior VAs? First, my results establish that a posterior source of the diffusible ligand, EGL-20/Wnt is required for expression of CEH-12/HB9 in VAs. Genetic results indicate that the frizzled receptors, MOM-5 and MIG-1, respond to EGL-20/Wnt and drive ceh-12 expression through a canonical Wnt pathway. This also revealed a parallel pathway, involving LIN-44/Wnt and LIN-17/Frz, which antagonizes ceh-12 expression and promotes the creation of VA-type inputs. We hypothesize that UNC-4 inhibits expression of MIG-1 and MOM-5 to prevent posterior VAs from responding to EGL-20/Wnt. UNC-4 effectively biases overall Wnt signaling to favor the LIN-44/LIN-17 pathway. Second, I utilized genetic screens designed to isolate mutants that function in parallel to ceh-12. This approach revealed 22 independent blr (backward locomotion restored) mutations that map to 16 genetic linkage groups. Detailed phenotypic characterization of six blr mutants confirmed at least three genetic loci, blr-1, blr-3, blr-15, function in parallel to ceh-12 to regulate the specificity of interneuron input to VAs. Future molecular identification of these unc-4 pathway genes should provide key insights into the mechanism of synaptic specificity.

The Wnt/beta-catenin signaling pathway is a well-conserved signal transduction pathway that is highly regulated during metazoan development and is associated with various human diseases. In the current model of Wnt/beta-catenin signaling, Wnt ligands bind to its receptors, Frizzled and LRP6 (low-density lipoprotein receptor-related protein 6). These receptors transmits an intracellular signal that ultimately increases steady-state levels of cytoplasmic beta-catenin by inhibiting the phosphorylation of beta-catenin by the kinase GSK3. Elevated beta-catenin translocates to the nucleus and induces Wnt target gene expression. A major question in the field is how a Wnt/beta-catenin signal is transmitted from the receptors to mediate transmission of the Wnt signal from the plasma membrane. To understand how signaling through LRP6 inhibits beta-catenin degradation we expressed and purified the LRP6 intracellular domain and found that it stimulated beta-catenin stabilization while also stimulating the degradation of the negative regulator, Axin. Through additional egg extract and biochemical reconstitution experiments we found that LRP6 stabilizes beta-catenin independently of Axin degradation by directly inhibiting GSK3's phosphorylation of beta-catenin. The Frizzled receptor is predicted to have seven transmembrane domains, a feature that is characteristic of G protein-coupled receptors. To identify a role for heterotrimeric G proteins in Wnt/beta-catenin signaling, we screened major families of recombinant G protein alpha subunits in our egg extract system and found that Galphao, Galphaq, Galphai2, and Gbetagamma inhibit β-catenin degradation. We find that Gbeta1gamma2 is required for and promotes LRP6 phosphorylation and activation by directly recruiting GSK3 to the membrane and enhancing its kinase activity. We propose that heterotrimeric G protein activation results in formation of free Gbetagamma and Galpha, which act cooperatively to inhibit beta-catenin degradation and activate beta-catenin-mediated transcription. Together, these studies provide insight on the molecular mechanism of the early intracellular events of Wnt/beta-catenin signaling.