Cadherin adhesion complexes direct cell aggregation in the epithelial transition of Wnt-induced nephron progenitor cells

The aggregation of nephron progenitor cells (NPC) is required to form the nephron precursor renal vesicle (RV) as they undergo the mesenchymal-to-epithelial transition (MET). Canonical Wnt signaling regulates the MET of NPCs via the effector molecule ß-catenin. ß-catenin acts as a transcriptional co-activator during the differentiation of NPCs, however, if it has any role as an adhesion regulating molecule via the cadherin-catenin complex is currently unknown. Using in vitro NPC culture system with gene editing and modulated Wnt signaling input, we investigated the morphological transition of NPCs and its underlying mechanism modeling the initial step of the MET in vivo. Increasing Wnt signaling input with the small molecule agonist CHIR resulted in the aggregation of NPCs similar to the generation of the nephron anlagen. By removing ß- and a-catenin from the NPCs, NPCs are sorted out from these aggregates, demonstrating the key role of the cadherin-catenin complex in the aggregation. ß-catenin was essential for the induction, however, the removal of a-catenin did not affect the transcriptional profile of NPCs. Removal of extracellular Ca2+ resulted in the transient loss of cell-cell contacts suggesting the role of cadherins in the aggregation process. The analysis of in vitro bulk RNA-seq cadherin expression profile matched the in vivo cadherin expression profile determined by single-cell RNA-seq. The combined removal of pre-existing and inducible cadherins phenocopied the results of ß- and a-catenin KO experiments highlighting the crucial role of the cadherin-catenin complex by multiple lines of evidence. Notably, the induction of NPCs is independent of their aggregation. The in vitro modeling of nephron development provides a mechanistic understanding how cell adhesion is regulated via the cadherin-catenin complex during nephrogenesis. Overall design: Primary nephron progenitors (NPCs)were isolated using the methods published in Brown et al., 2015 and were cultured in NPEM media. NPCs were then transfected using Lipofectamine™ MessengerMAX™ Transfection Reagent (Thermofisher, Cat # LMRNA015) with gene editing components (sgRNAs) and mCherry mRNA to distinguish transfected cells. Cells were cultured and lifted for FAC sorting. RNA was extracted from FAC sorted samples using mCherry+ NPCs were sorted on BD SORP FACS Aria IIu into RLT buffer with 1:100 beta-mercaptoethanol prior to RNA isolation. RNA was isolated using Qiagen RNeasy Micro Kit (74004). Total RNA integrity was determined using Agilent Bioanalyzer or 4200 Tapestation. Library preparation was performed with 10ng of total RNA with a Bioanalyzer RIN score greater than 8.0. ds-cDNA was prepared using the SMARTer Ultra Low RNA kit for Illumina Sequencing (Takara-Clontech) per manufacturer's protocol. cDNA was fragmented using a Covaris E220 sonicator using peak incident power 18, duty factor 20%, cycles per burst 50 for 120 seconds. cDNA was blunt ended, had an A base added to the 3' ends, and then had Illumina sequencing adapters ligated to the ends. Ligated fragments were then amplified for 12-15 cycles using primers incorporating unique dual index tags. Fragments were sequenced on an Illumina NovaSeq-6000 using paired end reads extending 150 bases.