(A-C) Schematic of CRISPR/Cas9 knock-out of taz. The target site (shown in blue) is located in exon 1 of taz, and the PAM region is shown in red (A). Two mutant alleles with small deletions, taz ^Δ10 and taz ^Δ1, were obtained (B), both of which result in frame-shifts of the taz open reading frame and truncated proteins (C). aa, amino acid; TEAD BD, Tead transcription factor binding domain (light blue); WW, dual tryptophan motif (green); TAD, transactivation domain (purple); PDZ domain (blue). (D) Western blot analysis shows Taz protein in wild type but not in taz^{Δ10/ Δ10} mutant embryos. β-Tubulin is used as a loading control. (E, F) Similar to wild type embryos (E), taz^Δ10/Δ10 embryos are largely normal at 4.5 dpf, except for a smaller inflated swim bladder and mild pericardial edema (F). (G) Survival curve of taz^Δ10/Δ10 in heterozygote intercrosses from larval to adult stages (shown as % survivors, from two independent experiments). The red dashed line indicates the expected 25% homozygous mutants in accordance with Mendelian segregation. Number of larvae/adults at each stage: 7 dpf (59/228), 15 dpf (37/200), 30 dpf (20/182), 60 dpf (22/216) and 120 dpf (10/174). Black arrow, swim bladder; black arrowhead, pericardium. Scale bar, 500 μm.

(A-C’”) Immunofluorescence of Taz and β-Catenin in sectioned wild type oocytes at various stages: late II/ early III (A-A’”, n = 14), mid III (B-B’”, n = 14) and late III (C-C’”, n = 13). β-Catenin and DAPI label the cell membrane and nucleus, respectively. While basal levels are detected in follicle cells, in the micropylar cell high levels of Taz are detected, predominantly in the nucleus (A-C, A”-C”). Taz expression levels decrease as the micropylar cell develops (A-C, A”-C”). The micropylar cell undergoes dramatic shape change from flattened to ‘nail’-like to perforate the developing vitelline envelope (A’-C’, A”-C”). Bright field images show the gradual invagination of the vitelline envelope by the protruding micropylar cell (A’”-C’”). (D-E”) Immunofluorescence of Taz and β-Catenin in sectioned wild type and taz^-/- oocytes at stage III after in situ hybridization to detect cyclinB. In wild type ovaries, the high Taz expressing micropylar cell sits on the top of the oocyte animal pole, marked by cyclinB, and perforates the vitelline envelope (D-D”, n = 9). No Taz is detectable in taz^-/- oocyte (E), and neither the micropylar cell around the animal pole nor an invagination on the vitelline envelope is observed in mutant oocytes (E-E”, n = 11). (F-G) Immunofluorescence to detect Taz and in situ hybridization for cyclinB in wild type and taz^-/- oocytes at stage III. A single high Taz expressing micropylar cell is located at the animal pole of wild type oocyte (F, n = 13), but is not found in taz^-/- ovaries (G, n = 17). Two stage I oocytes, ubiquitously expressing cyclinB, are found adjacent to the stage III oocyte in figure F. White arrow, the micropylar cell indicated by high Taz expression. Scale bar, 100 μm in F and G and 10 μm in others.

(A-C’”) Single confocal plane of immunofluorescence of Taz and β-Catenin in whole mount wild type oocytes at three stages: late II/ early III (A-A’”), mid III (B-B’”) and late III (C-C’”) shows β-Catenin and DAPI in the cell membrane and nucleus respectively. The nucleus in most micropylar cells (57/60) is composed of two closely juxtaposed nuclei. (D-F’”) Similarly, single confocal planes of immunofluorescence of Taz and Nup107 (nuclear membrane marker) in stage late II/ early III (D-D’”), mid III (E-E’”) and late III (F-F’”) oocytes show two closely juxtaposed nuclei surrounded by continuous nuclear membranes in the micropylar cell (n = 32 oocytes). Scale bar, 10 μm.

(A-F’”) Immunofluorescence shows Taz and F-actin (A-C’”, n = 32) or α-Tubulin (D-F’”, n = 19) in sectioned ovaries; besides the invagination on the vitelline envelope, the expression of Taz and the shape of the micropylar cell changes with the growth of the micropylar cell; Phalloidin labelled F-actin bundles are found gradually deposited in the leading tip in the cytoplasmic extension of the micropylar cell and the part of oocyte cortex contacting the micropylar cell (A-C’”), and α-Tubulin is enriched in the cytoplasmic extension of the micropylar cell (D-F’”). Scale bar, 10 μm.

Oocyte polarity is normal in taz^-/- zebrafish females.

In situ hybridization on sectioned ovaries showed that transcripts of pou2 (A-B) and brl (C-D) were normally located on the animal and vegetal pole, respectively, in both wild type and taz^-/-oocytes. Dark blue arrow, vegetal pole; pink arrow, animal pole. Scale bar, 50 μm.

Cytoskeleton in the micropylar cell.

Consecutive confocal sections at 2.5 μm intervals showing immunofluorescence of Taz, F-actin and α-Tubulin in a stage III wild type oocyte (n = 17). (A-B) show the micropylar cell body in which Taz is mainly expressed in the nucleus and α-Tubulin is in the cytoplasm. (C-F) show the cytoplasmic extension of the micropylar cell; α-Tubulin is enriched in the cytoplasm and F-actin is deposited at the leading tip (F). Dashed white circle, the micropylar cell. Scale bar, 10 μm.

Transient disturbance of actin or Myosin does not significantly affect morphology of the micropylar cell.

(A-C’”) Immunofluorescence shows Taz and F-actin in sectioned stage III oocytes after Latrunculin B (B-B’”, n = 7) or Blebbistain (C-C’”, n = 6) treatment. DMSO is the control (A-A’”, n = 7). Transient inhibition of actin polymerization (Latrunculin B) or Myosin II ATPase activity (Blebbistain) does not remarkably affect morphology of the micropylar cell (B”-C”). However, Latrunculin B treatment leads to cytoplasmic retention of Taz in the micropylar cell (6/7, B”), while Blebbistain does not (0/6, C”). Scale bar, 10 μm.