2-OST is necessary for epiboly in early zebrafish development. (A-L) Embryos (1- to 2-cell stage) were injected with 3 ng control MO (A-C), 3 ng of 2-OST MO1 (D-F), 3 ng of 2-OST MO2 (G-I), or mixture of 3 ng 2-OST MO1 and 0.2 ng 2-OST RNA (J-L), then incubated in embryo water at 28°C. They were examined at 2.5 hpf (A,D,G,J), 4 hpf (B,E,H,K) and 8 hpf (C,F,I,L). (M) Quantitative comparison of injections and rescue scored based on failure to initiate epiboly. The co-injection of 2-OST MO+2-OST RNA rescued the percentage of embryos that fail to initiate epiboly in the 2-OST MO alone or the 2-OST MO+control RNA by >50%. ^*P<0.05. Error bars represent s.d.

2-OST morphants display reduced levels of 2-OST protein and reduced levels of ³⁵S incorporation. (A) By western blot, levels of 2-OST protein were shown to be statistically significantly reduced by roughly half using both 2-OST MO1 and MO2 compared with WT and control embryos at 4 hpf. The antibody was validated using 2-OST RNA, with degradation product or altered post-translational modifications beneath the band, and 2-OST-EGFP RNA (two embryos/lane/sample, n=4 experiments). Quantification of protein levels was normalized to uninjected levels of 2-OST relative to the total levels of α-tubulin per sample. (B) WT, control MO and 2-OST MO embryos were injected with 1 nl 10 mCi ³⁵S, grown for five hours, and collected at 8 hpf (n=3, 90 embryos group/n). Protein purification was performed to isolate radiolabeled GAG chains and cpu per embryo was calculated based on liquid scintillation counting results. (C) Ratio of ³⁵S incorporation per embryo was normalized to WT. The 2-OST morphants exhibit a nearly 80% decreased rate of incorporation of ³⁵S compared with WT and control morphants. ^*P<0.05. Error bars represent s.d.

Molecular marker expression in WT and 2-OST morphant embryos. (A-P) Wild-type embryos were injected with 3 ng of either control MO or 2-OST MO, fixed at 8 hpf, and processed for whole-mount in situ hybridization. Expression patterns of mesoderm markers (A-F), mesendoderm markers (G-J), a DFC and endoderm marker (K,L), an ectoderm marker (M,N) and a YSN marker (O,P) were analyzed. The presence and organization of these markers in both the WT and morphant embryos suggest that 2-OST morphants were establishing the primary germ layers and axes. Furthermore, the presence of sox17 and shh, which are expressed later in development, indicate that the 2-OST morphants were not severely delayed in development.

β-Catenin accumulation is dependent on 2-OST. (A,D,G,J) Single planes of z-stack at 4 hpf of β-catenin for control MO+control RNA- (A), 2-OST MO+control RNA- (D), control MO+stabilized β-catenin RNA-(G), and 2-OST MO+stabilized β-catenin RNA (J)-injected embryos. (B,E,H,K) Single planes of z-stack of E-cadherin control MO+ control RNA- (B), 2-OST MO+control RNA- (E), control MO +stabilized β-catenin RNA- (H), and 2-OST MO+stabilized β-catenin RNA (K)-injected embryos. (C,F,I,L) Merged images of β-catenin and E-cadherin for control MO+control RNA (C), 2-OST MO+control RNA- (F), control MO+stabilized β-catenin RNA- (I), and 2-OST MO +stabilized β-catenin RNA (L)-injected embryos (n=21 for each group). The purple arrows indicate regions in which adhesion is completely lost between deep cells based on dark spaces and altered cellular morphology. Scale bars: 100 μm. (M) Western blot results, comparing band densitometry at 4 hpf. β-catenin (green) levels per sample were normalized to α-tubulin levels per sample, and then WT, control MO and control RNA, 2-OST MO and control RNA, control MO and stabilized β-catenin RNA, and 2-OST MO and stabilized β-catenin RNA were each normalized to WT, using WT as a value of 1. The same was done for the E-cadherin (red) (two embryos/lane/sample, n=5 experiments). ^*P<0.05 for levels of protein statistically significantly different for the 2-OST MO and control RNA compared with all the other samples on the chart. Error bars represent s.d.

2-OST modulates Wnt signaling upstream of Gsk3 and downstream of Wnt8. (A,D,G,J,M,P) Single planes of z-stack at 4 hpf of β-catenin for control MO+control RNA- (A), 2-OST MO+control RNA- (D), control MO+Wnt8 RNA- (G), 2-OST MO+Wnt8 RNA- (J), control MO+dnGSK3 RNA- (M) and 2-OST MO+dnGSK3 RNA (P)-injected embryos. (B,E,H,K,N,Q) Cross-sections of E-cadherin for control MO+control RNA- (B), 2-OST MO+control RNA- (E), control MO+Wnt8 RNA- (H), 2-OST MO+Wnt8 RNA- (K), control MO+dnGSK3 RNA- (N) and 2-OST MO+dnGSK3 RNA (Q)-injected embryos. (C,F,I,L,O,R) Merged and magnified cross-sections of β-catenin and E-cadherin for control MO and control RNA- (C), 2-OST MO+control RNA- (F), control MO+Wnt8 RNA- (I), 2-OST MO+Wnt8 RNA- (L), control MO+dnGSK3 RNA- (O) and 2-OST MO+dnGSK3 RNA (R)-injected embryos. Arrows indicate regions in which adhesion is completely lost between deep cells based on dark spaces and altered cellular morphology. Scale bars: 100 μm.

2-OST contributes to regulation of cell proliferation via β-catenin. (A,E) Single planes of z-stack of Sytox Green nuclear stain for control MO+control RNA- (A) and 2-OST MO+control RNA (E)-injected embryos. (B,F) Anti-phospho-histone H3 immunostaining of control MO+control RNA- (B) and 2-OST MO+control RNA (F)-injected embryos. (C,G) Anti-BrdU immunostaining of control MO+control RNA- (C) and 2-OST MO+control RNA (G)-injected embryos. (D,H) Merged images of Sytox Green, anti-phospho-histone-H3 and anti-BrdU staining of control MO+control RNA- (D) and 2-OST MO+control RNA (H)- injected embryos. Scale bar: 100 μm. (I) Comparison of average cell area. The cell area for 2-OST MO+control RNA-injected samples was statistically significantly different from all the other samples cell areas. (J) Comparison of percentage of cells in M phase. (K) Comparison of rate of cell proliferation. The rate of proliferation for 2-OST MO+control RNA-injected embryos was statistically significantly different from all the other samples rates of proliferation and was nearly half the rate of the controls. n=15 per group. (L) Comparison of percentage of cells in M phase. (M) Comparison of rate of proliferation. The rate of proliferation for the 2-OST MO+control RNA- and 2-OST MO+Wnt8 RNA-injected embryos was nearly half the rate of the controls (n=15 for each group). All these experiments were conducted at 4 hpf. ^*P<0.05. Error bars represent s.d.

Adhesion defects in 2-OST MO are cell autonomous. (A-F) Single planes of z-stack at 4 hpf of mosaic embryos formed from injecting either control MO+lissamine-conjugated MO (B,C) or 2-OST MO+lissamine-conjugated MO (E,F) into a single cell at the 32-cell stage. Anti-β-catenin for control MO mosaic (A) or 2-OST MO (D). (G-I) Animal caps removed and immediately imaged from control MO (G), 2-OST MO (H), or 2-OST MO+2-OST RNA (I) embryos to examine adhesion defects. Minimal cells were observed around control MO and rescue-injected animal caps, but many cells were observed around 2-OST MO, indicating decreased adhesion.

WT and control morphants exhibit comparable levels of β-catenin and E-cadherin. (A-F) WT levels are comparable to control MO embryos for β-catenin (A,C,D,F) and E-cadherin (B,C,E,F) in stabilized β-catenin rescue experiments as well as Wnt epistasis experiments.

Unillustrated author statements