wtip expression is identical to PE-specific markers during zebrafish heart development. Lateral view of (A-D) 48 hpf and (E-H) 4 dpf whole-mount in situ hybridization of (A,E) wtip, (B,F) wt1a, (C,G) tcf21 and (D,H) tbx18. At 48 hpf, (A) wtip, and PE markers (B) wt1a-, (C) tcf21- and (D) tbx18- positive cells were in the clusters near the sinus venosus, between the atrium and the yolk and near the AV junction, in contact with the ventral surface of the heart (yellow arrowhead in A-D). At 48 hpf, tcf21 expression occurred in the pharyngeal arch (black arrows in C) and tbx18 expression occurred in the pectoral fin (black asterisk in D). By 4 dpf, the wt1a-, tcf21- and tbx18-positive cells spread over the heart to cover the myocardium (yellow arrowhead in F-H). By 4 dpf, wtip-, tcf21- and tbx18-positive cells appeared to spread over the heart to cover the myocardium along the dorsal surface of the heart (yellow arrowhead in E-H). (I,J) Double immunofluorescence for the cilia marker acetylated α-tubulin (green) and Wtip (red) in confocal projections of the zebrafish PE at 48 hpf. (J) No Wtip signal was detectable in wtipMO knockdown embryos, confirming antibody specificity. (K-P, red) Localization of Wtip in the basal bodies of cilia was confirmed by double immunostaining with Wtip antibody and either (L,M, green) anti-γ-tubulin or (O,P, green) anti-centrin, which are basal body markers in heart. 4′,6-diamidino-2-phenylindole was used to counterstain nuclei (blue). Scale bar=10 µm. wtip, Wilms tumor 1 interacting protein; PE, proepicardial organ; hpf, hours post-fertilization; dpf, days post fertilization; wt1a, Wilms tumor 1a; tcf21, transcription factor 21; tbx18, Tbox 18; wtipMO, wtip morpholino oligonucleotide; conMO, control MO; AT, acetylated α-tubulin.

Wtip is required for PE specification. Ventral view of 48 hpf whole-mount in situ hybridization of (A-C) tcf21 and (D-F) tbx18 in (A,D,G,H)control (1.035 pmol/embryo) and (B,C,E-H) wtip morphant embryos (1.035 pmol/embryo). Both tcf21 and tbx18 express specifically in the PE in the control embryos (yellow arrowhead in A, D), but is reduced or absent in wtip morphant embryos. tcf21 expression in the pharyngeal arch (black arrow in A-C) and tbx18 expression in the pectoral fin (black asterisk in D-F) in wtip morphants are unaffected. wtip, Wilms tumor 1 interacting protein; PE, proepicardial organ; hpf, hours post fertilization; tcf21, transcription factor 21; tbx18, Tbox 18; conMO, control morpholino oligonucleotide.

Ectopic expression of tcf21 and tbx18 in the cardiac and pharyngeal arch regions by wtip mRNA overexpression. Ventral view of 48 hpf whole-mount in situ hybridization of (A,B) tcf21 and (C,D) tbx18) in (A,C) control and (B.D) wtip mRNA-injected embryos (92 pg/embryo). The ectopic expression of tcf21 (black arrows in B) and tbx18 (black arrows in D) expression in the heart region compared with control tcf21 (yellow arrowhead in A) and tbx18 (yellow arrowhead in C) expression in the PE region of the heart. tbx18 expression in the pectoral fin (black asterisk in C) was unaffected in wtip mRNA-injected embryos. tcf21, transcription factor 21; tbx18, Tbox 18; wtip, Wilm′s tumor 1 interating protein; hpf, hours post fertilization; PE, proepicardial organ.

Wt1a and Wtip cooperate for PE formation. Ventral view of 48 hpf whole-mount in situ hybridization of (A,C,E,G,I,K) tcf21 and (B,D,F,H,J,L) tbx18 expression in control (yellow arrowhead, A,B), high concentration of wtipMO (black arrow, C,D; 1.035 pmol/embryo), sub-threshold concentration of wtipMO (yellow arrowhead, E,F; 0.259 pmol/embryo), high concentration of wt1aMO (black arrow, G,H; 1.15 pmol/embryo), sub-threshold concentration of wt1aMO (yellow arrowhead, I,J; wt1aMO, 0.288 pmol/embryo), sub-threshold concentrations of wt1aMO (1.15 pmol/embryo)+wtipMO (0.259 pmol/embryo; black arrow, K,L) in PE at 48 hpf. With high concentrations of wt1aMO, PE marker expressions were reduced or absent (black arrow, for tcf21; G,M, and for tbx18; H,N). With wtipMO, PE marker expressions were reduced (for tcf21, M and for tbx18, N) or absent (black arrow, for tcf21; C,M, and for tbx18; D,N). Sub-threshold doses for each MO had no effect on tcf21 (yellow arrowhead, E,I,M) or tbx18 (yellow arrowhead, F,J,N) expression in PE. In the wtip and wt1a double morphants, tcf21 and tbx18 were severely reduced (black arrow, for tcf21; K,M and for tbx18; L,N) or no longer expressed (for tcf21 and for tbx18) in the cardiac region. These data indicate that Wtip is associated with Wt1a and influences PE specification. Wt1a, Wilm′s tumor 1a; Wtip, Wilm′s tumor 1 interacting protein; PR, proepicardial organ; hpf, hours post fertilization; tcf21, transcription factor 21; tbx18, Tbox 18; wtipMO, wtip morpholino oligonucleotide.

Wtip is required for AV boundary formation, but does not affect chamber patterning. Hearts of transgenic Tg(myl7:EGFP) embryos were visualized in 48 hpf (A) control or (B) wtip morphant embryos (1.035 pmol/embryo) to assess the extent of cardiac looping. (C) Diagram depicting the cardiac LA, defined as the degree of difference in the anterior/posterior body axis and the plane of the AV junction. (D) Wtip depletion decreases the mean LA by 48 hpf. The mean ± standard error is shown. *P<0.000359. n=10 embryos/treatment. 48 hpf whole-mount in situ hybridization in (E,F,I,J,M,N,Q) control and (G,H,K,L,O,P,L) wtip morphant embryos (1.035 pmol/embryo) of (E-H) myl7, (I-L) amhc, (M-P) vmhc and (Q,R) nppa. (E,G,I,K,M,O,Q,R) Ventral view and (F,H,J,L,N,P) lateral view. Wtip does not regulate chamber patterning, but regulates nppa expression to maintain chamber myocardium expression and exclusion from the myocardium of the AV boundary. Wtip, Wilm′s tumor 1 interacting protein; AV, atrioventricular; myl7, myosin light chain 7; EGFP, enhanced green fluorescent protein; hpf, hours post fertilization; LA, looping angle; amhc, α myosin heavy chain; vmhc, ventricular myosin heavy chain; nppa, natriuretic peptide A; conMO, control morpholino oligonucleotide; A, anterior; P, posterios; avj, AV junction.

Wtip is required for proper valve development. Sagittal histological section of (A-C) 48 hpf and (D-F) 72 hpf zebrafish embryo hearts. (B,E) wtip morphants (1.035 pmol/embryo) display pericardial edema and (C,F) wtip mRNA (92 pg/embryo) can rescue pericardial edema. Scale bar=10 µm. Wtip, Wilm′s tumor 1 interacting protein; hpf, hours post fertilization; conMO, control morpholino oligonucleotide.

Wtip is required for early left-right patterning. Dorsal views, anterior to the bottom of the panels. (A-D,K) lefty1 and lefty2, (E-H,L) spaw and (I,J,M) bmp4 expressions in (A-J) 22-somite stage embryos. Normally, (A,E,I) left1+2, spaw and bmp4 transcripts expressed in the left cardiac lateral plate mesoderm, (B,F,J) have bilateral, (C,G) right and (D,H) absent expression in wtip morphants (1.035 pmol/embryo). Bar chart details the left-right patterning for control and wtip morphants. Wtip, Wilm′s tumor 1 interacting protein; lefty1, left right determination factor 1; spaw, southpaw; bmp4, bone morphogenic factor 4; conMO, control morpholino oligonucleotide.