Regulation of Kidney Field Size by Transcriptional Regulation of microRNAs

BERMUDEZ ESPIRITU, EUGENEL; PHUA, YU LENG; SANTOS, DMCS; HUKRIEDE, NEIL A.; CIRIO, MARIA CECILIA; HO, J

Chicago, IL

Congreso; Kidney Week; 2016

American Society of Nephrology

Background: Renal progenitors arise from the intermediate mesoderm (IM), a germlayer that lies between the lateral & paraxial mesoderm. The transcription factor lhx1 isexpressed in the lateral & IM. Eventually lhx1 becomes restricted to the IM becomingone of the earliest genes expressed in the kidney field. Lhx1 is essential to specify kidney;the molecular mechanism remains undefined and very few targets have been identified.Methods: In Xenopus kidney cells, we identified proteins using tandem-affinity purified(TAP) proteins that could interact with constitutively active form of Lhx1 (TAP-LL-CA) ¬ a version lacking the C-terminal region (TAP-LL-delC). The C-terminal region of Lhx1contains the functional domains necessary to confer transcriptional activity. Furry (Fry)was identified as a potential Lhx1 interacting protein that bound only to the TAP-LL-CA.In Xenopus, Fry acts as a transcriptional co-repressor of microRNA (miRNA) expression.We depleted Fry & Lhx1 utilizing antisense oligonucleotides & visualized their effectson the kidney by staining for pronephric kidney markers. We conducted miRNA deepsequencing on these embryos. miRNA mimics were injected into embryos and their effectson the kidney were analyzed as before.Results: Here we show fry is expressed in the IM & pronephric anlagen. We determinedthat embryos depleted of fry show loss of the kidney field, phenocopying the lhx1 depletion.We observed a synergistic requirement of these two proteins in the IM indicating the potential interaction in the specification of the kidney field. To gain further insight of themechanism for Fry & Lhx1 in the kidney field, we identified miRNAs regulated by theproteins using miRNA deep sequencing, & perturbed specification of the kidney field byexpressing mimics for the identified miRNAs.Conclusions: These results support a role of a Lhx1/Fry complex in specification ofthe kidney field by regulation of miRNAs expression.Funding: NIDDK Support, Other NIH Support ‑ 5T32DK061296-13