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Examination of the effects of siRNA to six2 on embryonic kidneys ex vivo

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Item Summary

Title: Examination of the effects of siRNA to six2 on embryonic kidneys ex vivo
Authors: Phillips, Nora Alicia
Keywords: kidneys
Issue Date: May 2011
Publisher: [Honolulu] : [University of Hawaii at Manoa], [May 2011]
Abstract: Abnormal kidney development can have lifelong consequences that predispose one to increased risk of kidney disease and its associated morbidity and mortality. Evidence suggests that reduced nephron endowment at birth may be a contributing factor in the pathogenesis of renal disease. Studying the development of the embryonic kidney can lead to understanding one of the processes that may lead to congenitally-associated kidney disease.
Our lab previously identified a heritable mutation in the Brachyrrhine (Br) mouse that displays a severe renal hypoplasia phenotype and thus can provide a model of abnormal kidney development. It was found that the Br mouse carries a mutation affecting the embryonic expression of the gene sine oculis 2 (six2) indicating the critical role of six2 in renal morphogenesis. By studying the Br mouse, more can be learned about renal development. In this thesis work, RNAi directed towards six2 expression was used to knockdown six2 expression to determine what the role of six2 is in renal organogenesis. Although it is known that six2 plays a role in development of the renal organs in the embryo, it is unknown what the specific mechanisms and molecular players are in this process.
The present study was designed to identify cellular mechanisms that drive successful nephron differentiation and to examine the genetic basis of reduced nephron endowment, particularly with respect to six2 expression. The specific aims of this study are (1) to develop an organotypic kidney explant culture system that facilitates the study of renal organogenesis ex vivo and (2) to examine the effects on UB branching caused by reduced expression of six2 in the Br mouse (3) to utilize RNAi technology to knockdown six2 expression in order to examine the consequences on UB branching in the developing embryonic kidney. I hypothesize that siRNA-induced knockdown of six2 in kidney explant culture system would produce effects similar to those seen in the Br kidneys including a reduced level of six2 expression, renal hypoplasia and a reduced number of ureteric buds (UB) when compared to untreated wildtype controls.
Because the degree of UB branching during nephrogenesis could determine nephron endowment for life, it is crucial that the embryonic kidney develops enough buds during embryogenesis to function properly in the adult. Based on the pattern of six2 expression and the number of UB tips observed among the wild-type, the heterozygous Br/+, and the homozygous Br/Br kidneys, results indicate that reduced six2 expression is associated with reduced UB branching and thus the deficient nephron number in the developing kidney. This is further supported by the knockdown of six2 transcription by siRNA, leading to a significant reduction in the amount of six2-expressing cells and the number of UB tips and branching morphogenesis. Although incubation with siRNA to six2 provided for these results, using non-targeting siRNA or siRNA to other genes produced results similar to what is observed in siRNA to six2. I believe the down-regulation in six2 expression observed was unlikely due to apoptotic cell death because TUNEL staining was not observed in explants treated with siRNA reagents, but my experiments cannot account for the reduction of six2 expression seen in explants treated with siRNA not specific to six2.
(1) Embryonic E12.5 and E13.5 kidney explants were cultured and stained in a variety of ways to determine the optimal growing conditions for the developing kidney. (2) Following development of a successful organ culture, wild-type kidney explants as well as organs from Br animals were allowed to grow for 72h. Explants were then either stained for six2 and calbindin and examined for UB branching or evaluated for six2 expression using qRT-PCR (3) In the subsequent experiment, gene silencing technology via siRNA was used to inhibit the expression of six2 transcription early in the developing kidney culture. The effect of siRNA to six2 on UB branching was determined using immunohistochemistry or qRT-PCR. Explants were incubated with vehicle or 2-3μM of siRNA oligos selective to six2. After 72h explants were either processed and stained for six2 and calbindin and UB tips were counted or explants were harvested and processed for quantitative PCR analysis to determine the level of six2 mRNA expression.
Results (1) Explants grown in the developed culture system were able to grow in a comparable way to explants dissected and immediately processed and stained for UB branching at the same age. During time course trials, left and right kidneys from the same animal exhibited growth from 0h thru 72h in the culture system. (2) Cultured kidney rudiments prepared from the Br/+ and Br/Br animals were substantially smaller in size and exhibited fewer UB tips compared to those from the wild-type explants suggesting a disruption of kidney development in the absence of six2 expression. In the wild-type tissue, six2 was expressed predominantly in the metanephric mesenchyme along the nephrogenic zone, typical of the developing kidney. The expression of six2 corresponds well with the branching pattern and number of UB tips, indicating successful kidney development in our preparation. In contrast, six2 expression was markedly reduced in Br/+ explants and absent from Br/Br kidneys. Correspondingly, the number of UB tips decreases significantly in the Br/+ and are least amongst Br/Br kidneys. (3) Explants that were treated with exogenous six2-siRNA oligos exhibit a significant decrease in six2-positive cells, relative to the non-siRNA controls. There was a significant amount of depletion in UB tips and reduction in six2 transcript level in response to the siRNA treatment.
In summary, this study suggests that six2 functions to influence embryonic renal development and that its absence leads to reduced UB numbers and impaired nephron differentiation. Our results strongly implicate the role of six2 in early nephrogenesis and suggest that six2 may constitute as the genetic basis for renal nephron endowment.
Description: M.S. University of Hawaii at Manoa 2011.
Includes bibliographical references.
Appears in Collections:M.S. - Developmental and Reproductive Biology

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