Abnormalities affecting orofacial development are some of the most common, expensive, and devastating birth defects. Children born with such defects may experience difficulties with eating, breathing, and speech and in addition, these defects often require multiple surgeries to correct them. Therefore, it is critical to understand how the orofacial region develops in order to better treat and prevent these types of birth defects. Xenopus laevis has emerged as a strong model in which to examine orofacial development and was utilized here to investigate the cellular and molecular mechanisms underlying the complex development of the orofacial region. Retinoic acid is one signal involved in orchestrating orofacial development and accomplishes this in part by regulating the nucleosome structure of target genes. The work presented here characterizes the role of an ATP-dependent chromatin remodeler, chromodomain helicase DNA binding protein 1 (Chd1), in orofacial development in X. laevis. The spatial expression of Chd1 supports its role in orofacial development and reduced expression of Chd1 resulted in abnormal facial development. Closer examination of Chd1 morphant embryos revealed that Chd1 is required for the expression of important neural crest and cartilage genes that are necessary for proper development of the face. In addition, there was an increase in apoptosis in regions consistent with migrating neural crest and neural crest derived structures. As a consequence, many of the facial cartilages do not form properly in morphant embryos resulting in a smaller face. Further, this work presents evidence that Chd1 may cooperate with retinoic acid to regulate orofacial development in X. laevis.

No description available.

Neuropilin1 (Npn1) is a transmembrane receptor that is critical for development of both the nervous and vascular systems. It is a ligand for both the chemorepulsive Semaphorin3s and for vascular endothelial growth factor (VEGF), which is a protein critical for proper development, particularly for angiogenesis. Npn1 knockout mice die during early development due to cardiovascular abnormalities, and mice lacking Npn1 in endothelial cells (ECs) die perinatally with similar cardiovascular deficits. Because of the known importance of VEGF in cardiovascular development, it had been thought that the VEGF- Npn1 interaction was responsible for the premature death seen in Npn1 mutants. We identified one amino acid residue (D320) in the b1 domain of Npn1 that is necessary for VEGF-Npn1 binding. By mutating this site, we eliminated VEGF-Npn1 binding in vitro. We then made a knock-in mouse containing the D320K mutation, thus creating a mouse with no VEGF-Npn1 binding \((Npn1^{VEGF-} mouse)\). Surprisingly, the \(Npn1^{VEGF-}\) mutant mouse does not have the premature death or vascular phenotype seen in the Npn1-null. In particular, it does not recapitulate the decreased vascular density or decreased endothelial cell number seen in the Npn1 null. This indicates that the vascular phenotype seen in the Npn1 null is not a result of VEGF-Npn1 binding, and instead implicates that the phenotype is the result of the interaction of Npn1 with its VEGF co-receptor VEGFR2.

Directed differentiation of human pluripotent stem cells (hPSCs) has the potential to produce human cell types that can be used for disease modeling and cell transplantation. Two key challenges in the differentiation from hPSCs to \(\beta\) cells are the specification from pancreatic progenitors to insulin-expressing \((INS^+ )\) cells and the maturation of \(INS^+\) cells into glucose responsive β cells. To address the first, two high-content chemical screens identified PKC inhibitors as inducers of \(INS^+\) cells from pancreatic progenitors. PKC inhibition generated up to tenfold more \(INS^+\) cells while PKC agonists blocked differentiation into \(INS^+\) cells. Transplantation of \(PKC\beta\) inhibitor-treated pancreatic progenitors, containing higher proportions of endocrine progenitors and endocrine cells, resulted in mature \(\beta\) cells showing higher levels of glucose-stimulated human c-peptide production in vivo. This indicates that in vitro derived \(INS^+\) cells might be competent to mature into functional \(\beta\) cells. To address the second challenge, we first studied mouse and human \(\beta\) cell maturation in vivo. Postnatal mouse \(\beta\) cell maturation was marked by an increase in the glucose threshold for insulin secretion and by expression of the gene urocortin 3. To study human \(\beta\) cell maturation, a Method for Analyzing RNA following Intracellular Sorting (MARIS) was developed and used for transcriptional profiling of sorted human fetal and adult \(\beta\) cells. Surprisingly, transcriptional differences between human fetal and adult \(\beta\) cells did not resemble differences between mouse fetal and adult \(\beta\) cells, calling into question inter-species homology at the late stages of development. A direct comparison between hPSC-derived \(INS^+\) cells, and \(\beta\) cells produced during human development is essential to validate directed differentiation and provide a roadmap for maturation of hPSC-derived \(INS^+\) cells. Genome-wide transcriptional analysis of sorted \(INS^+\) cells derived from three hPSC-lines suggest that different lines produce highly similar \(INS^+\) cells, confirming robustness of directed differentiation protocols. Furthermore, nonfunctional hPSC-derived \(INS^+\) cells resemble human fetal \(\beta\) cells, which are distinct from adult \(\beta\) cells. We therefore suggest that in vitro directed differentiation mimics normal human development and reveal differences in gene expression that may account for the functional differences between hPSC-derived \(INS^+\) cells and true \(\beta\) cells.

The vertebrate small intestine is responsible for nutrient absorption during digestion. To this end, the surface area of the gut tube is maximally expanded, both through a series of loops extending its length and via the development of a complex luminal topography. Here, I first examine the mechanism behind the formation of intestinal loops. I demonstrate that looping morphogenesis is driven by mechanical forces that arise from differential growth between the gut tube and the anchoring dorsal mesenteric sheet. A computational model based on measured parameters not only quantitatively predicts the looping pattern in chick, verifying that these physical forces are sufficient to explain the process, but also accounts for the variation in the gut looping patterns seen in other species. Second, I explore the formation of intestinal villi in chick. I find that intestinal villi form in a stepwise process as a result of physical forces generated as proliferating endodermal and mesenchymal tissues are constrained by sequentially differentiating layers of smooth muscle. A computational model incorporating measured differential growth and the geometric and physical properties of the developing chick gut recapitulates the morphological patterns seen during chick villi formation. I also demonstrate that the same basic biophysical processes underlie the formation of intestinal folds in frog and villi in mice. Finally, I focus on the process by which intestinal stem cells are ultimately localized to the base of each villus. The endoderm expresses the morphogen, Sonic hedgehog (Shh). As the luminal surface of the gut is deformed during villus formation there are resulting local maxima of Shh signaling in the mesenchyme. This results, at high threshold, in the induction of a new signaling center under the villus tip termed the villus cluster. This, in turn, feeds back to restrict proliferating progenitors in the endoderm, the presumptive precursors of the stem cells, to the base of each villus. Together, these studies provide new insight into the formation of the small intestine as a functional organ and highlight the interplay between physical forces, tissue-level growth, and signaling during development.

<p>Basement membrane (BM) is a dense, conserved sheet-like extracellular matrix that provides structural support, compartmentalizes tissues, and regulates cell behaviors. Despite the barrier-like properties of BM, cell invasion through BM takes place normally in many developmental and physiological processes. Deregulation of cell invasion causes a variety of human diseases, most notably, cancer metastasis. A better understanding of cell invasion would help in the design more effective therapeutic strategies for those diseases.</p><p> Cell invasion through BM is a dynamic process comprising multiple intertwined steps, including acquirement of polarized cellular morphology, BM breaching, and BM remodeling. Despite much effort on investigating cellular invasive programs used for BM penetration, little is know about how cells detect invasive cues that polarize the invasive responses. Although the establishment of invasive polarity is critical as it initiates subsequent invasive behavior, the invasion process would not be completed without effective BM remodeling. Given that BM remodeling is often an integral part of tissue morphogenesis, the underlying interactions among cells and surrounding tissues make it challenging to understand the individual contributions of cells to changes in BM structure. </p><p> To gain insight into these two questions requires simple, experimental in vivo models. Anchor cell (AC) invasion into the vulval epithelium in C. elegans provides a visually accessible and experimentally tractable invasion model that is particularly suitable for cell biological and genetic analysis of the complicated interplay among local BM, an invading cell and the surrounding tissues. Using this model, I have investigated (1) how the AC detects dynamically expressed and localized netrin, a polarizing invasive cue for the AC; (2) what the functional contribution of the AC (as an invading cell) is to BM remodeling during uterine-vulval attachment, a post-embryonic organogenesis process. </p><p> First, I found that localized netrin polarizes the cellular invasive response towards the BM by stabilizing and spatially orienting a novel receptor-induced polarity oscillation. This oscillation is characterized by periodic F-actin assembly and disassembly at random sites of the plasma membrane of the AC. I have found F-actin assembly is accompanied by the formation of cellular protrusions. Strikingly, when these protrusions contact localized netrin, they are stabilized. Thus, I propose a mechanistic model where the ligand-independent activity of the receptor generates exploratory behavior. This mechanism orients the invasive polarity of the AC towards its BM target where netrin is normally localized. Second, taking advantage of an unbiased mutagenesis screen, I characterized a mutant with defects in BM sliding, a newly uncovered BM remodeling mechanism. I found that the invading AC utilizes a conserved transcription factor to control the initiation of BM sliding, which involves the regulation of integrin-mediated cell-matrix adhesion. Thus, my study revealed a novel functional role for the AC in BM remodeling during tissue restructuring.</p> / Dissertation

Bioelectricity refers to differential membrane voltage and cytoplasmic ion concentrations in tissues or cells which persist over long periods of time. Differences in these steady-state ionic conditions are responsible for large-scale axial patterning and morphogenesis in developing embryos. The sea urchin embryo is an excellent model organism for studying embryonic development, yet a comprehensive study of bioelectricity in sea urchin development has not been reported. Differential ion channel activity is a primary means by which bioelectricity is controlled; thus, we hypothesized that disrupting ion channel activity would reveal the requirements for bioelectricity in the sea urchin embryo. We performed a screen of ion channel inhibitors and discovered that their activities are required for many processes in sea urchin development. We chose two interesting phenotypes to investigate further. First, we demonstrate that H+/K+ ATPase (HKA) activity is required for biomineralization of the sea urchin larval skeleton. We determined that embryos raised with HKA inhibitors initially exhibit voltage and pH changes, then revert to normal voltage and pH during biomineralization via compensatory changes in sodium and chloride ions; it is likely that these compensatory changes lead to defects in transport of carbonate ions, that in turn, inhibit biomineralization of the calcium carbonate skeleton. We hypothesize that similar mechanisms are at play in human patients on long-term HKA inhibitors to treat acid reflux, in whom biomineralization is also decreased. Next, we demonstrate that V-type H+ ATPase (VHA) activity is required for specification of the dorsal-ventral (DV) axis, for the normal inactivation of p38 MAPK in the presumptive dorsal region, and for the subsequent asymmetric onset of expression of the TGFβ family member Nodal, that locally specifies the ventral territory. Embryos treated with VHA inhibitors exhibit global p38 MAPK activity and Nodal expression, and are ventralized. We describe previously unknown gradients of voltage and pH across the DV axis, the sharpness of which requires VHA activity. We propose that the voltage and pH gradients encode spatial information which confers asymmetry on p38 MAPK activity. Overall, we demonstrate that bioelectrical changes are essential for development of the sea urchin embryo, specifically via roles in biomineralization and DV axis specification. / 2019-01-25

<p>Cell signaling determines cellular behavior through the regulation of complex biochemical networks, slight disruptions in which can lead to a plethora of pathologies. The key to curing such diseases lies in part in gaining a comprehensive understanding of the mechanisms and molecules involved. The aim of this thesis was to characterize the role of A Kinase Anchoring Protein 200 (AKAP200), to expand our current understanding of signaling pathways in the context of development. AKAP200, a scaffolding protein previously known for its role in the spatial and temporal regulation of Protein Kinase A (PKA), was identified in our laboratory in a dominant modifier screen as a novel regulator of Planar Cell Polarity (PCP), which refers to the polarization of cells across the plane of an epithelium. Here, I demonstrate a novel role of AKAP200 in promoting Notch protein stability. In Drosophila, AKAP200 mutants show phenotypes that resemble Notch loss-of-function defects, including eye patterning and sensory organ specification defects, and its overexpression affects wing venation. Importantly, Notch signaling is downstream of the PCP pathway in the eye, the context, where AKAP200 was identified. AKAP200 shows a strong genetic interaction with Notch in the eye and thorax, and appears to promote Notch activity. Interestingly, these interactions are independent of AKAP200?s role in PKA signaling, linking AKAP200 to other functions. AKAP200 physically interacts with Notch, stabilizes endogenous Notch protein, and limits its ubiquitination. I provide genetic and molecular evidence that AKAP200 protects Notch from the E3-ubiquitin ligase Cbl and the lysosomal pathway, thereby promoting Notch signaling. In this thesis, I have discovered a novel role of AKAP200 as a post-translational regulator of Notch signaling that functions to achieve optimal Notch protein levels.

The large quantity and ready availability of developmental-genetic data, coupled with increased rigor and detail in the characterization of morphological phenotypes, has made the genotype-phenotype map of whole organisms a central challenge in evolutionary developmental biology. This in turn necessitates more general modeling strategies that can efficiently represent different types of biological knowledge and systematically applied across levels of organization, spatiotemporal scales, and taxonomic groups. Graph-based models appear useful in this context but have been remarkably underutilized in biology. Simulation of ontogenetic and evolutionary change by means of graphrewriting algorithms has been explored as a means of providing a coordinate-free approach to form transformation in time and space. A finite set of rules describing generic graph transformations is used to encode knowledge about morphogenetic steps. Their application to skeletal growth in sea urchins effectively models ontogenesis in terms of topology rather than specific geometry, suggesting a promising approach to general modeling of developmental evolution.

No description available.