DEV024406 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 (Adobe PDF) -

  Fig. S1. The sq181 mutation alters a conserved motif in Mypt1. (A) Integrated genetic/physical map summarizing the map-based cloning of the sq181 mutant gene. Genetic markers are shown above with their corresponding number of recombinants shown below. The mutant gene was located between SNP24 and SNP41 spanning a 45 kb genomic fragment contained in two BACs, DKEY-66D18 and DKEY-28J4. m (red), sq181 mutation locus. (B,C) Sequence traces (B) showing the G-to-A change (yellow) in the mypt1 gene in the sq181 mutant that results in a GTG codon to ATG codon change (underlined in gray) converting V³⁶ to M in the conserved KV³⁶xF motif in Mypt1 (underlined in green in C). (D) The liverless phenotype of the sq181 mutant (mu) was fully or partially rescued by the WT mypt1 mRNA but not by the G-to-A mutant mRNA (mypt1^m). Embryos 3 days post-injection were examined with the lfabp probe.

• Supplemental Figure S2 (Adobe PDF) -

  Fig. S2. Loss-of-function of the myosin phosphatase complex causes a liverless phenotype. (A) Diagram showing the viral vector insertion in the mypt1 gene in the hi2653 zebrafish line obtained from Nancy Hopkins' laboratory. (B) Injection of a morpholino specifically targeting the junction of exon 2 and intron 2 of pp1c (PP1c-MO, 5′-AAGGAGAGAATCTAACCTACCACAG-3′) caused a liverless or small liver phenotype (see Fig. 4C) due to the formation of an aberrant splicing product that contains the entire intron 2. The last 50 bases of exon 2 and the first 50 bases of exon 3 of pp1c are shown in capital letters. The entire intron 2 is shown in lowercase. The premature stop codon TAA introduced by intron 2 is highlighted in red.

• Supplemental Figure S3 (Adobe PDF) -

  Fig. S3. The V³⁶ to M³⁶ change in Mypt1 compromises the binding of Mypt1 to PP1c. Interactions of the KVxF motif (cyan) of Mypt1 with PP1c (gray) in the crystal structure of the chicken Mypt1-PP1c complex (A; PDB code 1S70). Molecular modeling shows that all five possible conformations of methionine in KM³⁶xF are involved in steric clashes with the KVxF-binding pocket of PP1c (B). For simplicity, only three conformations of M³⁶ are shown − in yellow, red and green (B).

• Supplemental Figure S4 (Adobe PDF) -

  Fig. S4. The mypt1^sq181 mutation does not affect the polarization of LPM cells. Sections from WT (A) and mutant (mu) (B) embryos at 29 hpf were stained with a monoclonal antibody to αPKC.

• Supplemental Figure S5 (Adobe PDF) -

  Fig. S5. Bmp signaling is essential for liver development in zebrafish. (A) Immunoblot analyses of dominant-negative BmpR1a-GFP expression using an anti-GFP antibody after heat-shock treatment. Total protein lysates were prepared from heat-shocked embryos at different time intervals as indicated. (B-D) WISH using the lfabp (B), prox1 (C) or ifabp (D) probes and F59 antibody (C) on heat-shocked embryos. Embryos in B and D were heat shocked at 18-21 hpf, in C at 24 hpf.

• Supplemental Table S1 - Adobe PDF