Rhythmic nephron formation in the developing kidney [E14_shRNA_bulkRNAseq]

The mammalian kidney achieves massive parallelization of function by exponentially duplicating nephron-forming niches during development. Each niche caps a tip of the ureteric bud epithelium (the future urinary collecting duct tree) as it undergoes branching morphogenesis, while nephron progenitors within niches balance self-renewal and differentiation to early nephron cells. Nephron formation rate approximately matches branching rate over a large fraction of mouse gestation, yet the nature of this apparent pace-maker is unknown. Here we correlate spatial transcriptomics data with branching 'life-cycle' to discover rhythmically alternating signatures of nephron progenitor differentiation and renewal across Wnt, Hippo/Yap, retinoic acid (RA), and other pathways. Our data bring temporal resolution to the renewal vs. differentiation balance in the nephrogenic niche and inform new strategies to achieve self-sustaining nephron formation in synthetic human kidney tissues. Overall design: RNA-seq of E14 wild-type CD1 kidneys transduced with AAV2 carrying cag driven mir30 shRNA knockdown cassettes an upstream mCherry transduction reporter prior to culturing for 72 hours in a 3D-culture format. Knockdown conditions include shRNA sequences targeting the Mylk gene (Mylk-shRNA), the Rock1 and Rock2 genes (Rock1/2-shRNA), or a scrambled negative control (scramble-shRNA). Samples include FACS enriched mCherry-negative and mCherry-positive fractions of cells isolated from pooled kidneys. Kidneys for each replicate come from the same litter.