Chemical Treatments for Distal Nephron Segmentation

Chemical treatments were completed as previously described, with chemicals as listed in Table 1 [34 (link),38 ,39 (link)]. Chemicals were dissolved in DMSO to make a 10 mM stock solution. Stocks were aliquoted and stored at −80 °C. Aliquots were thawed at room temperature and protected from light. Working solutions were diluted in E3 and distributed to 6- or 12-well plates. Chemical treatments were completed beginning at the shield stage (6 h post-fertilization (hpf)) until the 24 hpf stage, unless otherwise noted. We chose to treat at the shield stage, as the animals were already undergoing gastrulation. Thus, this timepoint prevents interference with the onset of gastrulation. The dose for each chemical was decided by treating at doses consistent with previous studies or slightly increased concentrations to maximize penetrance while minimizing morphological defects. Animals treated with E2 exhibited distal segmentation phenotypes at both 20 µM and 25 µM. As most animals had a curved body axis at 25 µM, we proceeded with 20 µM treatments. We treated with 400 µM DPN, 400 µM MPP, and 75 µM PPT, however the animals did not exhibit changes in distal nephron segmentation. When exposed to higher doses, the animals exhibited morphological defects or mortality. PHTPP exhibited the highest penetrance of distal segmentation without morphological defects at 18 µM. Xenoestrogens genistein and ethinylestradiol exhibited the highest penetrance of distal segmentation without morphological defects at 20 µM. Treatments were conducted in triplicate with at least n > 20 embryos per replicate at various doses (Table 2). All experiments were conducted with a DMSO vehicle control. DMSO control animals are demarcated as “WT” in all graphics and schematics.