FIGURE SUMMARY
Title

tbx6l and tbx16 are redundantly required for posterior paraxial mesoderm formation during zebrafish embryogenesis

Authors
Morrow, Z.T., Maxwell, A.M., Hoshijima, K., Talbot, J.C., Grunwald, D.J., Amacher, S.L.
Source
Full text @ Dev. Dyn.

Zebrafish tbx6l mutants do not express Tbx6l protein and are homozygous viable. A: Sequence of tbx6l showing left and right TALEN binding sites (blue) and the spacer region (black) for wild-type, tbx6lz34, and tbx6lz35 alleles. Uppercase letters indicate exon 3 coding sequence; lowercase letters indicate intron sequence. Dashes indicate deleted nucleotides. A PvuII site (underlined) is deleted in both mutant alleles. B: Diagram of wild-type Tbx6l protein and predicted truncated Tbx6l proteins encoded by each mutant allele, showing the T-box domain (black box and black text), aberrant sequence caused by the introduced frameshift in mutant alleles (gray box and gray text), and the immunogen peptide (green box). C,D: Dorsal views of 14- to 16-somite stage wild-type (C) and tbx6lz34 mutant (D) embryos processed by immunofluorescence with Tbx6l antibody. E: Western blot of protein extracts prepared from wild-type (WT) and tbx6z35 homozygous mutant embryos, probed sequentially with anti-Tbx6l antibody (left blot), then with anti-alpha Tubulin antibody as a loading control (right blot). The inferred position of Tbx6l, which runs slightly higher than the predicted 55-kD mass, is marked by a single asterisk, and alpha-Tubulin (50 kD) by double asterisks. F,G: Live images of 24-hpf wild-type (F) and tbx6lz35 mutant (G) embryos. Scale bar in D (for C,D) = 25 μm. Scale bar in G (for F,G) = 250 μm.

Loss of tbx6l does not enhance the msgn1 mutant phenotype. A–D: In situ hybridization for the cb1045/xirp2a somite boundary marker gene in 36-hpf wild-type (A), tbx6l mutant (B), msgn1 mutant (C), and tbx6l; msgn1 double-mutant (D) embryos that were raised together at standard temperature and fixed several hours after somitogenesis is normally completed. Lateral views of posterior tail are shown. E: Graph showing distribution of somite number per embryo for each genotype. F: Graph showing average somite number per embryo for each genotype. * P < 0.0005, when compared to wild-type.

tbx6l; tbx16 double mutants have an enlarged tail bud and lack tail somites. A–C′: Brightfield images of 24-hpf live wild-type (A, A′), tbx16 (spt) mutant (B,B′), and tbx6l; tbx16 double-mutant (C,C′) embryos. tbx6l single-mutant embryos are not shown here as they are indistinguishable from wild-type embryos (see Fig. 1F,G). Black dots in the magnified views (A′–C′) indicate morphologically visible tail somites. D–F: In situ hybridization for msgn1 at the 16-somite stage. Scale bar in A (for A–C) = 250 μm. Scale bar in A′ (for A′–C′) = 100 μm. Scale bar in F (for D–F) = 50 μm.

tbx6l and tbx16 have partially redundant roles in formation of tail paraxial mesoderm. A–R: In situ hybridization for ta (A–C), tbx6 (fss) (D–F), myoD (G–I), pax2a (J–L), fli1a (M–O), and lmo2 (P–R) at 24 hpf (A–C;G–R) and the 14- to 16-somite stage (D–F). Not shown are tbx6l single-mutant embryos as they are indistinguishable from wild-type embryos. In panel J, the row of pax2a-expressing pronephros cells is indicated by an asterisk (*), and the row of pax2a-expressing spinal cord neurons is indicated by a double asterisk (**). Scale bars in C (for A–C) and R (for G–R) = 100 μm. Scale bar in F (for D–F) = 50 μm.

A single neural tube forms in tbx6l; tbx16 double mutants. A–F: In situ hybridization for isl1 (A–C) and sox2 (D–F) at 24 hpf. G–L: Confocal projections of embryos labeled for acetylated tubulin (green), fast muscle (F310; red), and myosin heavy chain (A4.1025; blue) in lateral (G–I) and dorsal (J–L) views. Not shown are tbx6l single-mutant embryos as they are indistinguishable from wild-type embryos. Scale bars in A (for A–F) and G (for G–I) = 100 μm. Scale bar in J (for J–L) = 50 μm.

Acknowledgments
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