Fig. S1. Conservation of Fosl2 proteins across vertebrate species. Shown is a boxshaded ClustalW2
alignment of human (H. sapiens), mouse (M. musculus), chicken (G. gallus), frog (X. laevis), and zebrafish
(D. rerio) Fosl2 proteins. Black and grey boxes highlight identical and similar amino acids, respectively.
The zebrafish and human proteins are 65% and 82% identical and similar, respectively. These
percentages jump to 85% and 93% within the basic-leucine zipper (bZIP) domain (underlined).
Development • Supplementary information
Development 143: doi:10.1242/dev.126136: Supplementary information
Development 143: doi:10.1242/dev.126136: Supplementary information
Development • Supplementary information
Fig. S2. Hepatobiliary morphogenesis proceeds at a normal rate between 36 hpf and 48 hpf in fosl2
mutants. (A-D) In situ hybridization analysis of nkx2.5 transcripts in control sibling (CTRL; A,B) and fosl2/- (C,D) embryos at 36 hours post fertilization (hpf) (A, C; n=>9 embryos) and 48 hpf (B,D). (E) Bar graph
showing the average sizes of the nkx2.5 signal in 48 hpf control (n=18) and mutant (n=26) embryos.
Error bars represent one standard deviation. n.s., not significant. H, heart. HB, hepatobiliary system.
Fig. S3. Analysis of proliferation and apoptosis in fosl2 mutants. (A,B) Confocal images of 36 hours
post-fertilization (hpf) control sibling (CTRL; A; n=4) and fosl2-/- (B; n=4) Tg(nkx2.5:ZsYellow) embryos
processed for the detection of apoptotic cells (red) using the TUNEL assay. Embryos were coimmunostained to detect the ZsYellow fluorescent protein (green). Although apoptotic cells were
detected within the Z-stacks (arrowheads), they were never found overlapping ZsYellow+ myocardium in
either experimental group. (C,D) Confocal images of the ZsYellow+ SHF in 48 hpf control (C; n=4) and
fosl2fb16-/- (D; n=4) Tg(nkx2.5:ZsYellow) embryos double immunostained for ZsYellow (green) and EdU
(red) following exposure to EdU at 36 hpf for 30 minutes. Embryos were co-stained with DAPI (not
shown) to visualize all nuclei. (E) Bar graph showing the average percentages of DAPI+, ZsYellow+ cells
that were also EdU+. Error bars represent one standard devision. n.s., not significant. Scale bars: 50m
in (A,B) and 25m in (C,D).
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Development 143: doi:10.1242/dev.126136: Supplementary information
Fig. S4. Developmental expression profiles of AP-1 components fosl2, c-jun, and c-fos. (A, B) In situ
hybridization analysis of fosl2 transcripts at the 12 somites stage (ss) (A) and 42 hours post fertilization
(hpf) (B). The embryo shown in (B) is co-stained for the myocardial transcript cmlc2. The arrowhead in
(B) highlights fosl2 expression in pharyngeal mesoderm on the extra-cardiac side of the arterial pole
where SHF progenitors reside. (C-F) Double in situ hybridization analysis of c-jun and cmlc2 transcripts at
26 hpf (C-E) and 36 hpf (F). Double stained embryos shown in (D) were washed with methanol to
remove the cmlc2 signal and photographed again (E). C-jun+ cells were observed on either side of the
arterial pole. Arrowhead in (F) highlights c-jun expression in pharyngeal mesoderm on the extra cardiac
side of the arterial pole where SHF progenitors reside. In all cases, greater than 20 embryos were
analyzed.
Development • Supplementary information
Development 143: doi:10.1242/dev.126136: Supplementary information
Fig. S5. fosl2-/- adults are indistinguishable from siblings with regard to animal size, heart size, heart
morphology, and cardiac regenerative capacity. (A,B) Photographs of 6-month old sibling control (CTRL;
A; n=6) and fosl2 null (B; n=6) zebrafish showing their equivalent sizes. (C,D) Photographs of hearts
dissected from 6 month old control (C; n=6) and fosl2 null (D, n=6) zebrafish showing their equivalent
sizes and morphologies. (E,F) AFOG-stained cardiac sections from 6 month-old control (E; n=4 hearts)
and fosl2 null (F; n=4 hearts) hearts on day 45 post apical resection. Mutant hearts regenerated (*)
resected myocardium normally. A, atrium. V, ventricle. BA, bulbus arteriosus. Scale bars: 1mm in (A,B),
200m in (C-F).
Development • Supplementary information
Development 143: doi:10.1242/dev.126136: Supplementary information
Fig. S6. Analysis of fosl2 morphants. (A,B,D,E) Confocal images of hearts in 48 hpf (A,B) and 72 hpf (D,E)
control sibling (CTRL; A; n=7 and D; n=12) and fosl2 morphant (B; n=6 and E; n=7) Tg(cmlc2:dsRed2-nuc)
embryos. (C,F) Bar graphs showing average total, atrial, and ventricular cardiomyocyte numbers in each
experimental group at 48 hpf (C) and 72 hpf (F). Error bars represent one standard deviation. n.s., not
significant. ***p<0.001. (G-I) Confocal images of ventricular and outflow tract (OFT) regions of 72 hpf
control (G) and fosl2 morphant (H, I) embryos co-stained with antibodies recognizing striated muscle
(red) or Eln2+ OFT smooth muscle (green) that demonstrate normal (G), reduced (H), or absent (I) OFT
Eln2. White lines approximate the sizes of the OFTs. (J) Bar graph showing the percentages of morphant
(WT embryos injected with the fosl2 morpholino; n=18 total) and morphant-mutant (fosl2-/- embryos
injected with the morpholino; n=25 total) embryos that fall into each phenotypic class shown in (G-I).
Uninjected (Un) WT (n=22) and fosl2-/- embryos (n=22) were used as controls. (K) Western blots of 30
hpf whole-embryo lysates from control and morphant embryos probed with Fosl2 (zFosl2) or -Tubulin
(-Tub) antiserum. V, ventricle, A, atrium.
Development • Supplementary information
Development 143: doi:10.1242/dev.126136: Supplementary information
Development 143: doi:10.1242/dev.126136: Supplementary information
Development • Supplementary information
Fig. S7. Analysis of SHF-mediated cardiomyocyte accretion between 36 and 48 hours post-fertilization
in fosl2-/- embryos. (A-G) Cardiomyocyte conversion assay. Control sibling (n=10) and fosl2-/- (n=11)
Tg(myl7:nlsKiKGR) embryos were photoconverted at 36 hours post fertilization (hpf) and imaged by
confocal microscopy at 48 hpf in the red (A, B) and green (B, E) channels. Merged images are shown in
(C, F). (G) Bar graph showing the average numbers of green and red positive ventricular cardiomyocytes
and green only cardiomyocytes. Error bars represent one standard deviation. ***p<0.001. n.s., not
significant.
Fig. S8. Analysis of ventricular cardiomyocyte proliferation in fosl2-/- animals. (A,B) Confocal images of
72 hpf control sibling (CTRL; A, n=5) and fosl2fb16-/- (B; n=4) Tg(nkx2.5:ZsYellow-nuc) embryos exposed to
BrdU between 48 hpf and 72 hpf and double immunostained for ZsYellow protein (green) and BrdU
(red). The arrows highlight double positive nuclei. (C) Bar graph showing the average percentages of
ZsYellow+ ventricular nuclei that were BrdU+. Error bars represent one standard deviation. n.s., not
significant. Scale Bars: 25m.
Development • Supplementary information
Development 143: doi:10.1242/dev.126136: Supplementary information
Development 143: doi:10.1242/dev.126136: Supplementary information
Development • Supplementary information
Fig. S9. In situ hybridization analysis of c-fos. Double in situ hybridization analysis of c-fos and cmlc2
transcripts at 36 hpf (A), 42 hpf (B) and 48 hpf (C). Greater than 20 embryos were evaluated at each
developmental stage.