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. 2018 Nov 5;7:e38911. doi: 10.7554/eLife.38911

Figure 6. Notch inhibition precludes CS gland formation.

(A) Panels show a stage series of fluorescent images of the TP1:VenusPEST transgenic line in the trunk region (arrows mark fluorescence in the region of the CS gland). (B) Left panels show lateral views of the posterior trunk showing stc1 expression after the indicated treatments. Middle panels show transverse sections of the pronephric tubule and position of the CS gland showing Hnf1b staining (±DAPI) after the same treatments. Right panels show lateral views of the trunk in the region of the CS gland (highlighted by unfilled arrow). (C) Histogram showing ‘corrected total nuclear fluorescence’ (CTNF) measurements of nuclei in the positions indicated. (D) Histograms showing the number of embryos with fluorescence in the CS gland in progeny from TP1:VenusPEST in-crosses with the indicated treatments.

Figure 6.

Figure 6—figure supplement 1. Notch components are expressed in the CS gland.

Figure 6—figure supplement 1.

Lateral views of wild-type embryos stained for notch3 and jagged2b (jag2b) at the stages shown, black arrows indicate expression of notch factors in the CS gland.
Figure 6—figure supplement 2. Notch activity in the pronephros is not observed early in development in the TP1:VenusPEST reporter line.

Figure 6—figure supplement 2.

Top panels show lateral views of the trunk of an 18 hpf embryo, bottom panels show lateral views of the trunk region at 24 hpf. Venus+ cells in the region of the pronephros (arrows) were in the epidermis when analysed along the Z-axis.
Figure 6—figure supplement 3. Early Notch inhibition promotes CS gland formation but later treatment inhibits CS development.

Figure 6—figure supplement 3.

(A) Panels show lateral views of the trunk of 50 hpf embryos stained for stc1 after early (from 10 hpf) treatments with cpdE and DAPT. (B) Lateral views of stc1 stained embryos treated with DMSO and DAPT from 18 hpf.
Figure 6—figure supplement 4. irx3b crispants and stable mutants recapitulate the renal phenotypes associated with irx3b morpholino knock down.

Figure 6—figure supplement 4.

(A) Panel shows the gel image of the irx3b PCR product amplified from genomic DNA and processed through the T7 endonuclease one protocol (see materials and methods section). (B) Panels show lateral views of Tg(cdh17:egfp) embryos in wild-type, irx3b morphant, irx3b crispant and germline stable irx3b mutant embryos. Sanger sequencing analysis (right) shows a 29 bp deletion that causes a frame-shift mutation in exon 1 of the irx3b gene. (C) Panels show lateral views of the posterior trunk of stable irx3b mutants stained for stc1, slc12a1 and cdh17 transcripts.