Fig. 4

Dchs1b Regulates Ttc28 Subcellular Localization, and Ttc28 Is Sufficient to Influence YCL Microtubule Dynamics and Embryonic Cleavages

(A) Representative images showing the subcellular localizations of HA-Ttc28-mCherry (magenta) with membrane GFP (green) or Dchs1b-sfGFP (green) in the WT and MZdchs1b at 3–4 hpf in embryos injected with synthetic RNAs encoding HA-Ttc28-mCherry (600 pg), membrane GFP (25 pg), and Dchs1b-sfGFP (600 pg). The arrow points to a centrosome marked by HA-Ttc28-mCherry accumulation. Scale bar, 30 μm.

(B) Quantification of the relative HA-Ttc28-mCherry intensity at the plasma membrane and centrosome normalized to the cytoplasmic signal in the same cell in the various injection conditions. n, number of cells quantified. ns, not significant. ^∗∗∗∗p < 0.0001, Tukey's multiple comparisons test.

(C) Cartoon of the experimental design and confocal images of mosaic analyses of HA-Ttc28-mCherry and Dchs1b-sfGFP interaction in vivo. HA-ttc28-mCherry RNA was injected at one-cell stage and dchs1b-sfGFP RNA was subsequently injected into one of the blastomeres at eight-cell stage. Confocal imaging was performed at 3–4 hpf. Scale bar, 30 μm.

(D) Representative time-lapse still images of YCL microtubules in mCherry and HA-Ttc28-mCherry RNA (900 pg) injected Tg[ef1α:dclk-GFP] WT embryos at 1.75–2.5 hpf. Scale bar, 30 μm.

(E) Quantification of YCL microtubule density in mCherry control and HA-Ttc28-mCherry overexpressing embryos at 1.75–2.5 hpf. Error bars represent SEM. N, number of embryos.

(F) Representative images of blastomeres stained with γ-tubulin (green) and counterstained with DAPI (blue) in WT and MZdchs1b embryos at 4–5 hpf injected with either mCherry or HA-ttc28-mCherry RNA (900 pg) at one-cell stage. Arrows indicate multiple nuclei, and arrowheads indicate chromatin bridges. Scale bar, 30 μm.

(G) Quantification of mitotic events at 4–5 hpf in WT and MZdchs1b blastulae injected with mCherry or HA-ttc28-mCherry RNA (900 pg) at one-cell stage. N, number of embryos; n, number of cells.

See also Figures S2 and S4, and Video S5.