PPAR agonist bezafibrate alters zebrafish pronephros segmentation, and the PPAR coactivator ppargc1a exhibits a dynamic expression pattern in renal progenitors. (A) Double WISH at the 28 ss for the PST segment marker trpm7 (top), and the DL segment marker slc12a3 (bottom), with smyhc (red) to mark somites in DMSO control (left) and PPAR agonist, 150 μM Bezafibrate-treated (right) samples confirmed the initial hit from the chemical screen. Scale bar = 90 μm. (B) Absolute length measurements of the changes to pronephros segment lengths in bezafibrate treated (grey) and control samples (black). (C) Double WISH for ppargc1a (purple) expression at the 8 ss, 20 ss, and 28 ss with somites stained (red) (8 ss = deltaC, 20 ss and 28 ss = smyhc). (D) Double FISH at the 28 ss showing colocalization of ppargc1a (red) with slc12a1 (distal early, left) and slc12a3 (DL, right). Expression boundaries are indicated with blue (DE, left and DL, right) and white (ppargc1a) arrowheads. Scale bars = 35 μm. Data are represented as ±SD, significant by t test comparing the drug treatment to the DMSO vehicle control, n.s. = not significant, *** = p < 0.001.

Loss-of-function studies show ppargc1a is necessary for proper PST and DL formation. (A) Exon map of zebrafish ppargc1a and the target sites (X) for morpholinos (MO) and the location of the ppargc1asa13186 mutant allele. (B) WISH images of 28 ss ppargc1asa13186 WT siblings (WT), ppargc1asa13186-/-, ppargc1a MO1 injected, and ppargc1asa13186-/- + ppargc1a cRNA illustrating the changes in PST (trpm7-purple, top) and DL (slc12a3-red, bottom) formation in the ppargc1asa13186-/- and ppargc1a morphants, and the subsequent rescue when ppargc1a cRNA was added to the ppargc1asa13186-/-. Scale bars = 100 μm. Absolute length measurements of the PST (C), and DL (D) segments. (E) ppargc1a expression in DMSO control (top) and 150 μM bezafibrate-treated (bottom) zebrafish at the 28 ss following vehicle or vehicle/drug addition at the 5 hpf stage. Scale bars = 65 μm. (F) Absolute length measurements of the ppargc1a expression domain at 28 ss in DMSO control and bezafibrate-treated embryos from panel E. (G) qRT-PCR results showing ppargc1a RNA expression levels in bezafibrate-treated samples compared to DMSO controls. Data are represented as ±SD, significant by t test, n.s. = not significant, ** = p < 0.01, *** = p < 0.001.

Loss of ppargc1a does result in segment changes at the 20 ss but no change in cellular turnover is observed. (A) WISH at the 20 ss for trpm7 (left) and (B) slc12a3 (right) in WT (top) and ppargc1a MO (bottom). Blue arrowheads indicate trpm7 expression in the developing pronephros. Scale bar = 100 μm. The representative graphs showing absolute length measurements of trpm7 (C) and slc12a3 (D). (E) FISH/IHC for trpm7 (red) and anti-Caspase-3 (green) with DAPI (blue) in WT (top) and ppargc1a MO1 (bottom). (F) FISH/IHC for trpm7 (red) and anti-phospho-Histone H3 (green) in WT (top) and ppargc1a MO1 (bottom). (G) FISH/IHC for slc12a3 (red) and anti-Caspase-3 (green) with DAPI (blue) in WT (top) and ppargc1a MO1 (bottom). (H) FISH/IHC for slc12a3 (red) and anti-phospho-Histone H3 (green) with DAPI (blue) in WT (top) and ppargc1a MO1 (bottom). (I) The number of trpm7/Caspase-3 or trpm7/pH3 double-positive cells is depicted. (J) The number of slc12a3/Caspase-3 or slc12a3/pH3 double positive cells is depicted. (WT quantifications = black bars, ppargc1a-deficient quantifications = white bars.) Data are represented as ±SD, significant by t test, n.s. = not significant, *** = p < 0.001.

ppargc1a acts upstream of tbx2b to form the DL segment. (A) WISH for tbx2b (purple) expression in 28 ss WT and ppargc1asa13186-/-zebrafish. Scale bars = 75 μm. (B) Absolute length measurements of tbx2b mRNA expression domains in WT and ppargc1asa13186-/- zebrafish. (C) ppargc1a expression in WT and tbx2b MO injected 28 ss zebrafish. Scale bars = 65 μm. (D) Absolute length measurements of ppargc1a expression domain in WT and tbx2b MO injected zebrafish. (E) WISH at 28 ss for slc12a3 (purple) in WT, ppargc1asa13186-/-, and ppargc1asa13186-/- injected with tbx2b cRNA. Scale bars = 75 μm. (F) Absolute length measurements of slc12a3 mRNA expression domains in WT, ppargc1asa13186-/-, and ppargc1asa13186-/- + tbx2b cRNA. Data are represented as ±SD, significant by t test, n.s. = not significant, ** = p < 0.01, and *** = p < 0.001.

ppargc1a and sim1a have a reciprocal antagonistic relationship that is necessary to negotiate the PST segment boundary. (A) WISH for ppargc1a (purple) expression at 28 ss in WT, sim1a MO, and sim1a cRNA injected zebrafish. The green and red dashed lines indicate the beginning and the end of WT expression, respectively, the expansion of the ppargc1a domain in sim1a MO is denoted with asterisks, and the reduction in the ppargc1a domain in sim1a cRNA injected is denoted with arrowheads. Scale bars = 60 μm. (B) WISH for sim1a (purple) and smyhc (red) in 20 ss WT, ppargc1asa13186-/-, and ppargc1a cRNA injected zebrafish. Arrows and the dashed green line represent the beginning of the sim1a domain expression, the red dashed line indicate the end of the WT sim1a expression domain to illustrate the expansion of the segment boundary in ppargc1asa13186-/- is denoted with asterisks, and the reduction of the sim1a domain in ppargc1a cRNA injected is denoted with arrowheads. Scale bar = 40 μm. (C) WISH for trpm7 at 28 ss in WT, sim1a cRNA, and ppargc1a cRNA injected zebrafish. Scale bar = 50 μm (D) Absolute length measurements of the ppargc1a domain in 28 ss in WT, sim1a MO injected, and sim1a cRNA injected zebrafish. (E) Absolute length measurements of the 20 ss sim1a domain in WT, ppargc1asa13186-/-, and ppargc1a cRNA injected zebrafish. (F) Absolute length measurements of the trpm7 domain in the 28 ss WT, sim1a cRNA injected, and ppargc1a cRNA injected zebrafish. (G) WISH for trpm7 (PST segment) in WT, WT + sim1 a MO, ppargc1asa13186-/-, and ppargc1asa13186-/- + sim1a MO zebrafish. The green and red dashed lines indicate the beginning and end of WT trpm7 expression domains respectively. Asterisks represent expanded expression domain. Scale bars = 100 μm. (H) Absolute length measurements for the trpm7 domain in WT, sim1a MO injected, ppargc1asa13186-/-, and ppargc1asa13186-/- sim1a MO injected zebrafish. (I) Expression summary table depicting the segment boundaries and expression domains of ppargc1a and sim1a in WT, sim1a-deficient, ppargc1a-deficient, and double-deficient zebrafish. (J) Genetic model illustrating the relationships supported in this study that ppargc1a works upstream of tbx2b to form the DL and has a cross-repressive relationship with sim1a to properly form the segment boundary of the PST. Data are represented as ±SD, significant by t test, n.s. = not significant, *** = p < 0.001.