GSE106085
HNF1B controls epithelial organization and cell polarity during ureteric bud branching and collecting duct morphogenesis
Kidney development depends critically on proper ureteric bud branching giving rise to the entire collecting duct system. The transcription factor HNF1B is required for the early steps of ureteric bud branching. Yet, the molecular and cellular events regulated by HNF1B are poorly understood. We report that specific removal of Hnf1b from the ureteric bud leads to defective cell-cell contacts and apico-basal polarity during the early branching events. High resolution ex vivo imaging combined with a membranous fluorescent reporter strategy show decreased mutant cell-rearrangements during mitosis-associated cell dispersal and severe epithelial disorganisation. Molecular analysis reveals downregulation of Gdnf-Ret pathway components and suggests that HNF1B acts both upstream and downstream of Ret-signaling by directly regulating Gfra1 and Etv5. Subsequently, Hnf1b-deletion leads to massively mispatterned ureteric tree network, defective collecting duct differentiation and disrupted tissue architecture leading to cystogenesis. Consistently, mRNA-seq analysis performed from E15.5 control and mutant kidneys shows that the most impacted genes encode intrinsic cell-membrane components with transporter activity. Our study uncovers a fundamental and recurrring role of HNF1B in epithelial organization during early ureteric bud branching and further patterning and differentiation of the collecting duct system. Article submitted to Development. Authors: Audrey Desgrange, Claire Heliot,Ilya Skovorodkin, Saad U. Akram, Janne Heikkilä, Veli-Pekka Ronkainen, Ilkka Miinalainen I., Seppo J. Vainio and Silvia Cereghini Overall design: We examine the function of Hnf1b during renal branching morphogenesis by conditional inactivation in the UB and its derivatives using the Hoxb7cre reporter mouse line. We crossed mice homozygous for a Hnf1b floxed allele (Hnf1bfl/fl ) (Heliot el al., Development 2013 (4):873-885.) with males HoxB7-Cre;Hnf1bLacZ/+ heterozygous for the Hoxb7-cre and for the Hnf1b null allele (Hnf1blacZ/+), (Barbacci et al., Development 1999 21, 4795-805.) to generate HoxB7-Cre;Hnf1bLacZ/fl embryos designed as mutants.  Hnf1bLacZ/fl and Hnf1b+/fl embryos without the HoxB7-Cre transgene were considered as controls, due to the lack of a phenotype in heterozygous embryos for the Hnf1b LacZ-null allele (Barbacci et al., Development 1999 21, 4795-805.).The two kidneys from E15.5 mutant and control embryos were microdissected from the same litter. This requirement limited the number of samples used to 2 controls and 2 mutants, independently of the sex since we found a similar phenotype in males and females. RNA was extracted by Tryzol using the miRNA mini kit Qiagen for extraction of total and miRNAs. The quality of the RNA samples was assessed on the Agilent Bioanalyzer system using the Agilent RNA 6000 Nano Kit  (Agilent Technologies) and RNA with a RIN higher than 8 was used. 1-2 µg of total RNA were used for mRNA isolation using the Dynabeads® mRNA DIRECT™ Micro Kit (ThermoFisher Scientific). mRNA was digested with RNase III, purified, hybridized and ligated to Ion Adaptors, reverse transcribed, barcoded and amplified, using the Ion Total RNA-Seq Kit v2 (ThermoFisher Scientific).