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.

Keywords: Corpuscles of Stannius; Kidney; Nephrogenesis; Nephron; Pronephros; Proximal tubule; Retinoic acid; Segmentation; Zebrafish; sim1a.

Figure 1. sim1a expression is dynamic during nephrogenesis

(A) Schematics depict the IM and PM domains in a young zebrafish embryo. At 24 hpf, the zebrafish pronephros consists of two segmented nephrons as indicated by the colored regions. Corresponding numbers indicate the boundaries of each segment respective to the somites. (B) Illustrations denote the developmental time course of sim1a expression, which first appears within the IM (pax2a+ domain) at the 2 ss. Dark purple indicates strong expression, while light purple signifies weak expression. (C) Flat mounted embryos staged at young developmental time points were assayed by WISH for the kidney markers pax2a, sim1a, or mecom (purple). The somites and proximal renal progenitors were labeled by dlc or myod1 (red). Solid lines (purple and red) demarcate renal progenitors regions where high transcript expression was observed. The dotted line (purple) indicates a weak expression domain, with line color corresponding to the gene identity indicated in figure. (D) Embryos at older stages were analyzed by WISH for sim1a expression (purple). Embryo anterior is located to the left in all panels. Abbreviations: CS – corpuscles of Stannius; DE – distal early; DL – distal late; hpf – hours post fertilization; IM – intermediate mesoderm; N – neck; P – podocytes; PCT – proximal convoluted tubule; PD – pronephric duct; PM – paraxial mesoderm; PST – proximal straight tubule; ss – somite stage; WISH – whole mount in situ hybridization.

Figure 2. sim1a morphants display drastic developmental defects and abnormal kidney function

(A) A live time course shows the typical morphological phenotypes exhibited by sim1a morphants at different time points – open arrowhead demarcates darkened regions within the head; solid arrowhead indicates small head and eye phenotypes; solid arrow marks the presence of edema. (B) Kidney function in sim1a morphants was analyzed by a 40 kDa dextran-FITC uptake assay where injections were performed at approximately 38 hpf. sim1a morphants displayed partial PCT reabsorption (dorsal inset view) and reduced renal clearance of dextran-FITC over time, with persistent fluorescence throughout the head, trunk and pericardium when compared to wild-types. White lines indicate either the PCT domain in relation to the somites or the PCT morphology at given time points. Embryo anterior is to the left. Abbreviations: hpf – hours post fertilization; hpi – hours post injection; PCT – proximal convoluted tubule.

Figure 3. The loss of sim1a results in the abrogation of the PST segment and the CS

(A) Segmentation patterning changes within the pronephros following sim1a knockdown were independently assessed by the utilization of MOs targeting the sim1a ATG and splice sites. Control 5 bp ATG mmMO injections were performed to further confirm the specificity of the sim1a translation-targeting MO. WISH of embryos at 24 hpf indicates the expression of specific kidney segment markers (purple) in relation to the somites, which were labeled with smyhc1 (red). Segment domains are further demarcated by black lines with corresponding somite numbers as indicated. Embryo anterior is to the left. (B) Diagram indicates the dominant segmentation phenotypes displayed by sim1a morphants in comparison to the wild-type controls at 24 hpf. Colored bars indicate the respective domains of each nephron segment in relation to the somite numbers located above. The striped regions within the colored bars signify areas with lower transcript expression. Abbreviations: CS – corpuscles of Stannius; DE – distal early; DL – distal late; hpf – hours post fertilization; mmMO – mismatch morpholino; MO – morpholino; PCT – proximal convoluted tubule; PST – proximal straight tubule; WISH – whole mount in situ hybridization.

Figure 4. sim1a overexpression is partially sufficient to promote the PST and CS while inhibiting PCT fate

(A) Wild-type embryos injected with 200 pg of sim1a cRNA were assayed by WISH at 24 hpf with specific kidney segment markers, (purple), and with smyhc1 to demarcate the somites (red). Black lines indicate segment domains relative to somite numbers. Embryo anterior is to the left. Quantitative analyses utilizing the two-tailed Student’s t-test were performed to assess changes in the expression domains of (B) slc20a1a, (C) trpm7, (D) the size of individual stc1+ CS cells and (E) the size of the stc1+ area. (F) Summary of the sim1a overexpression phenotype. Colored bars indicate segment domains in relation to the corresponding somite numbers indicated above. Abbreviations: cRNA – capped RNA; CS – corpuscles of Stannius; DE – distal early; DL – distal late; hpf – hours post fertilization; OE – overexpression; PCT – proximal convoluted tubule; PST – proximal straight tubule; WISH – whole mount in situ hybridization.

Figure 5. RA mediates the sim1a domain within the pronephric renal progenitor field

(A) Wild-type embryos were treated with either a DMSO control at 75% epi, exogenous RA (1×10⁻⁸ M, denoted as + RA) at 50-60% epi, or 1.67×10⁻⁵ M DEAB at 75% epi. Groups were treated with their respective chemical until the 5-7 ss and then analyzed for sim1a expression (purple) by WISH at the 18 and 28 ss. Transcripts encoding smyhc1 (red) mark the somites. Black lines with corresponding numbers indicate segment domains and somites, respectively. Embryo anterior is located to the left. Abbreviations: DEAB – 4-diethylaminobenzaldehyde; epi – epiboly; RA – retinoic acid; ss – somite stage; WISH – whole mount in situ hybridization.

Figure 6. Increased RA levels promote the proximalization of the nephron but are not sufficient to rescue PST formation in sim1a morphants

(A) Wild-type embryos or (B) sim1a morphants were treated with either a 1×10⁻⁷ M DMSO control, a low dose of RA (1×10⁻⁸ M; denoted as + RA), or with a higher RA concentration of 1×10⁻⁷ M (denoted as ++ RA). Both the DMSO control and ++ RA dose were given from the 90% epi to 5-7 ss while the + RA dose was applied from 90% epi to the 15 ss. Changes to nephron segmentation after the addition of RA was visualized by WISH in embryos at 24 hpf. The expression patterns of segment specific transcripts (purple) and smyhc1 transcripts, which marks the somites (red), are shown. Black lines with corresponding somite numbers were used to further illustrate segment domains. Embryo anterior is located to the left. (C) Schematic delineating the effects of low (+) and high (++) RA concentrations on nephron segmentation in wild-type embryos and sim1a morphants at 24 hpf. Nephron segments are represented by colored bars in relation to their corresponding somite numbers. Regions with lower transcript expression are indicated by stripes. Abbreviations: CS – corpuscles of Stannius; DE – distal early; DL – distal late; epi – epiboly; hpf – hours post fertilization; PCT – proximal convoluted tubule; PST – proximal straight tubule; RA – retinoic acid (+ RA – low; ++ RA – high dose); ss – somite stage; WISH – whole mount in situ hybridization.

Figure 7. The inhibition of RA biosynthesis by DEAB induces distal fates but fails to rescue CS formation in sim1a morphants

DMSO control or 1.67×10⁻⁵ M DEAB treatments of (A) wild-types or (B) sim1a morphant embryos from 75% epi to the 5-7 ss were performed. WISH analysis in embryos at 24 hpf for distal segment markers (slc12a1, stc1, and slc12a3) (purple) and the somites (smyhc1) (red). Black bars represent segment domains in correlation to somite numbers. Embryo anterior is located to the left. Insets show dorsal view of the CS. Abbreviations: CS – corpuscles of Stannius; DE – distal early; DEAB – 4-diethylaminobenzaldehyde; DL – distal late; epi – epiboly; hpf – hours post fertilization; ss – somite stage; WISH – whole mount in situ hybridization.

Figure 8. The roles of sim1a during nephrogenesis in the zebrafish embryo

Interactions between RA, sim1a, and additional transcription factors and mechanisms are vital for establishing normal nephron segmentation pattern by 24 hpf in the zebrafish pronephros. sim1a is essential for the formation of both the PST and the CS. It is possible that sim1a mediates the PCT/PST boundary by inhibiting the PCT domain as well. RA could also be modulating the expression of sim1a during the establishment of the PST while acting to restrict sim1a during CS formation. While sim1a appears to function downstream of RA, interactions between RA and sim1a may be indirect or direct during nephrogenesis. Abbreviations: CS – corpuscles of Stannius; DE – distal early; DL – distal late; hpf – hours post fertilization; N – neck; P – podocytes; PCT – proximal convoluted tubule; PD – pronephric duct; PST – proximal straight tubule; RA – retinoic acid.