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. 2020 Oct 28;9:e61111. doi: 10.7554/eLife.61111

Figure 8. Shot and Tau functionally overlap during subcellular lumen formation.

(A–B) DB (A) and GB (B) embryonic TCs expressing tauGFP in the tracheal system, stained with GFP (green), CBP (white) and DSRF (magenta), showing the ESL phenotype induced by Tau overexpression. In A’ in B’ lumen and TC nuclei are shown, anterior side on the left, dorsal side is up; scale bar 5 μm. (C) Quantification of TCs with ESL in embryos overexpressing GFP (n = 240); ShotA (n = 400); one copy of btl >tauGFP (n = 440) or two copies of btl >tauGFP (n = 300). ***p-value<0.001; ns refers to a p-value>0.1. Statistics by two-tailed Student’s t-test. (D) Quantification of TCs without subcellular lumen in control (n = 820), shot3(n = 600), tau[MR22] (n = 180), shot3; tau[MR22](n = 180), shot3; btl >Tau (n = 440) and shot3; btl >Tau btl >Tau (n = 260) embryos. ***p-value<0.001; ns refers to a p-value>0.1. Statistics by two-tailed Student’s t-test. (E–J) Dorsal view of TCs from st. 16 embryos (genotype indicated) stained with anti-Gasp. tau deletion mutant does not display a subcellular lumen phenotype (D and F) but enhances the effect of shot mutation in the double mutant shot3; tau[MR22]. One copy of Tau is not sufficient to rescue shot3 (D and I, n = 400) but two copies rescues the shot LOF TC phenotype (D and J n = 260). Scale bars 10 µm. (K) Tau is detected in embryonic TCs. Embryonic shot::GFP dorsal TC stained with GFP (green in K, grey in K’), anti-Tau antibody (magenta in K, grey in K’) and CBP (blue in K grey in K’). Scale bar 5 μm.

Figure 8—source data 1. Quantification of tau mutant and overrexpression phenotypes and rescues.

Figure 8.

Figure 8—figure supplement 1. MT disorganisation in shot mutant TCs is rescued by ShotA and Tau, but not ShotC.

Figure 8—figure supplement 1.

Dorsal TCs from shot3;btl >GFP (A–A’’’); shot3; btl >shotAGFP (B–B’’’) shot3; 2Xbtl > TauGFP; (C–C’’’); and shot3; btl >shotCGFP (D–D’’’) embryos, stained with anti-GFP antibody (green in A–D’), CBP blue in A–D and grey (A’’–D’’) and anti-acetylated-tubulin antibody (magenta in A–D and grey in A’’’–D’’’). The MT network (control embryos Figure 5, panel E’’) disrupted in shot3 mutant TCs (A’’’), is partially restored in shot3;btl >shotA (B’’’) TCs and well reorganised in shot3 embryos overexpressing two copies of Tau-GFP (C’’’) but not in shot3,btl >shotC embryos (D’’’) (blue arrows). Anterior side is on the left and dorsal midline is up. The outline of TC is sketched in blue. Scale bar 5 μm.
Figure 8—figure supplement 2. Actin disorganisation in shot mutant TCs is rescued by ShotA and Tau, but not ShotC.

Figure 8—figure supplement 2.

Dorsal TCs from btl >GFP used as control (A–A’’’); shot3; btl >GFP (B–B’’’); shot3; btl >shotAGFP (C–C’’’); shot3;2Xbtl > TauGFP (D–D’’’) and shot3; btl >shotCGFP embryos, stained with anti-GFP antibody (green in A –E’), CBP (blue in A –E and grey in A’’–E’’) and anti-actin antibody (magenta in A – E and grey in A’’’–E’’’). The actin accumulation at the tip of TC (A’’’), disrupted in shot3 mutant TCs (B’’’), is restored in shot3 TCs overexpressing ShotA (C’’’) or two copies of Tau (D’’’), but not in shot3;btl >shotC embryos (E’’’), (see blue arrows). Anterior side is on the left and dorsal midline is up. The outline of TC is sketched in blue. Scale bar 5 μm.
Figure 8—figure supplement 3. Double shot3;tauMR22 mutant embryos display defects in lumen formation but not in tracheal cell number or TC fate.

Figure 8—figure supplement 3.

Dorsal branches of wt (A) and shot3; tau [MR22] embryos st.15 (B) stained with trachealess (trh) antibody to visualise all tracheal cell nuclei (magenta in A and B, grey in A’ and B’) and CBP to visualise the lumen (green in A and B, grey in A’’ and B’’) (n = 17 TCs) or wt (C) and shot3; tau [MR22] (D) stained with DSRF magenta in C and D (grey in C’ and D’) and CBP green in C and D and grey in C’’ and D’’ (n = 8 TCs). Dorsal view. Scale bar 10 μm.