The spatial patterning of neurons is a fundamental problem in neuroscience. The functions of the brain are rooted in the cellular architecture that underlies the structure of the brain. In the cerebral cortex, the functions of the cortex depend on the proper assembly of circuits made up of long-range excitatory neurons and locally-projecting inhibitory interneurons. Interneurons are incredibly diverse from a morphological and functional perspective and are found in every cortical area. Unlike excitatory cortical neurons, interneurons are born outside of the cortex and migrate long distances into the cortex and distribute across the cortex broadly. How do these diverse cells that essentially invade the cortex properly distribute? How do different developmental stages contribute to the final patterning of interneuron subtypes, and what are the molecules that influence this process? In this dissertation, I will present my original research which has advanced our knowledge of the answers to these fundamental questions in the field of developmental neuroscience. I addressed these questions by applying a range of techniques including mouse genetics, immunohistochemistry, confocal microscopy, and point pattern analysis. My research has shown that cortical interneuron subtypes are spatially independent. Spatial patterns of cortical interneuron subtypes are non-random within subtypes, but are randomly positioned with respect to other subtypes. I also explored the effects of loss of diversity within the clustered protocadherin family of adhesion molecules. Though these molecules do not appear to play a role in subtype specific spatial independence, I found that loss of clustered protocadherin diversity alters the density and laminar distribution of cortical interneuron subtypes. I also contributed to the development of genetic tools which could help us further understand how developmental stages contribute to final interneuron distribution. My original research has collectively advanced our knowledge of how cortical interneurons achieve their final distributions during development and has opened up new avenues of scientific inquiry for future research in developmental neuroscience.

Abnormal kidney development is a relatively prevalent health issue; however, the genetic basis is mostly unknown. The aim of this thesis is to identify genes important in kidney development and disease and to study their molecular functions. We hypothesized that human diseases associated with kidney anomalies can uncover novel genes important in kidney development and disease. The thesis is divided into three independent projects that examined three genes (i.e. Zeb2, Ilk, Robo2) at three stages of mouse kidney development: nephrogenesis, glomerular podocyte, and early ureteric bud outgrowth. In the first project, we identified Zeb2, a gene encoding the zinc finger E-box binding homeobox 2 transcription factor that is mutated in the Mowat Wilson syndrome, as a novel gene important in nephrogenesis. Zeb2 conditional knockout mice (Zeb2 cKO) develop glomerulocystic kidney disease with many atubular glomeruli and decreased expression of proximal tubular markers before cyst formation. These data suggest that abnormal nephrogenesis leads to the congenital atubular glomeruli and primary glomerular cysts in the Zeb2 cKO mice. This study implies that ZEB2 is a novel candidate gene for glomerular cystic disease in patients. Additionally we found that Pkd1, the gene mutated in autosomal dominant polycystic kidney disease, is upregulated in non-cystic glomeruli and knockout of one copy of the Pkd1 gene exacerbates the cystic phenotype of the Zeb2 cKO mice. These findings suggest a genetic interaction between Zeb2 and Pkd1 and that Zeb2 might be a novel PKD1 modifier. In the second project, we studied the roles of integrin-linked kinase (ILK) and roundabout 2 (ROBO2) in glomerular podocytes. We found that ILK and ROBO2 form a protein complex, and that loss of Robo2 improves survival and alleviates the podocyte and basement membrane abnormalities seen in Ilk knockout mice. In the third project, using microarray gene expression analysis, we found lower gene expression levels of extracellular matrix proteins during early ureteric bud outgrowth in the Robo2 homozygous knockout embryos as compared to wild type controls. These findings suggest that ROBO2 may regulate extracellular matrix components in the kidney. In conclusion, we found a new role for Zeb2 in nephrogenesis, and identified a novel function of Robo2 in regulating extracellular matrix gene expression in podocytes and during early kidney development. / 2017-11-03T00:00:00Z

No description available.

Mouse epiblast stem cells (EpiSC) are pluripotent cells derived of the epiblast of post-implantation blastocysts that can self-renew indefinitely in culture, display lineage-restricted differentiation, and appear to closely resemble human embryonic stem cells (ESC). Despite significant advances in the last decade, the precise molecular mechanisms and many master regulator (MR) genes underlying stem cell self-renewal, pluripotency, interactions with surrounding cells, and lineage-specific differentiation still remain elusive. The goal of this thesis is to address these gaps of knowledge using a systematic approach to identify novel MR genes and functionally validate them using genetically modified mouse models.In order to elucidate MR genes that control understudied biological processes, previous work in the Shen lab have computationally reconstructed the regulatory network of EpiSC and interrogated the EpiSC interactome with pluripotency signatures of EpiSC lines. One MR gene of interest from the previous analysis is ZC3H13, which encodes a protein that has been previously shown to be a crucial for N6-methyladenosine modification in RNA (m⁶A). This suggests a novel connection between m⁶A epitranscriptional modifications and primed state pluripotency. In my thesis research, I have shown that Zc3h13 is essential for proper trophoblast lineage differentiation and the importance of m6A modifications in early embryonic development. Using two Zc3h13 knockout mouse lines, I have found that Zc3h13 null embryos are embryonic lethal at the peri-implantation stage due to a failure to implant into the uterus. In vitro outgrowth analysis revealed a lack of trophoblast giant cells in Zc3h13 null outgrowths, and the lack of enlarged nuclei in the Zc3h13 null outgrowth suggests a failure in endoreduplication. Immunofluorescence analysis of Zc3h13 null blastocysts showed that the trophectoderm cells of Zc3h13 null blastocyst expressed trophectoderm specific factors at abnormal levels, indicating a severe dysregulation of the trophectoderm regulatory network. To elucidate the effects of Zc3h13 knockout on pluripotency, I also performed a detailed immunofluorescence analysis of Zc3h13 null inner cell mass (ICM), which expressed pluripotency factors at normal levels. However, Zc3h13 null blastocysts were less efficient at generating ESC lines and the Zc3h13 KO ESC generated were morphologically abnormal. Dot blot and mass spectrometry analysis showed that Zc3h13 KO ESC had a dramatically lower level of m⁶A modification, suggesting a connection between m6A epitranscriptional modification and endoreduplication. Interestingly, chimera and teratoma analysis showed that while Zc3h13 KO ESC can contribute to derivatives of the three primary lineages, Zc3h13 KO ESC has a bias towards neuroectoderm differentiation. In this thesis, I have shown the importance of m6A transcriptional regulation in trophoblast giant cell differentiation. Taken together, my studies can help further the understanding of the biological functions of m⁶A modifications as well as the relationship between transcriptional regulation and cell fate transition. My work highlights another level of gene regulation through epitranscriptional modification and the importance of the epitranscriptomic landscape in cell fate transition and development.

No description available.

CK2α is a serine-threonine kinase that is involved in a large number of biological processes, including embryonic development, cancer, and cell proliferation. Recently, it has been found that mutations in CK2α results in a developmental condition known as Okur-Chung neurodevelopmental syndrome (OCNDS). This disorder commonly results in intellectual disability, congenital heart defects (CHDs), gross motor delay, and facial abnormalities. CK2α inhibition has so far primarily been studied in mice, through methods such as knockout, gene floxing, and CRISPR/Cas9 mutations. In this thesis, we provide a proof of principle that chemical inhibition of CK2 in Xenopus laevis embryos can induce a phenocopy similar to the heart phenotype of the CK2α knockout mouse model, and demonstrate the potential of Xenopus laevis as an animal model to study molecular mechanisms that may underlie OCNDS. Here we carefully examined whole embryos, sections stained with multiple antibodies, sections stained with hematoxylin and eosin, and assessment of proliferation and apoptosis rates. The phenotypes observed in the Xenopus laevis model were analyzed and compared to both the CK2α knockout mouse model and OCNDS patients. Results found commonalities among facial features, heart deformities, and muscle patterning between the animal models, which overlapped heavily with patient symptoms. Thus, this work has established Xenopus laevis treated with chemical inhibitors as an appropriate animal model for further characterization of the mechanisms that may underlie OCNDS. / 2023-11-18T00:00:00Z

The pharyngeal foregut endoderm (PFE) gives rise to several important organs including the thyroid, thymus and parathyroid glands. In mice and humans, defects in the development of PFE can lead to thymic aplasia and aberrations in thymic epithelial cell (TEC) function can lead to immunodeficiency or autoimmune disease. Successful differentiation of pluripotent stem cells (PSCs) to PFE could provide a renewable cell source that enables the study of human diseases that originate in the PFE. Here, I identify signaling pathways that influence the differentiation of PSCs to PFE. Firstly, using a novel mouse reporter PSC line we develop a protocol that generates a Pax9 expressing population that is enriched for PFE markers and upon transplantation can form organized epithelial structures. However, since this protocol was inefficient for human PSCs, we subsequently identified additional signaling pathways required for the efficient generation of human PFE and determined a key role for retinoic acid. Upon transplantation, the human PFE gives rise to TECs, a ventral PFE derivative. Finally, to facilitate future investigation into the gene regulatory networks in PFE, we develop a CRISPR-effector system to modulate endogenous gene expression in PSCs. We demonstrate that developmentally relevant genes can be repressed or induced, thereby influencing the cellular state. These data present strategies to generate cells of the PFE lineage from PSCs, facilitating the production of cells for patient-specific disease modeling or cell replacement therapies, and a method to interrogate gene and regulatory element function in PFE and its derivatives.

lin-28 was first characterized as a developmental timing regulator in Caenorhabditis elegans. Loss of lin-28 function (lin-28(lf)) mutants skip the hypodermal cell fates specific to the 2nd larval stage. Here, we studied two aspects of lin-28 which had not yet been investigated. First, we show that lin-28(lf) mutants exhibit reduced fertility associated with abnormal somatic gonadal morphology. In particular, the abnormal spermatheca-uterine valve morphology of lin-28(lf) hermaphrodites traps embryos in the spermatheca, which disrupts ovulation and causes embryonic lethality. The same genes downstream of lin-28 in the regulation of hypodermal developmental timing also act downstream of lin-28 in somatic gonadal morphogenesis and fertility. Importantly, we find that hypodermal expression, but not somatic gonadal expression, of lin-28 is sufficient for restoring normal somatic gonadal morphology in lin-28(lf) mutants. We propose that the abnormal somatic gonadal morphogenesis of lin-28(lf) hermaphrodites results from temporal discoordination between the accelerated hypodermal development and normally timed somatic gonadal development. Thus, our findings exemplify how a cell-intrinsic developmental timing program can also control proper development of other interacting tissues, cell non-autonomously. We also investigated the expression patterns and functions of two lin-28 isoforms in C. elegans. Our analysis of spatial expression patterns suggests that lin-28a and lin-28b are co-expressed in diverse tissues. Consistently, neither of isoform specific knock-out mutant, lin-28a(lf) or lin-28b(lf), exhibits defects in hypodermal development, somatic gonad, or fertility, indicating functional redundancy of two isoforms. Our study will contribute to further investigation of lin-28 isoforms by providing the mutants of each isoform as well as the primary analysis of their phenotypes.

Etv2 is an endothelial-specific ETS transcription factor that is essential for endothelial differentiation and vascular morphogenesis in vertebrates. However, etv2 expression dynamics during development and the mechanisms regulating it are poorly understood. I found that etv2 transcript and protein expression are highly transient during zebrafish vascular development, with both expressed early during development and then subsequently downregulated. Inducible knockdown of Etv2 in zebrafish embryos prior to mid-somitogenesis, but not later, causes severe vascular defects, suggesting a role for Etv2 in specifying angioblasts from the lateral mesoderm. I further demonstrate that the 3’UTR of etv2 is post-transcriptionally regulated in part by the let-7 family of microRNAs. Ectopic expression of let-7a represses endogenous Etv2 transcript and protein expression with a concomitant reduction in endothelial cell gene expression. Additionally, overexpressed Etv2 in HEK293T cells is ubiquitinated and degraded by the proteasome. Accordingly, endogenous zebrafish Etv2 protein is rapidly degraded in the presence of the translation inhibitor cycloheximide in vivo. Taken together, our results suggest that etv2 acts during early development to specify endothelial lineages and is subsequently downregulated through post-transcriptional and post-translational mechanisms, to allow normal vascular development to proceed.

The lymphatic vasculature is composed of lymphatic endothelial cells (LECs) that coalesce into a branched hierarchy of small capillaries and larger collecting vessels that regulate interstitial fluids, lipid uptake and immunity. Few studies have focused on pulmonary lymphatic system. To fill these critical knowledge gaps, we interrogated the fetal maturation program of lymphatic endothelium, and we provide evidence that CSF1R-lineage progenitors contribute to LECs in the lung during a temporally defined period in early postnatal life. The pulmonary lymphatic system is required for fluid clearance and air breathing at birth, suggesting a prenatal maturation program. To interrogate this, we developed a cell sorting strategy to enrich pulmonary LECs by their unique cell surface immunophenotype (CD45-, EPCAM-, CD31+, VEGFR3+, PDPN+, LYVE1+) for transcriptional profiling. These experiments highlighted the coordinate down-regulation of genes involved in “cell cycle”, and “mRNA processing” along with coordinate upregulation of “complement/coagulation cascade”, “lipid metabolism”, and “angiogenesis” genes from embryonic day E16.5 to E18.5. The most significantly enriched gene set corresponded to the “interferon-alpha/beta signaling” pathway which was confirmed with qRT-PCR and in-situ hybridization. These data provide the first description of the transcriptional landscape of fetal pulmonary LEC maturation. During development, all LECs are thought to originate from embryonic veins, however multiple studies have suggested a myeloid origin for a subset of LECs. A relationship between myeloid cells and the pulmonary LECs has not been elucidated. Here, we used myeloid-specific inducible CSF1R-CreERtdTomato lineage tracing mice and identified rare, single cells that co-expressed CSF1R- CreERtdTomato and Prox1, the master lymphatic regulator, in the postnatal day 3 lung. This process was temporally restricted to the early postnatal period. Lineage tracing with additional myeloid-Cre mice (CSF1R-iCre and CX3CR1-Cre) also showed contribution to postnatal LECs. To determine the biological significance of CSF1R-derived LECs to postnatal lung biology, we performed conditional Prox1 loss of function experiments. CSF1R-CreER mediated deletion of Prox1 resulted in lymphatic hypoplasia, edematous foci and clotting. These findings suggest that early postnatal CSF1R+ progenitors contribute to the pulmonary lymphatic endothelium and that vascular clotting may result from lymphatic malformation/dysfunction. / 2021-06-14T00:00:00Z