A novel mechanism of gland formation in zebrafish involving transdifferentiation of renal epithelial cells and live cell extrusion
- University of Auckland, New Zealand
Figures
Figure 1
CS extrusion is achieved by apical extrusion of multiple renal epithelial cells.
(A) Lateral views of a stage series analysis of CS extrusion in a single live Tg(cdh17:egfp) embryo (top panels) and embryos fixed at the stages shown and stained for cdh17(purple)/stc1(red). (B) …
Figure 2 with 2 supplements
CS extrusion is mediated by apical constriction.
(A) Panels show lateral views of the markers indicated in the region of the trunk where the CS will be derived (at 22 hpf) and is extruded (at 50 hpf). (B) Panels show lateral views of the trunk …
Figure 2—figure supplement 1
lhx1a expression stage series.
Panels show lateral images of zebrafish embryos stained for lhx1a transcripts at the indicated stages. Arrows indicate the intermediate mesoderm.
Figure 2—figure supplement 2
Cell division is not required for CS formation.
(A) Panels show lateral views of Tg(cdh17:egfp) embryos stained for EdU labelling at 36 hpf (left panel) and 50 hpf (two right panels) after treatment with EdU between the stages shown. (B) Lateral …
Figure 3 with 2 supplements
Hnf1b is translocated to the cytoplasm in prospective CS cells.
(A) Panels show Hnf1b immunostains (graysale) on transverse cryosections of zebrafish embryos through the region of the trunk where CS glands form at 22 hpf, 36 hpf and 50 hpf. Arrows indicate CS …
Figure 3—figure supplement 1
hnf1ba is not expressed in the CS gland.
Panel shows a 50 hpf wild-type embryo stained for hnf1ba transcripts. Black arrow indicates the approximate position of the CS gland.
Figure 3—figure supplement 2
Hnf1b nuclear export at 50 hpf.
Lateral view of a 50 hpf Tg(cdh17:egfp) (green) embryo immunostained for Hnf1b (red) and counterstained for DAPI (blue). Hnf1b in the CS is largely non-nuclear (white arrow), in contrast to nuclear …
Figure 4
irx3b depletion converts the DE segment to CS fate.
(A) Panels show lateral views of 18 hpf, 20 hpf, 22 hpf and 24 hpf wild-type and irx3b morphant embryos co-stained for cdh17 (red) and slc12a1 (purple). Black arrowheads highlight the initiation of …
Figure 5
irx3b depletion causes aberrant pronephric morphogenesis.
(A) Lateral views of live un-injected and irx3b morphant Tg(cdh17::egfp) embryos with lower panels showing higher magnification of indicated pronephric regions. Histograms show the width of the …
Figure 6 with 4 supplements
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 …
Figure 6—figure supplement 1
Notch components are expressed in the CS gland.
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.
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 …
Figure 6—figure supplement 3
Early Notch inhibition promotes CS gland formation but later treatment inhibits CS development.
(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 …
Figure 6—figure supplement 4
irx3b crispants and stable mutants recapitulate the renal phenotypes associated with irx3b morpholino knock down.
(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 …
Videos
Video 1
Time-lapse imaging showing extrusion of the CS endocrine gland from the pronephric tubule Movie shows a lateral view of the posterior trunk region of a Tg(cdh17:egfp) embryo between 32 hpf and 48 hpf.
Green fluorescent cells label the pronephric tubule and the CS cells, with the latter observed as a bulge of cells that undergo extrusion during the time-lapse.
Tables
Table 1
Empirical results for the effect of irx3b morpholino on cdh17, stc1 and slc12a1 expression at 24 hpf, 36 hpf and 50 hpf Un-injected wild-type embryos showed no phenotypes in the pronephros.
The phenotypes for irx3b morphants are: slc12a1, reduction in staining; stc1, increased/ectopic staining; cdh17, expanded DL domain, reduced DE staining and reduced/stretched PST segment. …
| slc12a1/stc1 | 24 hpf | 36 hpf | 50 hpf |
|---|---|---|---|
| Un-injected | 33 | 34 | 33 |
| irx3bmo slc12a1 phenotype | 17/23 | 27/32 | 30/39 |
| irx3bmo stc1 phenotype | 18/23 | 27/32 | 33/39 |
| cdh17/stc1 | 24 hpf | 36 hpf | 50 hpf |
|---|---|---|---|
| Un-injected | 27 | 30 | 31 |
| irx3bmo cdh17 phenotype | 25/27 | 29/31 | 30/38 |
| irx3bmo stc1 phenotype | 22/27 | 26/31 | 31/38 |
| cdh17/slc12a1 | 24 hpf | 36 hpf | 50 hpf |
|---|---|---|---|
| Un-injected | 23 | 23 | 27 |
| irx3bmo cdh17 phenotype | 24/27 | 29/30 | 32/35 |
| irx3bmo slc12a1 phenotype | 26/27 | 27/30 | 34/35 |
Additional files
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Transparent reporting form
- https://doi.org/10.7554/eLife.38911.018
