Mutation of the zebrafish tmem88a gene.

(A) Schematic structure of the zebrafish tmem88a gene shows three exons and two introns. TALEN pairs TAL1 (green) and TAL2 (magenta) were designed and assembled to target the coding region of exon2. There is a ScaI endonuclease restriction site within the spacer region (orange). (B) A tmem88a mutant line with an 8 bp deletion (Δ8) was generated. (C) Predicted translated sequences of wild-type Tmem88a and Tmem88aΔ8 show frame-shift after residue 48 (red), causing truncation of the protein and loss of the trans-membrane domains (blue) and the Dishevelled-binding domain (teal). (D) Brightfield images of control Tg(fli1a:egfp) and tmem88aΔ8 embryos at 24 hpf (n = 67, n = 53 respectively), 2 dpf (n = 54, n = 35), and 5 dpf (n = 51, n = 18) as labelled. (E) Sequence alignment shows that the tmem88a e2i2 SBMO is specific to tmem88a and illustrates a 9 bp mismatch with tmem88b mRNA.

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Cardiovascular associated genes are downregulated in tmem88aΔ8 embryos.

(A) qRT-PCR showing the expression of key cardiovascular genes in tmem88Δ8 mutants compared to Tg(fli1a:egfp) controls at 13 hpf. (B) In situ hybridisation for fli1a, gata1a, and myoD in control and tmem88aΔ8 mutants as labelled. Gata1a expression is denoted with black error heads, reduced or absent expression is shown with grey or white arrowheads respectively. Somites are marked by asterisks. (C) qRT-PCR showing the expression of key cardiovascular genes in tmem88Δ8 mutants compared to Tg(fli1a:egfp) controls at 48 hpf. All P-values determined by Unpaired Student’s t test. Error bars denote the standard deviation of three biological replicates. P-values determined by Unpaired Students t test. * = p<0.05; ** = p<0.01; *** = p<0.001; unlabelled bars show no significant difference (p>0.05). fli1a, friend leukaemia integration 1a; gata1a, GATA-binding factor 1a; hbbe1, haemoglobin beta-embryonic 1; hpf, hours post fertilisation; lyz, lysozyme; runx1, runt-related transcription factor 1; scl, stem cell leukaemia; spi1, spleen focus forming virus (sffv) and proviral integration oncogene 1b (also known as pu.1).

Myelopoiesis was not affected in tmem88aΔ8 zebrafish mutants.

(A-B) Sudan Black B staining of neutrophils at 48 hpf in Tg(fli1a:egfp) controls (A, n = 21), with the highlighted tail region magnified (A′) and tmem88aΔ8 mutants (B, n = 21), magnified in (B′). (C) The number of neutrophils was counted for each condition represented in a Tukey box and whisker plot. Neutrophil numbers were not significantly changed between the two groups (p = 0.6769, Unpaired Student’s t test). A, anterior; h, hours post fertilisation; D, dorsal; n.s., not significant.

Erythropoiesis was not affected in tmem88aΔ8 zebrafish mutants.

(A-D) o-Dianisidine staining of erythrocytes at 48 hpf in uninjected Tg(fli1a:egfp) controls (A, n = 16) or controls injected with tmem88a SBMO (B, n = 12), and uninjected tmem88aΔ8 mutants (C, n = 18) or mutants injected with tmem88a SBMO (D, n = 15). (E) The area of o-Dianisine staining was quantified and presented as a percentage of the total yolk area, shown in a Tukey box and whisker plot. No significant difference was found between control embryos and tmem88aΔ8 mutants (p = 0.2710, Unpaired Student’s t test). (F) tmem88b expression is not affected in tmem88aΔ8 mutants. qRT-PCR showing the expression of tmem88b in tmem88Δ8 mutants compared to Tg(fli1a:egfp) controls at 13 and 48 hpf. P-value determined by Unpaired Student’s t test. Error bars denote the standard deviation of three biological replicates. A, anterior; h, hours post fertilisation; D, dorsal; L, lateral; V, ventral; n.s., not significant.