While the genetic control of ureteric bud (UB) morphogenesis has been extensively studied, the cellular basis of this process remains unclear. The renal organoid system is a novel technique in which embryonic kidneys are dissociated into single cells and then reaggregated, where they reassociate to form organotypic structures. This system may be very beneficial for investigating the cellular basis of ureteric bud development. Here, we first used a fluorescent UB marker, Hoxb7:myrVenus, and time-lapse microscopy to characterize the cellular and tissue-level events during self-organization and UB morphogenesis of E12.5 or E14.5 renal organoids. Briefly, we found that UB structures self-assembled by aggregation of individual cells that sent out long cell processes. The cellular aggregates grew and elongated into epithelial tubes that displayed characteristic ampullae, bifurcated, and appropriately expressed UB tip markers analogous to their in vivo counterparts. We also found that cap mesenchymal cells are attracted to newly formed epithelial structures early in renal organoid development, and were later found in cell clusters surrounding new branches. RET is a trans-membrane tyrosine kinase receptor (RTK), expressed in ureteric bud cells, whose expression is gradually restricted to the tips of the growing ureteric tree. We demonstrate that the renal organoid system can be used, as an alternative to the generation of in vivo chimeric embryos, to study Ret-dependent cell rearrangements previously shown to establish and maintain the UB tip progenitor domain. Chimeric renal organoids that juxtaposed wild-type cells with Sprouty1–/– mutant cells (higher Ret-signaling) or with Ret51/cre (lower Ret-signaling) mutant cells recapitulated the cell sorting pattern observed in similar in vivo chimeras. The cells with higher Ret-signaling preferentially sorted to, and were maintained in, the forming and growing tips of these mosaic ureteric bud structures, out-competing cells with lower Ret-signaling. We then used the mosaic organoid system to ask if fibroblast growth factor receptor 2 (Fgfr2), another RTK expressed in the ureteric bud and important for its development, also mediates individual cell rearrangements that generate and maintain the UB tips. UB cells null for Fgfr2 were largely unable to compete with wild-type cells for occupancy of the UB tips in chimeric renal organoids. Using the innovative MASTR (Mosaic Mutant Analysis with Spatial and Temporal Control of Recombination) technique in vivo, mosaic homozygous deletion of Fgfr2 in newly formed ureteric buds also revealed that mutant cells were slightly deficient in their ability to contribute to Fgfr2 heterozygous UB tips. This demonstrates a novel, cell-autonomous role of Fgfr2 in ureteric bud development. Matrix metalloproteinase 14 (MMP14) is a membrane-bound protein known to participate in a wide variety of cell functions including degradation of the extracellular matrix (ECM), cell signaling, and cell-autonomous cell migration. It is expressed in the UB and was discovered to act downstream of Ret-signaling. Although needed in the ureteric epithelium for ECM degradation and proper UB morphogenesis, its specific function in the UB has not been thoroughly investigated. In generating in vivo chimeras, we discovered that Mmp14 null cells could contribute to wild-type ureteric bud tips at E12.5 and E14.5, demonstrating that, despite its documented role in UB branching, Mmp14 does not have a cell-autonomous role in the cell rearrangements observed during UB morphogenesis.

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The mechanisms that genes use to direct patterns of development are of fundamental interest. Using Arabidopsis thaliana as a model, I have investigated aspects of these mechanisms in the separate processes of vascular and floral development. Specifically, I conducted a screen for vascular-defective mutants, and analyzed a region of the genome that regulates the expression of the floral homeotic gene, AGAMOUS ( AG). / In this report, I describe the identification of over forty mutants that are abnormal in tracheary element development or vein patterning. The spectrum of mutant phenotypes that I observed indicates that the mechanisms that pattern primary and secondary veins of leaves or cotyledons are at least partially separable; that among the genes that affect vascular development, a significant proportion are repressors of vascular differentiation; and that the majority of vascular mutants that can be identified in this type of screen have pleiotropic phenotypes. / I characterized two of the mutants, varicose ( vcs) and scarface (sfc), in more detail. vcs mutants are temperature sensitive, and at the non-permissive temperature, accumulate distended tracheary elements around veins. VCS is also required at an early stage of leaf development for normal vein patterning, and interacts with the AUXIN RESISTANT 1 gene in this process. sfc mutants fail to develop normal, contiguous vein networks in cotyledons, leaves, sepals, and petals. It is specifically the secondary and higher order veins in these organs that are affected by the mutation. sfc mutants have exaggerated responses to exogenous auxin, and the SFC gene overlaps in primary and secondary vein patterning functions with an auxin-response factor gene MONOPTEROUS. / This report also includes an analysis of the cis-regulatory regions that control expression of AGAMOUS, a gene that when properly expressed in two central domains of the developing flower, directs the formation of carpels and stamens. My dissection of an AG intragenic region demonstrated that AG expression in stamens can be activated independently of carpels. Moreover, the stamen-specific expression pattern was found to be independent of APETALA2, a known negative regulator of AG, while the carpel-specific expression pattern was shown to be independent of LEUNIG, another negative regulator of AG.

The gene capicua is required for the establishment of dorsal-ventral polarity in the Drosophila melanogaster ovary. Loss of capicua function in the follicle cells results in dorsalization of both the embryo and eggshell. The most prominent dorsal features of the Drosophila eggshell are the dorsal appendages. We show that loss of capicua function results in the ventral ectopic specification of dorsal appendage-producing follicle cell fate. This cell fate change is due in part to the ectopic expression of genes such as mirror and Broad-Complex in capicua mutant ovaries. When either mirror or Broad-Complex are ectopically expressed independently of loss of capicua function, they generate a phenotype similar to the capicua mutant phenotype. We propose that Capicua normally acts in the ventral follicle cells to repress the expression of genes that pattern the dorsal follicle cells. EGF receptor signaling may normally inactivate Capicua repression in the dorsal follicle cells.

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Morphogenesis is the fundamental and remarkable biological process that produces elaborate and diverse tissues and organs from simple groups of cells, which can happen on timescales as short as minutes or as long as days. One of the biggest challenges in understanding morphogenesis is the gap between our knowledge of the molecular-scale activities of genes and proteins, and the large-scale behaviors of cells and tissues. To fill this gap, a complete understanding of both biochemical and mechanical factors involved in morphogenesis is needed. Morphogenesis is naturally a mechanical process in which tissues are physically sculpted by mechanical stress, strain, and movements of cells that are induced by these genetic and molecular programs. However, many of the mechanical factors involved in morphogenesis remain poorly understood partially due to the strong coupling of mechanical factors and biological factors, the active responses of living tissues to the environment, and the lack of experimental methods to study the mechanics of tissues in vivo. Epithelial tissues play crucial roles in shaping early embryos and are widely spread in mature animals to serve as boundaries and barriers. They are robust tissues that not only support the structure of embryos and organs, but also actively change shape and structure, displaying a fluid behavior during morphogenesis. Contractile tension and cell-cell adhesion are thought to be the main mechanical factors involved in epithelial tissue morphogenesis, but how the balance between these two determines epithelial tissue mechanics remains unclear. To build a fundamental understanding of the mechanical mechanisms underlying epithelial tissue morphogenesis, this dissertation studies the germband epithelial tissue in the early Drosophila melanogaster embryo and addresses two important open questions in the field of mechanics in morphogenesis: (1) what mechanical factors are involved in the morphogenesis of epithelial tissues; (2) how does a cell control these factors to tune tissue mechanical behaviors. In this dissertation, we developed a systematic, quantitative, in vivo experimental approach to explore mechanics of epithelial tissue morphogenesis in the Drosophila embryo by integrating molecular genetics approaches, live confocal fluorescence imaging, and quantitative image analysis. Combining our experimental studies in the Drosophila embryo with our collaborators’ theoretical modeling approaches, we showed that the shapes and alignment of cells within tissues can help us understand and predict epithelial tissue mechanical behaviors, such as tissue fluidity, during morphogenesis and how defects in these processes can result in abnormalities in embryo shape. We also observed that the Drosophila germband tissue transitions from more solid-like to more fluid-like behavior to help accommodate dramatic tissue flows during convergent extension, which indicates that the mechanical properties of developing tissues might be tuned during morphogenetic events. To elucidate molecular mechanisms underlying how tissue mechanical properties may be regulated during morphogenesis, this dissertation explores the role of cell-cell adhesion in controlling epithelial tissue mechanics. By systematically modulating cell-cell adhesion levels in the Drosophila germband tissue and combining live imaging and quantitative image analysis, we studied the effects of cell-cell adhesion levels on cellular and tissue behaviors. We found biphasic dependencies of cell rearrangements, cell shape, and tissue fluidity on cell-cell adhesion levels, which are surprisingly linked to each other by cell patterns in the tissue. In particular, tissues comprising cells with either lower or higher cell-cell adhesion levels tend to rearrange faster and show cell patterns indicating more fluid-like tissue behaviors. Further studies suggested that cell-cell adhesion works with cytoskeletal molecules to achieve these effects. The experimental approaches developed for exploring mechanics in 2-D in the Drosophila germband epithelial tissue are expanded upon in order to investigate germband tissue mechanics in 3-D. These approaches are also used to study mechanics in the inner ear round window membrane of the guinea pig for clinical application. This dissertation advances our understanding of mechanics of epithelial tissue morphogenesis in vivo and provides a practical, quantitative, and appealing platform for exploring mechanics in living tissues during morphogenesis. This helps fill the gap in our knowledge of molecular-scale activities and tissue-level behaviors, provides insight into building tissues with precise shapes and structures in the lab, and sheds light on human diseases associated with improper regulation of tissue mechanics such as birth defects, aberrant wound healing, and cancer metastasis.

Mechanisms of post-transcriptional control are critical to deploy RNAs and proteins asymmetrically to a discrete region of cytoplasm at the posterior of the Drosophila oocyte and embryo, called the pole plasm and thus allow differentiation of the germline. Research presented in this thesis investigates the post-transcriptional control of Drosophila pole plasm component germ cell-less (gcl ). Maternal gcl activity is required for germ cell specification and gcl RNA and protein accumulate asymmetrically in the pole plasm. gcl RNA, but not Gcl protein, is also detected in somatic regions of the embryo, and ectopic expression of Gcl in the soma causes repression of somatic patterning genes suggesting that gcl RNA is subject to translational control. I find that Gcl is expressed during oogenesis, where its expression is regulated by translational repressor Bruno (Bru). Increased levels of Gcl are observed in the oocyte when Bru is reduced (i.e., in an arrest heterozygote) and Bru overexpression reduces the amount of Gcl. Consistent with this, reduction of the maternal dosage of Bru leads to ectopic Gcl expression in the embryo, which, in turn, causes repression of anterior huckebein RNA expression. Bruno binds directly to the gcl3'UTR in vitro, but surprisingly, this binding is largely independent of a Bruno Response Element (BRE) in the gcl 3'UTR and depends upon a novel site. Furthermore, the gcl BRE-like region is not required to repress Gcl expression during oogenesis or embryogenesis. I concluded that Bru regulates gcl translation in a BRE-independent manner. In addition, I established the role of the gcl 3'UTR in gcl RNA localization and translation using transgenes that replace the endogenous 3'UTR with the alpha-tubulin 3'UTR or place it in tandem to the bicoid 3'UTR. I find that accumulation of gcl RNA in the embryonic pole plasm requires the gcl 3'UTR. Moreover, Gel is restricted to the pole plasm by translational repression mediated by the gcl 3'UTR and a limiting pool of trans-acting translational repressors. The phenotypic consequences of loss of this translational control are relatively mild, suggesting that gcl translation does not require stringent repression in the soma.

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Indiana University-Purdue University Indianapolis (IUPUI) / The heart is a complex organ that is composed of numerous cell types, which must integrate their programs for proper specification, differentiation, and cardiac morphogenesis. During cardiac development the basic helix-loop-helix transcription factor Hand2 is dynamically expressed within the endocardium and extra-cardiac lineages such as the epicardium, cardiac neural crest cells (cNCCs), and NCC derived components of the autonomic nervous system. To investigate Hand2 function within these populations we utilized multiple murine Hand2 Conditional Knockout (H2CKO) genetic models. These studies establish for the first time a functional requirement for Hand2 within the endocardium, as several distinct phenotypes including hypotrabeculation, tricuspid atresia, aberrant septation, and precocious coronary development are observed in endocardial H2CKOs. Molecular analyses reveal that endocardial Hand2 functions within the Notch signaling pathway to regulate expression of Nrg1, which encodes a crucial secreted growth factor. Furthermore, we demonstrate that Notch signaling regulates coronary angiogenesis via Hand2 mediated modulation of Vegf signaling. Hand2 is strongly expressed within midgestation NCC and endocardium derived cardiac cushion mesenchyme. To ascertain the function of Hand2 within these cells we employed the Periostin Cre (Postn-Cre), which marks cushion mesenchyme, a small subset of the epicardium, and components of the autonomic nervous system, to conditionally ablate Hand2. We find that Postn-Cre H2CKOs die shortly after birth despite a lack of cardiac structural defects. Gene expression analyses demonstrate that Postn-Cre ablates Hand2 from the adrenal medulla, causing downregulation of Dopamine Beta Hydroxylase (Dbh), a gene encoding a crucial catecholaminergic biosynthetic enzyme. Electrocardiograms demonstrate that 3-day postnatal Postn-Cre H2CKO pups exhibit significantly slower heart rates than control littermates. In conjunction with the aforementioned gene expression analyses, these results indicate that loss of Hand2 function within the adrenal medulla results in a catecholamine deficiency and subsequent heart failure.