Nephron proximal tubule patterning and corpuscles of Stannius formation are regulated by the sim1a transcription factor and retinoic acid in zebrafish

Abstract

The mechanisms that establish nephron segments are poorly understood. The zebrafish embryonic kidney, or pronephros, is a simplified yet conserved genetic model to study this renal development process because its nephrons contain segments akin to other vertebrates, including the proximal convoluted and straight tubules (PCT, PST). The zebrafish pronephros is also associated with the corpuscles of Stannius (CS), endocrine glands that regulate calcium and phosphate homeostasis, but whose ontogeny from renal progenitors is largely mysterious. Initial patterning of zebrafish renal progenitors in the intermediate mesoderm (IM) involves the formation of rostral and caudal domains, the former being reliant on retinoic acid (RA) signaling, and the latter being repressed by elevated RA levels. Here, using expression profiling to gain new insights into nephrogenesis, we discovered that the gene single minded family bHLH transcription factor 1a (sim1a) is dynamically expressed in the renal progenitors—first marking the caudal domain, then becoming restricted to the proximal segments, and finally exhibiting specific CS expression. In loss of function studies, sim1a knockdown expanded the PCT and abrogated both the PST and CS populations. Conversely, overexpression of sim1a modestly expanded the PST and CS, while it reduced the PCT. These results show that sim1a activity is necessary and partially sufficient to induce PST and CS fates, and suggest that sim1a may inhibit PCT fate and/or negotiate the PCT/PST boundary. Interestingly, the sim1a expression domain in renal progenitors is responsive to altered levels of RA, suggesting that RA regulates sim1a, directly or indirectly, during nephrogenesis. sim1a deficient embryos treated with exogenous RA formed nephrons that were predominantly composed of PCT segments, but lacked the enlarged PST observed in RA treated wild-types, indicating that RA is not sufficient to rescue the PST in the absence of sim1a expression. Alternately, when sim1a knockdowns were exposed to the RA inhibitor diethylaminobenzaldehyde (DEAB), the CS was abrogated rather than expanded as seen in DEAB treated wild-types, revealing that CS formation in the absence of sim1a cannot be rescued by RA biosynthesis abrogation. Taken together, these data reveal previously unappreciated roles for sim1a in zebrafish pronephric proximal tubule and CS patterning, and are consistent with the model that sim1a acts downstream of RA to mitigate the formation of these lineages. These findings provide new insights into the genetic pathways that direct nephron development, and may have implications for understanding renal birth defects and kidney reprogramming.