Expression of ppp1r12a splice variants during early embryonic development in zebrafish. (A) Transcript specific primers were used to detect ppp1r12a-tv202 or to detect the long transcript variants tv-201 and tv-203, labelled tv-long. Embryos were collected immediately after fertilization (0 hpf), 64-cell stage (2 hpf), sphere stage (4 hpf), shield stage (6 hpf), bud stage (10 hpf), 6 somite stage (12 hpf), 10 somite stage (14 hpf), 22 somite stage (20 hpf), Prim-5 stage (24 hpf), long-pec stage (48 hpf) and protruding-month stage (72 hpf). Amplification of eF1a and total RNA without addition of reverse transcriptase were used as controls. The PCR products amplified by tv-long, tv202 and eF1a primers were 769 bp, 206 bp and 720 bp in length respectively. (B-D) The spatial expression of ppp1r12a-tv202 was analyzed by whole-mount in situ hybridization using a probe targeted at the unique regions of exon 12 and 13 in tv202 at the 64 cell stage (B), bud stage (C) and 24 hpf (D). Quantitative analysis of mRNA expression levels of long-tv (E) and tv202 (F) during zebrafish development. Values are means of three biological replicates performed in duplicate and error bars are standard deviation. All values are normalized the eF1a and reported as % of expression of the long-tv expression at 0 hpf.

Mypt1-202 expression can reduce Mypt1 loss-of-function in zebrafish embryos. Lateral views of representative 48 hpf zebrafish embryos injected with (A) control morpholino, (B) 1.0 ng ppp1r12a MO, (C) a partially rescued embryo injected with 25 pg ppp1r12a-tv202 mRNA and 1.0 ng of ppp1r12a MO. (D–F) Embryos at bud stage stained with hgg1 (to mark the prechordal plate), shh (midline), pax2.1 (midbrain-hindbrain boundary) and dlx3 (neural plate) after injection of (D) control morpholino, (E) ppp1r12a MO or (F) ppp1r12a-tv202 mRNA and ppp1r12a MO. (G) Quantification of the truncated body axis phenotype in morphant and mRNA injected embryos. Error bars are standard error and a* indicates a statistically significant (p < 0.05) difference from control and a^# indicates a statistically significant (p < 0.05) rescue compared to the corresponding morpholino injected embryos. Injections were performed a minimum of three times with 50 total embryos measured. Embryos were collected by spawning heterozygotic hi2653 adult zebrafish (I and J) or from WT controls (H). Mutant embryos were injected with 25 pg of ppp1r12a-tv202 mRNA at the one cell stage (I). Embryos were collected at 36 hpf and probed with prox1 to mark the liver primordium (Marked with an arrowhead). Liver staining for prox1 was detected in 68 of 68 wild-type embryos, 57 of 77 uninjected embryos from the heterozygotic cross and 89/91 rescued embryos.

Overexpression of Mypt1-202 in zebrafish embryos results in constitutive dephosphorylation of Mlc2. Lateral views of representative 48 hpf zebrafish embryos injected with (A) 200 pg myc-long-mypt1 mRNA or (C) 100 pg myc-ppp1r12a-tv202 mRNA. Embryos at bud stage stained with hgg1 (to mark the prechordal plate), shh (midline), pax2.1 (midbrain-hindbrain boundary) and dlx3 (neural plate) after injection of (B) 200 pg myc-long-mypt1 mRNA or (D) 100 pg myc-ppp1r12a-tv202 mRNA. A representative field of presomitic and notochordal mesoderm at the bud stage in control embryos (E), embryos injected with 200 pg myc-long-mypt1 mRNA (F) or 100 pg ppp1r12a-tv202 mRNA (G). (H) The actomyosin driven elongation of mesodermal cells was determined by calculating the length width ratio (y-axis). The crossed bars indicate cells with the long axis length and the short axis width. Error bars are standard error and a * indicates a statistically significant difference from control. All calculations were made on 75 cells from 3 to 5 separate embryos. Statistical significance was calculated using a one-factor ANOVA with Tukey post hoc analysis and is defined as p < 0.05. (I) Embryo lysates from 25 control embryos or 25 embryos injected with either myc-long-mypt1 mRNA or myc-ppp1r12a-tv202 mRNA were analyzed by Western blot using anti-myc, anti-phospho-Mlc2 or anti-Mlc2 antibodies. This biochemical analysis was performed four times.