Decoding Kidney Development in Zebrafish: TFAP2A-KCTD15 Genetic Network Regulates Distal Nephron Differentiation

<p>Kidney disease is a devastating condition affecting millions of people worldwide, where over 100,000 patients in the United States alone remain waiting for a lifesaving organ transplant. Concomitant with a surge in personalized medicine, single-gene mutations and polygenic risk alleles have been brought to the forefront as core causes of a spectrum of renal disorders. Due to the increasing prevalence of kidney disease, it is imperative to make substantial strides in the field of kidney genetics. Nephrons, the core functional units of the kidney, are epithelial tubules that act as gatekeepers of body homeostasis by absorbing and secreting ions, water, and small molecules to filter the blood. Each nephron contains a series of proximal and distal segments comprised of distinct stretches of cells each expressing unique solute transporter surface proteins. These ion channels are tightly linked to specific nephron cell identities and confer explicit metabolic functions. To date, the transcriptional code driving nephron patterning, epithelial maturation, solute transporter program activation, and subsequent terminal differentiation of specialized nephron segments remains poorly understood. The embryonic zebrafish provides an ideal platform to dissect the genetic cues governing kidney development. This aquatic vertebrate possesses an architecturally simple two-nephron kidney (pronephros) and a conserved nephron segmentation pattern coupled with high fecundity, <i>ex utero</i> development, and optical transparency for easy visualization of organogenesis. </p><p>Here, we demonstrate that employing the zebrafish to perform genetic studies cultivates the identification of novel nephron regulators. By performing a forward haploid genetic screen, we discovered that the <i>transcription factor AP-2 alpha</i> (<i>tfap2a) </i>directs a genetic regulatory network that promotes the terminal differentiation of the distal early/thick ascending limb (DE/TAL) and distal late/distal convoluted tubule (DL/DCT) nephron segments. Tfap2a<i> </i>operates a circuit consisting of <i>tfap2b</i> and <i>irx1a</i> to activate the expression of distal nephron solute transporter genes <i>clcnk</i>, <i>slc12a1</i>, <i>kcnj1a.1</i>, and <i>slc12a3</i>. In a separate study, we reported for the first time that KCTD15 paralogs, <i>kctd15a</i> and <i>kctd15b,</i> are key components of the <i>tfap2a</i> distal nephron network. By employing CRISPR-Cas9 and knockdown strategies, we determined <i>kctd15a/b</i> loss primes nephron cells to adopt a DE/TAL cell signature. Mechanistically, <i>kctd15a/b</i> restricts DE/TAL differentiation by repressing Tfap2a activity in developing nephrons. Further interrogation of this signaling axis revealed Tfap2a can reciprocally promote <i>kctd15</i> transcription. Our data presents a new transcription factor-repressor feedback module where nephron segment fate is controlled by precise regulation of Tfap2a-Kctd15 kinetics. Lastly, we identified two discrete roles for the GRHL2 paralogs, <i>grhl2a</i> and <i>grhl2b</i>, in solute transporter program activation and epithelial maturation during distal nephron development. Loss of <i>grhl2a/b</i> produced developmental abnormalities in both the pronephric duct and otic vesicle. In addition to reductions in <i>clcnk </i>and <i>slc12a3 </i>expression, <i>grhl2a/b</i>-deficient nephrons exhibited defects in cell polarity, ciliogenesis, and basement membrane integrity. Significantly, <i>grhl2a/b</i> functions as part of the Tfap2 genetic network and promotes expression of <i>tfap2b</i> in the distal nephron. Taken together, key developmental insights from our zebrafish studies will support the assembly of the genetic blueprint required to fashion a nephron, and in turn support efforts to advance kidney organoid technology, further develop precision medicine, and deepen our understanding of congenital renal syndromes.</p>