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Regulation of kidney field specification by transcriptional regulation of microRNAs

ABSTRACT: The transcriptional events driving specification of the kidney field have been well characterized. However, it remains unknown how the initial field size is established, patterned, and proportioned. Lhx1 is a transcription factor expressed in the kidney anlage and is required for specification of the kidney field, but few Lhx1 interacting cofactors or downstream targets have been identified. By tandem-affinity purification, we isolated Furry (FRY), a multifunctional protein that acts as a transcriptional co-repressor of microRNAs. We found that Xenopus embryos depleted of fry exhibit loss of the kidney field, phenocopying the lhx1 depleted animals. In addition, we demonstrated a synergism between Fry and Lhx1, identified candidate microRNAs regulated by the protein pair, and show at least two microRNA clusters influence specification of the kidney field. Therefore, our data shows that a tissue-specific transcription factor, Lhx1, can interact with a broadly expressed microRNA repressor, Fry, to establish the kidney field.

ORGANISM(S): Xenopus laevis

PROVIDER: GSE100434 | GEO | 2018/10/10

REPOSITORIES: GEO

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Project description:The goal of this study is to investigate the molecular mechanism of lhx1 on regulation of pronephros formation during the early embryonic development. In the vertebrate embryo the kidney is derived from the intermediate mesoderm. The LIM-class homeobox transcription factor lhx1 is expressed early in the intermediate mesoderm and is one of the first genes to be expressed in the nephric mesenchyme. The animal cap cells can be induced by treatment of activin and retinoic acid to differentiate into pronephros tissue. In this study we investigated the role of Lhx1 in differentiation of pronephros by depleting lhx1 in the organ culture system. We generated the gene expression profile of early pronephros tissue, and demonstrated that expression of genes from all the kidney domains is affected by the absence of lhx1. Taken together our results highlight an essential role for Lhx1 in pronephros formation. lhx1 is involved in driving specification of intermediate mesoderm into nephrogenic mesenchyme. Lhx1 is initially expressed throughout the entire intermediate mesoderm. To determine the role of lhx1 pronephros formation, we performed a microarray analysis using an explant culture system. Xenopus tissue explants can be surgically isolated and cultured under specific conditions to be driven towards many distinct tissue types. Formation of pronephric cell fates is induced by culturing isolated explants in the presence of Activin and RA (AcRA). Treatment of dissected explants of stage 9 blastulae embryos with 10ng/ml Activin and 1x10-4 M retinoic acid can induce differentiation of the pluripotent ectoderm into pan-kidney tissue. For this experiment, both blastomeres of 2-cell embryos were injected with a total of 800pg lhx1 DEED-AS. Explants were dissected and treated with AcRA and expression of pax8 at stage 15 (based on timing of paired control whole embryos) was analyzed. We observed a lack of induction of pax8 expression in lhx1-depleted explants under AcRA treatment conditions in which expression of this gene is normally induced. Based on this observation, microarray analysis was carried out to identify genes whose expression is affected by the absence of lhx1. Explants of injected embryos with 800pg of lhx1 DEED-AS were dissected, treated with pronephric tissue inductive conditions (AcRA) and harvested after 24 hours incubation at 14C (Fig. S5B). The sibling control embryos reached stage 12.5. Explants from uninjected embryos +AcRA and -AcRA as well as explants from DEED injected embryos -AcRA were also harvested. Approximately 12 caps were pooled for each RNA preparation and the analysis was performed using triplicates.

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Regulation of kidney field specification by transcriptional regulation of microRNAs

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