Figure 1

Figure 1

Generation of abcc6a mutants and expression patterns in zebrafish. (A) Schematic of the zebrafish abcc6a gene. Left and right arms of TALEN targeting exon 2 are color-coded to represent 4 repeat-variable diresidues (RVDs) (red, NI = A; green, NN = G; blue, NG = T; orange, HD = C). Nucleotides in red letters and underlined indicate TALEN target sequence. Selected StuI site in the spacer is highlighted with violet. Genomic loci including StuI site is targeted using primer pairs P1 and P2. (B) Deletion mutations generated by TALEN at the abcc6a gene. The WT sequence is shown at the top. Deletions are indicated by the black dashes. Red letters represent partial TALEN target sequences. Sequences are organized as sets of three bases of in-frame codons. (C) Domain structure and predicted amino acid sequence of WT and TALEN mutants. Gray shade indicates regions missing in the mutant proteins. All mutants contain premature stop codon resulting in a lack of most of Abcc6a domains. (D1–D3) Lateral views showing embryo morphology of abcc6a^Δ1/Δ1 (D2) and abcc6a^Δ2/Δ2 mutants (D3) and WT siblings (D1) at 4 dpf. (E1–E3) Whole-mount in situ hybridization (WISH) analysis of abcc6a expression at 4 dpf in abcc6a^Δ1/Δ1 (E2) and abcc6a^Δ2/Δ2 mutants (E3) and wild-type siblings (E1). (F,G) abcc6a expression levels were examined using real-time PCR analysis in abcc6a^Δ1/Δ1 and abcc6a^Δ2/Δ2 mutant larvae at7 dpf (F) and adult hearts (G). Expression levels were normalized to the expression of β-actin. Data are mean ± SEM from 20 larvae and 6 hearts for each group. ** p < 0.01, *** p < 0.001, Student’s t-test (unpaired, two-tailed). (H) Western blot analysis showing ABCC6 protein levels in abcc6a^Δ1/Δ1 and abcc6a^Δ2/Δ2 mutants and WT larvae at 7 dpf. (I–K) WISH showing ubiquitous abcc6a expression at the two-cell stage (I), the 80%-epiboly stage (J), and 8 somite stages (K). Blue arrow indicates dorsal forerunner cells (J) and black arrow indicates Kupffer’s vesicle (K). (L,M) abcc6a is detected in the pronephric duct (L; lime arrowhead), aorta-gonad-mesonephros (AGM) region (L; green arrow), midbrain–hindbrain boundary and hindbrain (L, M; green bracket) and otic vesicle (L, M; orange arrow). (N) At 48 hpf, abcc6a expression is detected in the heart (red arrow), opercula (green arrow), cleithrum (white arrow), and ear (yellow arrow). (O,P) Lateral views of the abcc6a transcripts expression in frozen sections of heart (O) and notochord (P) at 4 dpf. Pink dashes indicate heart outlines. V, ventricle. A, atrium. (Q,R) abcc6a^Δ1/Δ1 mutants exhibit malformed adult body axis curvature and short body length at 8 months post fertilization (mpf). Dotted red line indicates extent of the skull uplift. Small compartment represents 1 mm. (S–V) Micro-CT(MCT) scan (S,U) and Alizarin Red staining (ARS; T, V) of WT and abcc6a^Δ1/Δ1 mutants showing vertebral hyperossification. (W,X) Quantification of the centrum bone volume (W) and bone mineral density (X) in abcc6a^Δ1/Δ1 and abcc6a^Δ2/Δ2 mutants. *** p < 0.001, Student’s t-test (unpaired, two-tailed). Scale bar: 1mm (D1–D3, E1–E3); 200 μm (I–K, O, P, S–V); 500 μm (L,N); 500 μm (M).