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.

Prostaglandin signaling regulates nephron segment patterning of renal progenitors during zebrafish kidney development

“…Modulating levels of RA affects the specification of renal progenitors, inducing proximal segment lineage formation over distal, which can be accentuated by the addition of exogenous all-trans RA, while distal fates are induced over proximal by inhibiting endogenous production of RA through the application of the biosynthesis inhibitor N,N-diethlyaminobenzaldehyde (DEAB) (Wingert et al, 2007; Wingert and Davidson, 2011). Through expression profiling and subsequent functional studies, several transcription factors have been mapped as acting downstream of RA signaling to regulate pronephros segmentation and epithelial fate choice, including hepatocyte nuclear factor-1 beta (paralogues hnf1ba and hnf1bb ), iroquois homeobox 3b ( irx3b ), mds1/evi1 complex ( mecom ), single minded family bHLH transcription factor 1a ( sim1a ), and t-box 2 (paralogues tbx2a and tbx2b ), among others (Wingert and Davidson, 2011; Naylor et al, 2013; Li et al, 2014; Kroeger and Wingert, 2014; Cheng and Wingert, 2015; Marra and Wingert, 2016; Marra et al, 2016; Drummond et al, 2017). Despite these advances, the identity of the other essential signals that control renal progenitor fate decisions has remained elusive (Cheng et al, 2015).…”

Nephrotoxin Microinjection in Zebrafish to Model Acute Kidney Injury

“…Zebrafish development occurs ex utero and with minor pigmentation, allowing for direct observation of renal organogenesis and monitoring of responses to external cues 34 . Tools for molecular analysis, such as gene expression 35–37 , have been utilized with high resolution to determine the features of nephrogenesis 38–40 . Experimental manipulations for testing of small molecules through chemical genetics are well established in zebrafish and applicable to the kidney 41–43 .…”

Insights into kidney stem cell development and regeneration using zebrafish

“…Despite these differences, it has been shown that the segment pattern in the nephrons of the zebrafish pronephros [61] and mesonephros [62][63][64] is remarkably similar to the segment pattern in nephrons of other vertebrates, supporting the notion that nephron composition is the evolutionary link across species with diverse forms that inhabit a wide range of environments [60] . This has spurred a number of segmentation studies utilizing the zebrafish pronephros [65][66][67][68][69][70] , which further suggest nephrogenesis mechanisms have been conserved during evolution [60] . The zebrafish pronephros and mesonephros are therefore useful for studying developmental and regenerative pathways.…”