Browsing by Subject "Organogenesis"
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Item Investigating Vascular Patterning and Regression in Kidney Development and Organoids(December 2021) Ryan, Anne Regina; Marciano, Denise; Munshi, Nikhil; Dellinger, Michael T.; Cleaver, OndineChronic kidney disease (CKD) and end stage renal disease (ESRD) are increasingly frequent and devastating conditions that have driven a surge in the need for kidney transplantation. A stark shortage of organs has fueled interest in generating viable replacement tissues ex vivo for transplantation. One promising approach has been self-organizing organoids, which mimic developmental processes and yield multicellular, organ-specific tissues. However, a recognized roadblock to this approach is that many organoid cell types fail to acquire full maturity and function. I comprehensively assessed the vasculature in two distinct kidney organoid models as well as in explanted embryonic kidneys. Using a variety of methods, my work shows that while organoids can develop a wide range of kidney cell types, as previously shown, endothelial cells (ECs) initially arise but then rapidly regress overtime in culture. Vasculature of cultured embryonic kidneys exhibit similar regression. By contrast, engraftment of kidney organoids under the kidney capsule results in the formation of a stable, perfused vasculature that integrates into the organoid. This work demonstrates that kidney organoids offer a promising model system to define the complexities of vascular-nephron interactions, but the establishment and maintenance of a vascular network present unique challenges when grown ex vivo. The future of the field necessitates the inclusion of flow and perhaps additional factors into in vitro culture methods. Future studies investigating endothelial heterogeneity in the developing kidney will aid in forwarding our mission of creating a functional organoid vasculature.Item The Organization, Structure, and Role of Endothelial Cells During Organogenesis(2019-05-28) Daniel, Edward; Buszczak, Michael; Cleaver, Ondine; Carroll, Thomas J.; Dellinger, Michael T.Cellular crosstalk is essential for proper organogenesis. Although originally assumed to be simple conduits for blood, endothelial cells (ECs) actively coordinate organ development through communicating with nearby cell types to generate properly patterned tissues. Despite extensive studies detailing the signaling pathways important for organ development, little is known about how exactly ECs influence these processes. Furthermore, attempts to study ECs in this context have been hampered, in part, by an incomplete understanding of normal endothelial development in specific organs. Here, I characterized the organization, structure, and function of ECs during organ development in the kidney and lung. I first began with an in depth anatomical analysis of the kidney vasculature over the course of fetal development. I showed that the vasculature grows coordinately with the developing epithelium and stroma from the onset of kidney development and that it forms highly stereotyped structures around these other populations. I then carried out transcriptomic analysis of renal ECs and identified many novel EC-enriched genes expressed in the fetal kidney. From this analysis, I focused on the retinoic acid (RA)-catabolizing enzyme, Cyp26b1, which is enriched in ECs in both the kidney and the lung throughout development. Surprisingly, deletion of Cyp26b1 abrogated development of the lung, but not the kidney. Cyp26b1-null lungs exhibited defects in differentiation of the distal epithelium, leading to neonatal lethality. Transcriptomic and functional analyses suggested that the phenotype observed is due to RA-dependent and RA-independent mechanisms. On the other hand, the kidney appeared to be protected from loss of Cyp26b1 by upregulation of Dhrs3 to maintain normal RA levels. Bypassing Dhrs3 by exogenously administering RA interrupted kidney epithelial development only in mice lacking at least one allele of Cyp26b1. This body of work highlights the importance of EC-mediated cellular communication during development and identifies organ-specific mechanisms that regulate this crosstalk.Item Transcription of the Long Noncoding RNA, UpperHand, Is Required for Heart Development(2016-10-03) Anderson, Kelly Marie; Mendell, Joshua T.; Olson, Eric N.; Graff, Jonathan M.; Conrad, Nicholas; Stull, JamesThe basic helix-loop-helix transcription factor HAND2 is an ancestral regulator of heart development and one of four cardiac transcription factors that controls the reprogramming of fibroblasts to cardiomyocytes. Genetic deletion of Hand2 in mice results in hypoplasia of the right ventricle and embryonic lethality. The embryonic expression of Hand2 is tightly regulated by well-characterized upstream enhancer elements, which reside within a super-enhancer delineated by H3K27ac histone modifications. Here, I show that transcription of a long noncoding RNA upstream of Hand2, which I named UpperHand (UPH), is required to maintain the H3K27ac super-enhancer signature and elongation of RNA polymerase II through the Hand2 locus. Blockade of UPH transcription by insertion of a transcription termination cassette, but not knockdown of the mature transcript, abolished Hand2 expression, causing right ventricular hypoplasia and embryonic lethality in mice. Given the substantial number of uncharacterized promoter-associated lncRNAs encoded by the mammalian genome, the UPH-Hand2 regulatory partnership offers a mechanism by which noncoding transcription can establish a permissive chromatin environment necessary for the expression of a neighboring protein-coding gene.Item Transcriptional Regulation of Cardio-Pulmonary Development(2004-01-14) Aiyer, Aparna R.; Srivastava, DeepakOrganogenesis is a complex process, disruption of which results in developmental anomalies. In recent years, genetic dissection of the pathways involved in cardiogenesis, have shown a striking similarity in molecular mechanisms across species. One conserved protein is dHAND, a basic helix-loop-helix (bHLH) transcription factor that is required for normal development of the right ventricle, the pharyngeal arches and limb buds. Loss of dHAND leads to apoptosis in the aforementioned tissues and to embryonic lethality at E10.0. A differential display analysis was performed to identify genes dysregulated in dHAND-/- hearts. Characterization of such genes could potentially shed light on the molecular mechanisms involved in the defects seen in dHAND mutants, while also identifying genes required for normal embryonic development. This thesis represents work on two molecules that were identified in this screen. Bnip3, a hypoxia inducible, pro-apoptotic molecule that can induce mitochondrial damage, was upregulated in the dHAND-/- pharyngeal arches and heart, suggesting a role for mitochondrial damage in the observed apoptosis. I have shown that while Apaf-1, a downstream mediator of mitochondrial-induced apoptosis, is required for the apoptosis observed in dHAND-null pharyngeal and aortic arch mesenchyme, cardiomyocyte apoptosis in dHAND mutants is Apaf-1 independent. Rescue of pharyngeal arches revealed that premature closure of the pharyngeal arch arteries likely contributes to the early lethality observed in dHAND-/- embryos. The mouse ortholog of Bcl-2 associated transcription factor (Btf), which was similar to thyroid hormone receptor associated protein 150 (TRAP150), was down regulated in dHAND mutants. TRAPs are a family of transcriptional co-activators that are required for normal cardiac and embryonic development. Mice lacking Btf showed normal cardiac development, however, the animals had hypercellular lungs and died within 24 hours after birth. Analysis of lung ultrastructure and cell specific markers showed presence of immature secretory cells in the proximal airways of the lung and aberrant proximal-distal patterning. The ectopic presence of stem cell-like proximal epithelial cells (Clara cells) in the distal epithelium may explain the hypercellularity observed in btf-null lungs. These results show that Btf is required for normal maturation and patterning of the pulmonary epithelium and survival of the animal.