The Ca2+-binding protein recoverin may regulate visual transduction in retinal rods and cones, but its functional role and mechanism of action remain controversial. We compared the photoresponses of rods from control mice and from mice in which the recoverin gene was knocked out. Our analysis indicates that Ca2+-recoverin prolongs the dark-adapted flash response and increases the rod's sensitivity to dim steady light. Knockout rods had faster Ca2+ dynamics, indicating that recoverin is a significant Ca2+ buffer in the outer segment, but incorporation of exogenous buffer did not restore wild-type behavior. We infer that Ca2+-recoverin potentiates light-triggered phosphodiesterase activity, probably by effectively prolonging the catalytic activity of photoexcited rhodopsin.

Over 100 rhodopsin mutation alleles have been associated with autosomal dominant retinitis pigmentosa (ADRP). These mutations appear to cause photoreceptor cell death through diverse molecular mechanisms. We show that K296E, a rhodopsin mutation associated with ADRP, forms a stable complex with arrestin that is toxic to mouse rod photoreceptors. This cell death pathway appears to be conserved from flies to mammals. A genetics approach to eliminate arrestin unmasked the constitutive activity of K296E and caused photoreceptor cell death through a transducin-dependent mechanism that is similar to light damage. Expressing K296E in the arrestin/ transducin double knock-out background prevented transducin signaling and led to substantially improved retinal morphology but did not fully prevent cell death caused by K296E. The adverse effect of K296E in the arrestin/transducin knock-out background can be mimicked by constant exposure to low light. Furthermore, we found that arrestin binding causes K296E to mislocalize to the wrong cellular compartment. Accumulation of stable rhodopsin/arrestin complex in the inner segment may be an important mechanism for triggering the cell death pathway in the mammalian photoreceptor cell.

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Nephron progenitor cells (NPCs) self-renew and differentiate into nephrons, the functional units of the kidney. Here we report manipulation of p38 and YAP activity creates a synthetic niche that allows the long-term clonal expansion of primary mouse and human NPCs, and induced NPCs (iNPCs) from human pluripotent stem cells. Cultured iNPCs resemble closely primary human NPCs, generating nephron organoids with abundant distal convoluted tubule cells, which are not observed in published kidney organoids. The synthetic niche reprograms differentiated nephron cells into NPC state, recapitulating the plasticity of developing nephron in vivo. Scalability and ease of genome-editing in the cultured NPCs allow for genome-wide CRISPR screening, identify-ing novel genes associated with kidney development and disease. A rapid, efficient, and scalable organoid model for polycystic kidney disease was derived directly from genome-edited NPCs, and validated in drug screen. These technological platforms have broad applications to kidney development, disease, plasticity, and regeneration.

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