Proper development of the nephron, the functional unit of the kidney, is essential for kidney function. The nephron develops from a pool of cap mesenchymal cells, as defined by a cluster of cells adjacent to the ureteric bud tips of branching ureteric epithelium, giving rise to two subset populations: the self renewing cells and the nephron progenitors. These nephron progenitors undergo mesenchymal-epithelial transition (MET) to develop into polarized renal vesicles (RV), and eventually fuse with the epithelial tubule to develop into a mature nephron. Although these processes are essential for the formation of functional kidneys, little is known about the molecular mechanisms that regulate them. In this study, we characterize several steps during cap mesenchyme and renal vesicle formation using our Shroom3 knockout mouse kidney as our model. Previous researchers have associated Shroom3 with chronic kidney disease. Detecting and analyzing the genetic components of CKD is needed to improve our understanding of its pathogenesis. Shroom3 encodes an actin-binding protein that regulates cell shape changes through induction of apical constriction. However, there is a lack of evidence about Shroom3’s expression pattern and functional role upstream of developed nephrons. Here, I defined the spatial and temporal expression of Shroom3 within the cap mesenchyme region. I investigated the nephron progenitors between Shroom3 wildtypes and mutants. Lastly, I analyzed the renal vesicle polarity in mutants, by analyzing apical membrane markers on RVs to characterize any abnormalities in their orientation and establishment of polarity. / Thesis / Master of Science in Medical Sciences (MSMS)

How tissues and organs develop into their final shape during embryogenesis is a fascinating and long-standing question in developmental biology. Tissue morphogenesis is driven by a variety of events at the cellular level and individual cell shape change is one of the central morphogenetic engines. Thus, it is crucial to understand what signals specify the correct cell behavior in specific groups of cells during development. For my doctoral studies, I have focused on two cell shape change events, apical constriction and cilia assembly. First, we present data demonstrating that Shroom3 is essential for cell shape changes and morphogenesis in the developing vertebrate gut, where Shroom3 transcription requires the Pitx1 transcription factor. We identified a Pitx-responsive regulatory element in the genomic DNA upstream of Shroom3, and showed that Pitx proteins directly activated Shroom3 transcription in Xenopus. Moreover, we showed that ectopic expression of Pitx proteins was sufficient to induce Shroom3-dependent cytoskeletal reorganization and epithelial cell shape change. These data demonstrated new breadth to the requirements for Shroom3 in morphogenesis, and also provided a cell-biological mechanism for Pitx transcription factors action during morphogenesis. Next, we focused on understanding the transcriptional regulation of ciliogenesis. We first showed that Rfx2 transcription factor broadly controlled ciliogenesis, and by RNA- and ChIP-sequencing, we showed that Rfx2 directly regulated a wide range of genes encoding diverse ciliogenic machinery. Finally, in addition to ciliogenesis regulation, a large number of non-ciliary genes in our Rfx2 dataset led us to identify a novel role of Rfx2 in controlling insertion of multi-ciliated cells into the overlying mucociliary epithelium. Moreover, we showed here that Slit2, a target of Rfx2, was involved in multi-ciliated cell movements, possibly through mediating cortical E-cadherin level. This work allowed us to begin building a genetic network controlling multi-ciliated cells in mucociliary epithelium. Together, we showed a transcriptional regulation of apical constriction driving gut morphogenesis and a comprehensive transcriptional network that governs multi-ciliated cell development. / text

Chronic kidney disease (CKD), defined as an irreversible reduction in glomerular filtration rate, is a large public health concern. Dissecting the genetic components of CKD is required to improve our understanding of disease pathogenesis. Researchers have identified that SHROOM3, has very high associations with kidney disease and function. Shroom3 encodes an actin-binding protein important in regulating cell and tissue morphogenesis. However, there is a lack of evidence supporting a role for Shroom3 in kidney function or disease. Here, I investigated the developmental and functional role of Shroom3 in the mammalian kidney. For the first time, I described the expression pattern of Shroom3 in the embryonic and adult mouse kidneys. By performing in situ hybridization and immunohistochemistry, I demonstrated that Shroom3 is expressed in the condensing mesenchyme, podocytes, and collecting ducts. I further showed that Shroom3 protein is localized in the foot processes of podocytes, utilizing immunogold labeling and transmission electron microscopy. In order to uncover a potential role of Shroom3 in the kidney, we utilized Shroom3 knockout mice. Shroom3 mutants demonstrated marked glomerular abnormalities including cystic and degenerating glomeruli, and reduced glomerular number. Scanning and transmission electron microscopic analyses of Shroom3 mutant glomeruli revealed disruptions in podocyte morphology characterized by disorganized foot processes with less interdigitation and segmental foot processes effacement. Furthermore, immunofluorescence analysis of mutant kidneys revealed aberrant distribution of podocyte actin-associated proteins. Elucidating the underlying molecular mechanism of this abnormal podocyte architecture; v we demonstrated that in the absence of Shroom3, Rho kinase is mislocalized in the apical membrane of podocytes. As a result, mislocalized Rho kinase failed to phosphorylate non-muscle myosin and induce actomyosin contraction resulting in a patchy granular distribution of actin in the podocytes of Shroom3 mutants. Taken together, our findings established that Shroom3 is essential for proper actin organization in the podocytes through interaction with Rock. Furthermore, we took advantage of a haploinsufficiency phenotype of Shroom3 heterozygote adult mice and demonstrated these mice develop glomerulosclerosis and proteinuria. In conclusion, our studies provided evidence to support a role for Shroom3 in kidney development and disease and support the GWAS studies that suggested a correlation between SHROOM3 variants and kidney function in humans. / Thesis / Master of Science (MSc)

Nephrons develop from a specialized group of mesenchyme cells known as the nephron progenitors. Nephron progenitors can very dynamic as they can self-renew, migrate, and change their cell morphology. These alterations are essential for orientating and organizing select cells for progression through various stages of nephrogenesis. However, the underlying mechanisms that drive these dynamic morphological changes are not fully understood. Shroom3 is an actin-binding protein that regulates cell shape changes by modulating the actin cytoskeleton. In mice and humans, mutations in Shroom3 are associated with poor nephron function and chronic kidney disease. Despite these findings, the underlying mechanisms of Shroom3 function and how genetic mutations contribute to abnormal nephron formation are unclear. Here, we investigated functional roles for Shroom3 in the nephron progenitor population by analyzing E13.5 and E18.5 Wildtype and Shroom3 deficient mice (termed Shroom3-/-). First, using in-situ hybridization (ISH) and immunofluorescence (IF), we confirm Shroom3 expression in select nephron progenitors. Next, we demonstrated abnormal cell shape and abnormal nephron progenitor cell clustering using H&E staining and Pax2 immunofluorescence. We showed a reduction in nephron progenitor cell numbers and decreased cell length in E13.5 Shroom3-/- kidneys. Using markers of cell orientation, we discovered altered cell orientation in some but not all nephron progenitor cells. While analyzing the cell cytoskeleton, we also demonstrated the abnormal distribution of F-actin in Shroom-/- nephron progenitors. Lastly, immunofluorescence and transmission electron microscopy analysis of Shroom3-/- nephron progenitors confirmed the abnormal shape and reduced filopodia-like thin actin-based membrane protrusions. Our findings conclude that Shroom3 is essential for maintaining and regulating nephron progenitor cell morphology. Taken together, these findings could help explain why Shroom3 mutations are highly associated with kidney disease. / Thesis / Master of Science in Medical Sciences (MSMS)

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The Shroom family of proteins is currently comprised of four members, Shroom1, 2, 3 and 4. Since Shroom3 was shown to be a critical protein for neural tube closure, the other three proteins are also expected to play an important role for proper development. However, their functions during development were not clear. To address this, my study started with Shroom3 function in the neural plate. Shroom3 had been previously known to induce apical constriction by controlling actin filaments in neuroepithelial cells. My studies show that Shroom3 induces apico-basal cell heightening by controlling parallel microtubule assembly. Shroom3 is able to change the distribution of γ-tubulin, suggesting that Shroom3 controls apical constriction and apico-basal cell elongation via both actin filaments and microtubules. The ability to control γ-tubulin distribution is possessed not only by Shroom3, but also by all other Shroom proteins, although they can not induce apical constriction. In addition, they are expressed in tissues which contain apico-basally elongated cells. Data from functional assays with Shroom2 show that it induces cell elongation and is required for proper cell shape in deep layer neuroepithelial cells in Xenopus. These data suggest that Shroom family proteins control cell architecture during morphogenetic development. I have discovered another role for Shroom2. By comparative analysis with Xenopus and Physalaemus, which have different pigment patterns in eggs, I show that a high level of maternal Shroom2 mRNA is important for pigment polarity in Xenopus. Furthermore, Shroom2 controls the distribution of spectrin which plays a role in pigment granule movement. Thus, Shroom2 is suggested to be a key molecule to control the pigment polarity in amphibian eggs. Together all these data suggest that Shroom family proteins play a role in cell morphogenesis and polarization via controlling the cytoskeleton during Xenopus development. / text

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