Prostaglandin signaling regulates nephron segment patterning of renal progenitors during zebrafish kidney development
- University of Notre Dame, United States
Figures
Figure 1
A novel small molecule screen reveals that prostaglandins alter nephron patterning.
(A) A diagram detailing the segmentation of the pronephros in relation to somites within the zebrafish embryo. Arrows indicate the blood filter, duct, and cloaca. (B) A schematic of the chemical …
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Figure 1—source data 1
Compilation of chemical screen phenotypic data.
For each compound, the dosage tested is listed along with the known biological function and the effect on zebrafish when treated between 4 and 24 hpf. The scoring key of ± indicates segment expansion or reduction.
- https://doi.org/10.7554/eLife.17551.003
Figure 2 with 4 supplements
Exogenous prostaglandin activity promotes proximal straight tubule identity.
(A) Embryos were exposed to 100 µM dmPGE2 between 4 hpf and 24 hpf. WISH was used to stain for the PCT (slc20a1a), PST (trpm7), DE (slc12a1), and DL (slc12a3) (purple) and the somites (smyhc1) (red) …
Figure 2—figure supplement 1
Exogenous PGB2 treatment is sufficient to expand the proximal straight tubule.
(A) Embryos were treated with PGB2 at 100 μM from 4 hpf to 24 hpf. Using WISH, embryos were stained for the PCT (slc20a1a), PST (trpm7), DE (slc12a1), and DL (slc12a3) (purple) and the somites (smyhc…
Figure 2—figure supplement 2
dmPGE2 and PGB2 treatment have dosage-dependent effects on pronephros segmentation.
(A–D) Embryos were incubated from 4 hpf to 24 hpf in 30 μM, 50 μM, 80 μM, and 100 μM solutions of dmPGE2 or PGB2. The PCT (slc20a1a), PST (trpm7), DE (slc12a1), and DL (slc12a3) (purple) and the …
Figure 2—figure supplement 3
Embryos dimensions remain largely unchanged by dmPGE2 or indomethacin treatment.
(A) The pronephric domain (starting at the beginning somite five to the end of somite 18) was measured in microns for embryos treated with 1% DMSO, 100 μM dmPGE2, or 30 μM indomethacin from 4 hpf to …
Figure 2—figure supplement 4
Perturbation of the prostaglandin pathway fails to elicit gross blood or blood vessel abnormalities during development.
Embryos were treated with 1% DMSO, 30 μM indomethacin, or 100 μM dmPGE2 from 4 hpf to 24 hpf and WISH was performed to detect (A) vasculature (flk1) and (B) blood precursors (gata1) at the 24 hpf …
Figure 3 with 1 supplement
Prostaglandin production is required for normal distal cell fate specification.
(A) Embryos were treated with 0.3% DMSO or the nonselective Cox inhibitor indomethacin at 30 μM from 4 hpf to 24 hpf. WISH was used to stain for the PCT (slc20a1a), PST (trpm7), DE (slc12a1), and DL …
Figure 3—figure supplement 1
Indomethacin treatment has no effect on proximal cell-fate choice, yet shifts the balance between distal identities.
(A) The PCT (slc20a1a), PST (trpm7), DE (slc12a1), and DL (slc12a3) (purple) and the somites (smyhc1) (red) were quantified at the 24 hpf stage in triplicate according to changes in domain size …
Figure 4 with 10 supplements
Prostaglandin signal inhibition results in an expansion of the distal early domain, which is rescued by the addition of exogenous dmPGE2.
Embryos were treated with a (A) 1% DMSO control, the (B) Ptgs1 selective inhibitor SC-560 at 50 μM from 4 hpf to 24 hpf, microinjected with the ptgs1 MO alone, or microinjected with the ptgs1 MO and …
Figure 4—figure supplement 1
Inhibition of Ptgs1 or Ptgs2a did not alter proximal segment identity.
Embryos were treated with (A). 5% DMSO, (B) the selective Ptgs1 inhibitor SC-560 at 50 μM, and (E) the selective Ptgs2a inhibitor NS-398 at 50 μM from 4 hpf to 24 hpf. Embryos were microinjected …
Figure 4—figure supplement 2
Diminishing Ptgs1 or Ptgs2a function causes distal early cell-fate identity to be favored at the expense of the distal late domain.
(A) The PCT, (B) PST, (C) DE, and (D) DL segment phenotypes were quantified in triplicate at the 24 hpf stage according to comparisons to the somites for ptgs1/2a double MO, ptgs1 MO, ptgs2a MO, ptgs…
Figure 4—figure supplement 3
Inhibiting prostaglandin synthesis with splice morpholinos promotes distal early identity.
(A) WT embryos were microinjected with either ptgs1 MO splice or ptgs2a MO splice and grown until 24 hpf. WISH was used to assay for the PCT (slc20a1a), PST (trpm7), DE (slc12a1), and DL (slc12a3) …
Figure 4—figure supplement 4
The prostaglandin receptor transcripts ptger2a and ptger4a are expressed in the pronephros during development and co-localize with the nephron.
WT embryos from the 12 ss through 26 ss were stained for (A) ptger2a or (B) ptger4a transcript expression (purple) and the somites (red) using WISH. The ptger2a and ptger4a expression domains are …
Figure 4—figure supplement 5
Morpholino inhibition of ptger2a fails to affect proximal segment identity.
(A) Embryos were microinjected with ptger2a MO1 or MO2 and grown until 24 hpf. WISH was used to visualize the PCT (slc20a1a) and PST (trpm7) (purple) and the somites (red) at the 24 hpf stage. Black …
Figure 4—figure supplement 6
Inhibition of ptger2a using small molecule antagonists promotes distal early fate-choice.
(A) Embryos were exposed to 0.1% DMSO, 3 μM AH6809, or 3 μM PF04418948 from 4 hpf to 24 hpf. WISH was used to stain for the PCT (slc20a1a), PST (trpm7), DE (slc12a1), and DL (slc12a3) (purple) and …
Figure 4—figure supplement 7
ptger4a MO knockdown results in an expansion of the distal early segment.
(A) Embryos were microinjected with ptger4a MO1 or ptger4a MO2 and grown until 24 hpf. Embryos were stained for the PCT (slc20a1a), PST (trpm7), (purple) and the somites (smyhc1) (red) using WISH at …
Figure 4—figure supplement 8
Exogenous dmPGE2 is incapable of rescuing distal cell fates in Ptger2a or Ptger4a deficient morphant embryos.
Embryos were microinjected with either (A) ptger2a MO1 or (B) ptger4a MO1 and treated with. 5% DMSO or 50 μM dmPGE2 from 4 hpf to 24 hpf. WISH was used to label the DE (slc12a1), and DL (slc12a3) …
Figure 4—figure supplement 9
Inhibiting PGE2 production results in an expanded distal early domain at the expense of the distal late segment, which can be rescued by exogenous dmPGE2.
Embryos were microinjected with ptsgs1/2a MO, ptgs1 MO, ptgs2a MO, ptger2a MO1, ptger2a MO2, ptger4a MO1, and ptger4a MO2 and were allowed to develop until 24 hpf. Embryos were also microinjected …
Figure 4—figure supplement 10
Validation of transcriptional changes using splice morpholinos to target ptgs1, ptgs2a, ptger2a, and ptger4a.
Embryos were microinjected with morpholino and allowed develop until 24 hpf, where their RNA was then harvested for RT-PCR. Schematics illustrate each genetic locus, with the location targeted by …
Figure 5 with 1 supplement
Prostaglandin signaling influences nephron patterning after gastrulation.
Embryos were treated with 0.3% DMSO or 1% DMSO vehicle control, (A) 30 μM indomethacin, or (B) 100 μM dmPGE2 from the 12 ss to the 28 ss and stained for the PST (trpm7), DE (slc12a1), and DL (slc12a3…
Figure 5—figure supplement 1
Inhibiting Ptger2a after gastrulation induces an expansion of the distal early and a restriction of the distal late segment.
(A) Embryos were treated with 0.3% DMSO, 3 μM AH6809, or 3 μM PF04418948 from the 12 ss to the 24 hpf, then fixed and stained for the DE (slc12a1) and DL (slc12a3) using WISH at the 24 hpf stage. …
Figure 6 with 1 supplement
Prostaglandin signaling modulates key nephrogenesis transcription factors.
(A) Embryos were treated with a 1% DMSO control, dmPGE2 at 100 μM, or indomethacin at 30 μM from 4 hpf to the 20 ss. WISH was used to stain for the transcription factors sim1a, mecom, and irx3b …
Figure 6—figure supplement 1
Prostaglandin signaling alters the expression of transcription factors necessary for normal patterning of the nephron.
(A) Embryos were treated with 1% DMSO, 100 μM dmPGE2, and 30 μM indomethacin from 4 hpf to the 20 ss and then stained for sim1a, mecom, and irx3b using WISH. Embryos were categorized into phenotypes …
Figure 7 with 2 supplements
Prostaglandin signaling acts upstream of the transcription factors sim1a and irx3b.
(A) Embryos were microinjected with the irx3b MO and treated with 0.5% DMSO or 50 µM SC-560 from 4 hpf to 24 hpf and stained for the DE using WISH. WT control embryos were also treated with 0.5% …
Figure 7—figure supplement 1
Overexpression of mecom cRNA failed to rescue the distal late segment in indomethacin treated embryos.
(A) Embryos were microinjected with 70 pg of mecom cRNA then treated with either 0.3% DMSO or 30 µM indomethacin from 4 hpf to 24 hpf. Embryos were then stained for the DL (slc12a3) (purple) and the …
Figure 7—figure supplement 2
Prostaglandin treatment shifts the distal early and restricts the distal late in sim1a morphants.
(A) Embryos were microinjected with the sim1a MO and treated with 100 µM dmPGE2 or 1% DMSO from 4 hpf to 24 hpf. WISH was then used to label for the PCT (slc20a1a), DE (slc12a1), and DL (slc12a3) …
Figure 8
The role of prostaglandin signaling during zebrafish nephrogenesis.
(A) PGE2 is generated by the Ptgs1 and Ptgs2a enzymes, which interact with the Ptger2a and Ptger4a receptors to define the boundaries of the DE by negatively regulating the domain of irx3b …
