PhD, University of Cincinnati, 2018, Medicine: Molecular and Developmental Biology

The kidney is a complex organ that is made of many different cell types. In an effort to better understand the cell diversity within the developing kidney we have performed single cell RNA-seq on embryonic day 14.5 mouse kidneys using Drop-seq, Chromium 10X Genomics, and Fluidigm C1 platforms. AltAnalyze was used to identify sixteen cell clusters; medullary collecting duct, cortical collecting duct, ureteric bud tip, loop of Henle, distal comma shaped body, podocyte, mid S-shaped body, early proximal tubule, pre-tubular aggregate, three cap mesenchyme groups, endothelium, nephrogenic zone stroma, cortical stroma, and medullary stroma. In addition to the known identifier genes, novel specific gene associations were also discovered during analysis. One such example is the discovery of Gdnf expression from within the stromal population, which was previously thought of as being exclusively expressed from the cap mesenchyme. The wild type single cell RNA-seq data set was also used to identify Hox gene expression within the developing kidney. Interestingly there is an apparent lack of a Hox code within the developing kidney, with thirty-six of the thirty-nine Hox genes being ubiquitously expressed throughout the kidney. Previous studies have determined that Hox 10 and 11 paralogous groups have functional redundancies within kidney development. Based on the expression data it is feasible that flanking genes are also functionally redundant within kidney development. These redundancies can mask the specific functions of individual Hox genes. For this reason Hoxa9,10,11; Hoxd9,10,11 mutants were used to further elucidate the role of Hox genes during kidney development. Morphological analysis of the Hox mutants shows alterations in mature nephron segment identity, medullary zone specification, and the lack of a pelvic opening. Obtaining a comprehensive single cell RNA-seq data set allows for the visualization of the expression profile of many genes within the wild type develop (open full item for complete abstract)

Committee: Steven Potter Ph.D. (Committee Chair); Sudhansu Dey Ph.D. (Committee Member); Brian Gebelein Ph.D. (Committee Member); Ashish Kumar M.D. Ph.D. (Committee Member); Joo-Seop Park Ph.D. (Committee Member) Subjects: Developmental Biology

MS, University of Cincinnati, 2001, Arts and Sciences : Biological Sciences

Vertebrate Hox genes are activated sequentially, exhibiting specific spatial and temporal domains within developing embryos. This colinear relationship is a property of their clustered organization, however the underlying molecular mechanisms of regulation are unclear. Chromatin structural modifications have been implicated in the control of gene transcription and could function to sequentially release Hox genes into an active chromatin configuration. One form of chromatin modifications is mediated by acetylation of histones. Hyperacetylated histones are hallmarks of active genes. We hypothesize that sequential activation of HoxB genes will correlate with targeted histone H4 hyperacetylation and active chromatin states. To test this hypothesis, the expression of HoxB genes was induced by treatment with retinoic acid (RA) in mouse P19 embryonal carcinoma cells and the chromatin immunoprecipitated with anti-hyperacetylated H4 antibodies. HoxB genes were subsequently screened in the precipitated chromatin and the unprecipitated supernatants. Semi-quantitative PCR revealed the entire HoxB chromosomal domain becomes hyperacetylated upon RA induction while HoxB genes remain associated with hypoacetylated histones before RA induction. These data suggest that prior to retinoic acid induction, the HoxB cluster is in hypoacetylated chromatin configuration. Within 24 hours after retinoic acid induction, the entire cluster is presumed to be remodeled into an open chromatin state due to histone H4 acetylation. These data make histone acetylation at the HoxB locus a candidate in the global regulation of Hox gene activation.

Committee: Suzanne Bradshaw (Advisor) Subjects:

MS, University of Cincinnati, 2001, Arts and Sciences : Biological Sciences

Hox genes play a crucial role in controlling axial patterning during embryonic development. They function as transcription factors to control a wide range of developmental processes and all contain a highly conserved sequence motif referred to as the homeobox. Comparative analysis of this homeobox DNA sequence reveals that they are highly conserved across species. One of the most striking features of Hox genes is their organization in gene clusters. Their arrangement on these clusters is directly related to their spatially restricted domains of expression along the embryonic anteroposterior axis (colinearity), as well as their timing of expression during development. The fact that Hox genes exhibit such colinearity, have overlapping expression domains, and maintain arrangement within these chromosomal clusters, suggests an organizational requirement for proper Hox gene regulation. One likely effect of this cluster organization is that cis-regulatory elements for individual Hox genes are widely scattered in the cluster and are shared between multiple genes. Hoxc-8 and Hoxc-6 are adjacent genes located 20 kb apart in the Hoxc cluster on mouse chromosome 15. The expression of these two genes overlaps with Hoxc-6 maintaining a more anterior limit of expression. It was hypothesized that the genomic region surrounding these two genes contains multiple independent and shared cis-regulatory elements. Therefore, it was the aim of this research to generate a single construct for the identification of the regulatory elements responsible for both Hoxc-8 and Hoxc-6 expression. Using homologous recombination in yeast, a 23 kb fragment of the Hoxc cluster from Hoxc-5 to Hoxc-9 was cloned into the yeast/bacteria shuttle vector, pClasper. The lacZ and human alkaline phosphatase reporter genes were subsequently introduced by homologous recombination into the coding regions of Hoxc-8 and Hoxc-6 respectively. This large reporter construct was utilized for transfection of the P19 embryo (open full item for complete abstract)

Committee: Suzanne Bradshaw (Advisor) Subjects:

PhD, University of Cincinnati, 2006, Medicine : Molecular and Developmental Biology

Renal morphogenesis involves the reciprocal inductive interactions between the ureteric bud and metanephric mesenchyme forming the collecting ducts and nephrons within adult kidney. We applied microarray technology to the study of renal morphogenesis in order to better understand the molecular mechanisms underlying development. Additionally, the techniques employed in the expression analysis of the embryonic kidney were extended to the study of renal disease. Embryonic kidneys representing different stages of renal development were analyzed using expression microarrays. Renal developmental analysis revealed many novel genes and genetic pathways involved in renal development. In addition, the normal renal development data provides a baseline for the analysis of gene targeted mice possessing disruptions in renal morphogenesis. Microarray analysis was also performed on the Hoxa11/Hoxd11 compound null renal defect throughout renal development. In conclusion, these microarray studies greatly advance our knowledge of gene expression within the normal renal morphogenesis and identify possible downstream candidate genes regulated by the Hox11 genes. Wnt signaling is crucial for normal renal morphogenesis. In Drosophila, the pygopus gene encodes a transcriptional co-activator required for canonical Wnt signaling. The targeted deletion of the mammalian orthologs of pygopus, Pygo1 and Pygo2, in mice was investigated in renal development. A disruption in ureteric number tip and morphology was identified in Pygo1/Pygo2 compound null kidneys. Additionally, canonical Wnt signaling as measure by the Bat-gal transgene is reduced within the ureteric compartment in Pygo1/Pygo2 null kidneys. Overall, these experiments suggest that Pygo function is required for activation of canonical Wnt signaling in the ureteric compartment of the developing kidney. Focal segmental glomerulosclerosis (FSGS) is characterized by the segmental scarring of the glomerulus, ultimately resulting loss of neph (open full item for complete abstract)

Committee: Dr. S. Potter (Advisor) Subjects:

Doctor of Philosophy, Case Western Reserve University, 2008, Neurosciences

Hox genes play critical roles in the patterning multiple regions of the vertebrate spinal cord, including. The function of Hox genes are modulated by two families of cofactors, Pbx and Meis proteins, that affect binding affinity and specificity by forming heteromultimers with members of the Hox family. The work that follows examines the role that the Hox cofactors play in the differentiation and diversification of spinal neurons. First, I report that the conditional loss of Pbx3 function in most tissues caudal to the hindbrain resulted in progressive deficits of posture, locomotion, and sensation that became apparent during adolescence. In adult mutants, the size of the dorsal horn of the spinal cord and the numbers of calbindin-, PKC-gamma-, and calretinin-expressing neurons in laminae I-III were markedly reduced, but the ventral cord and peripheral nervous system appeared normal. In the embryonic dorsal horn, Pbx3 expression was restricted to a subset of glutamatergic neurons, but its absence did not affect the initial balance of excitatory and inhibitory interneuron phenotypes. By embryonic day 15 a subset of Meis(+) glutamatergic neurons assumed abnormally superficial positions and the number of calbindin(+) neurons was increased three fold in the mutants. Loss of Pbx3 function thus leads to the incorrect specification of some glutamatergic neurons in the dorsal horn and alters the integration of peripheral sensation into the spinal circuitry regulating locomotion. I also found that members of the Pbx and Meis families are broadly expressed in spinal motoneurons as well. A striking exception existed in the brachial and lumbar expansions where spatially continuous groups, that were reminiscent of motor pools, failed to express Pbx or Meis proteins. Retrograde transport studies demonstrated that all of the motorneurons within any given pool either expressed or failed to express the cofactors, indicating that they are expressed in a motor pool specific manner. The (open full item for complete abstract)

Committee: Stephen O'Gorman (Advisor) Subjects: Biology, Neuroscience