Esrrγa regulates nephron and ciliary development by controlling prostaglandin synthesis

Abstract

Cilia are essential for the ontogeny and function of many tissues, including the kidney. Here, we report that transcription factor ERRγ ortholog estrogen related receptor gamma a (Esrrγa) is essential for renal cell fate choice and ciliogenesis in zebrafish. esrrγa deficiency altered proximodistal nephron patterning, decreased the multiciliated cell populace and disrupted ciliogenesis in the nephron, Kupffer's vesicle and otic vesicle. These phenotypes were consistent with interruptions in prostaglandin signaling, and we found that ciliogenesis was rescued by PGE2 or the cyclooxygenase enzyme Ptgs1. Genetic interaction revealed that peroxisome proliferator-activated receptor gamma, coactivator 1 alpha (Ppargc1a), which acts upstream of Ptgs1-mediated prostaglandin synthesis, has a synergistic relationship with Esrrγa in the ciliogenic pathway. These ciliopathic phenotypes were also observed in mice lacking renal epithelial cell (REC) ERRγ, where significantly shorter cilia formed on proximal and distal tubule cells. Decreased cilia length preceded cyst formation in REC-ERRγ knockout mice, suggesting that ciliary changes occur early during pathogenesis. These data position Esrrγa as a novel link between ciliogenesis and nephrogenesis through regulation of prostaglandin signaling and cooperation with Ppargc1a.

Emx2 is an essential regulator of ciliated cell development across embryonic tissues

“…To investigate the hypothesis that emx2 is involved broadly in ciliogenesis, we examined cilia formation across tissues using the emx2 el586/el586 mutant line, referred to subsequently here as emx2 −/− , which encodes a 10 basepair deletion that induces a frameshift after amino acid 74 (of 247) (Supplemental Figure 1B,E,F) (Askary et al, 2017). We performed whole-mount immunofluorescence (IF) to detect acetylated α-tubulin, which marks cilia (Wesselman et al, 2023). Compared to WT embryo controls, emx2 −/− mutants displayed dramatic reductions in cilia formation within the pronephros segments occupied by MCCs (Figure 1C), as well as in distal segment regions where only monociliated cells are formed (Supplemental Figure 1D).…”

“…At 48 hpf, the emx2 morphant, emx2 crispant and emx2 −/− embryos all exhibited pericardial edema and hydrocephalus, indicative of impaired kidney function (Figure 2A). We hypothesized that these emx2 deficient phenotypes could be due to the lack of fluid flow resulting from changes in pronephros development, such as alterations in MCC development which frequently lead to the reduction or abrogation of luminal flow (Marra et al, 2019a; Chambers et al, 2020; Wesselman et al, 2023). To investigate this, we performed WISH analysis using the specific MCC marker odf3b in WT and emx2 deficient embryos.…”

“…Whole-mount IF experiments were completed as previously described (Gerlach and Wingert, 2014; Marra et al, 2017, 2019a; Chambers et al, 2020; Wesselman et al, 2023). For cilia, anti-tubulin acetylated diluted 1:400 (Sigma) was used.…”

“…PGE 2 metabolite quantifications were performed according to the manufacturer’s protocol (Cayman Chemical, Item No. 500141) and adapted from previously reported studies (Chambers et al, 2020; Wesselman et al, 2023). Groups of 25 WT and emx2 MO embryos were pooled, anesthetized and flash frozen in 100% ethanol.…”

“…Using the zebrafish model, we detected the presence of emx2 transcripts across developing ciliated cells and found that emx2 deficiency disrupted cilia formation in the Kupffer’s Vesicle (KV), macula, neuromasts and the kidney. Further, emx2 loss of function led to abnormal renal fluid flow and reductions in kidney MCC and distal late (DL) cell lineages—together, a suite of hallmarks that characterize developmental defects in prostaglandin signaling (Marra et al, 2019a; Chambers et al, 2020; Wesselman et al, 2023). Indeed, we found that emx2 deficiency significantly reduced expression of key prostanoid biosynthesis regulators: the Ppargc1a transcription factor, encoded by peroxisome proliferator-activated receptor gamma, coactivator 1 alpha (ppargc1a), and the Ptgs1 cyclooxygenase enzyme, encoded by prostaglandin-endoperoxide synthase 1 ( ptgs1 ).…”