Epithelial cell fate in the nephron tubule is mediated by the ETS transcription factors etv5a and etv4 during zebrafish kidney development

Abstract

Kidney development requires the differentiation and organization of discrete nephron epithelial lineages, yet the genetic and molecular pathways involved in these events remain poorly understood. The embryonic zebrafish kidney, or pronephros, provides a simple and useful model to study nephrogenesis. The pronephros is primarily comprised of two types of epithelial cells: transportive and multiciliated cells (MCCs). Transportive cells occupy distinct tubule segments and are characterized by the expression of various solute transporters, while MCCs function in fluid propulsion and are dispersed in a “salt-and-pepper” fashion within the tubule. Epithelial cell identity is reliant on interplay between the Notch signaling pathway and retinoic acid (RA) signaling, where RA promotes MCC fate by inhibiting Notch activity in renal progenitors, while Notch acts downstream to trigger transportive cell formation and block adoption of an MCC identity. Previous research has shown that the transcription factor ets variant 5a (etv5a), and its closely related ETS family members, are required for ciliogenesis in other zebrafish tissues. Here, we mapped etv5a expression to renal progenitors that occupy domains where MCCs later emerge. Thus, we hypothesized that etv5a is required for normal development of MCCs in the nephron. etv5a loss of function caused a decline of MCC number as indicated by the reduced frequency of cells that expressed the MCC-specific markers outer dense fiber of sperm tails 3b (odf3b) and centrin 4 (cetn4), where rescue experiments partially restored MCC incidence. Interestingly, deficiency of ets variant 4 (etv4), a related gene that is broadly expressed in the posterior mesoderm during somitogenesis stages, also led to reduced MCC numbers, which were further reduced by dual etv5a/4 deficiency, suggesting that both of these ETS factors are essential for MCC formation and that they also might have redundant activities. In epistatic studies, exogenous RA treatment expanded the etv5a domain within the renal progenitor field and RA inhibition blocked etv5a in this populace, indicating that etv5a acts downstream of RA. Additionally, treatment with exogenous RA partially rescued the reduced MCC phenotype after loss of etv5a. Further, abrogation of Notch with the small molecule inhibitor DAPT increased the renal progenitor etv5a expression domain as well as MCC density in etv5a deficient embryos, suggesting Notch acts upstream to inhibit etv5a. In contrast, etv4 levels in renal progenitors were unaffected by changes in RA or Notch signaling levels, suggesting a possible non-cell autonomous role during pronephros formation. Taken together, these findings have revealed new insights about the genetic mechanisms of epithelial cell development during nephrogenesis.

The zebrafish kidney mutant zeppelin reveals that brca2/fancd1 is essential for pronephros development

“…Previous research from our lab and others has indicated the significant role of RA and Notch in patterning proximal cell fates within the kidney, including the podocyte lineage (Wingert et al, 2007; Bollig et al, 2009; Wingert and Davidson, 2011; Li et al, 2014; Cheng and Wingert, 2015; Marra and Wingert, 2016). Thus, one possible explanation for the dearth of podocytes in zep was a local reduction in RA levels or Notch signaling.…”

“…Therefore, we next examined whether a deficiency of RA might underlie the abrogation of podocyte development in zep . Previous studies have shown that the nephron patterning defects caused by RA deficiency can be rescued by the addition of exogenous all-trans RA to the embryo media beginning at gastrulation stages (Wingert and Davidson, 2011; Li et al, 2014; Cheng and Wingert, 2015; Marra and Wingert, 2016; Marra et al, 2016). To test whether zep fail to form podocytes due to insufficient RA levels, heterozygous zep zebrafish were mated, and their clutches were treated with varying levels of exogenous all-trans RA to interrogate whether supplementation of this metabolite was sufficient to rescue podocyte numbers in zep mutant embryos.…”

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

“…Compared to WTs, exposure to 1 mM 4-HPR led to an expansion of the PCT, caudal shift of the DE, and a dramatic expansion of the interval between these segments where the PST normally emerges, suggestive of an expanded PST segment (Figure 1D) (Poureetezadi et al, 2014). The observation that molecules which impact the RA pathway were flagged as hits in the screen provided an important positive control for our experimental system, given the well-established effects of RA levels on renal progenitors (Wingert et al, 2007; Wingert and Davidson, 2011; Li et al, 2014; Cheng and Wingert, 2015; Marra and Wingert, 2016; Drummond et al, 2017). …”

“…MO sequences and dosages used were: irx3b 5'-ATAGCCTAGCTGCGGGAGAGACATG-3', 1 ng (Wingert and Davidson, 2011); ptger2a MO1 5'-GATGTTGGCATGTTTGAGAGCATGC-3', 3 ng (North et al, 2007); ptger2a MO2 5'-ACTGTCAATACAGGTCCCATTTTC-3', 1.6 ng (North et al, 2007); ptger2a MO3 splice 5'-CAATAAATCTTACTATTAACGGCAG-3', 3 ng; ptger2a MO4 splice 5'-ATGTACACACGGATCTG-AAGAGAAG-3', 3 ng; ptger4a MO1 5'-CGCGCTGGAGGTCTGGAG-ATCGCGC-3', 3 ng (North et al, 2007); ptger4a MO2 5'-CACGGTGGGCTCCATGCTGCTGCTG-3', 3 ng (Cha et al, 2006); ptger4a MO3 splice 5'-CCTGGAACTTACAACAAGCGGGATT-3', 3 ng; ptger4a MO4 splice 5'-TGAGAAACA-CCTGGACCTGCCAGAA-3', 3 ng; ptgs1 MO 5'-TCAGCAAAAAGTTACACTCTCTCAT-3’, 3 ng (North et al, 2007); ptgs2a MO 5'-AACCAGTTTATTCATTCCAGAAGTG-3', 3 ng (Grosser et al, 2002); ptgs1 MO splice 5'-AACTTTCATTGCTC-ACCTCTCATTG-3', 2 ng; ptgs2a MO splice 5'-ATTCAACTTA-CACAACAGGATATAG-3', 2 ng (Yeh et al, 2009), sim1a MO 5'-TCGACTTCTCCTTCATGCTCTACGG-3', 1 ng (Cheng and Wingert, 2015). To assess knockdown efficacy, RT-PCR and sequence analysis was performed as previously described (Marra and Wingert, 2016) and using the following primers, where uppercase letters indicate location in an exon and lowercase indicates primer location in an intron: ptgs1-F1 5'-TTTATTTATTTGCAGCTTTTTCTT-3'; ptgs1-R1 5'-CAGTGTTTGATGAAGTCGGGCTTTC-3'; ptgs2a-F1 5'-CTGAGCTTCTCACACGCATCAAAT-3'; ptgs2a-R1 5'-GGCGAAGAAAGCAAACATGAGACT-3'; ptger2a-F1 5'-AGACCGAGCGTATGCCAATGT- 3'; ptger2a-R4 5'-caggagggctaataattcagactt-3'; ptger2a-F3 5'-ctgtttcagtgatcagtttgt-3'; ptger2a-R7 5'-CCGCAGAGCTATGAGATCAGTC-3'; ptger2a-R8 5'-GCTGAGGATGATGAACACCAAG-3'; ptger4a-F3 5'-ATGGTCATCCTGTTGATCGCC-3'; ptger4a-R2 5'-aatgagagtcctggaacttac-3'; ptger4a-F5 5'-gggtgtagtcatttatgttgagca-3'; ptger4a-R5 5'-CAGGACCGCTTTACGCAGTAAG-3'.…”

“…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).…”