Platelet-derived growth factor (PDGF) signaling directs cardiomyocyte movement toward the midline during heart tube assembly

Figures

• 

  Figure 1.. Cardiac fusion defects in refuse-to-fuse (ref) mutants. (A,B) Three-dimensional reconstructions depict wild-type (wt) and ref mutant hearts expressing the myocardial reporter transgene Tg(myl7:egfp) at 48 hpf. In contrast to the normal contours of the wt heart (A), the ref mutant heart (B) often displays a bifurcated, two-lobed ventricle and a misshapen atrium. See Figure 1—figure supplement 1 and Table 1 for more information on the range of cardiac phenotypes observed in ref mutants at 48 hpf. A: atrium; V: ventricle. (C–G) Dorsal (C–E) and ventral (F,G) views, anterior to the top, of wt (C,F) and ref (D,E,G) mutant embryos displaying the expression of myl7 at the 22 somite stage (22 s; C–E) and the localization of ZO-1 at the 18 somite stage (18 s; F,G). (C–E) In ref mutants at this stage, cardiomyocytes typically fail to fuse at the midline (D) or only fuse posteriorly (E). Note that the ref mutation is incompletely penetrant, although its penetrance is more evident at 20 s than at 48 hpf (Table 2), suggesting that some ref mutants recover as development proceeds. (F,G) ZO-1 localization highlights junctions forming within the maturing epithelium of the ALPM in both wt and ref…

• 

  Figure 1—figure supplement 1.. ref mutant embryos display a range of cardiac defects. (A,B) Lateral views of live wild-type (wt, A) and ref mutant (B) embryos at 48 hpf. ref mutants exhibit a mild pericardial edema. (C–F) Lateral views from the right side of wt (C) and ref mutant (D–F) hearts reveal a variety of abnormal cardiac morphologies in ref mutants. The most commonly observed phenotype was a bifurcated ventricle (D), some other embryos exhibited abnormal looping (E) or a severely shrunken heart (F), and a few embryos had cardia bifida (not shown). See Tables 1 and 2 for more information regarding the incomplete penetrance and variable expressivity of the ref mutant phenotype. (C'–F') Cartoons outline the cardiac morphologies shown in C–F, with the ventricle in magenta and the atrium in green. DOI: http://dx.doi.org/10.7554/eLife.21172.004

• 

  Figure 1—figure supplement 2.. The anterior endoderm forms normally in ref mutant embryos. (A–D) Lateral views, dorsal to the right, depict the expression of axial at 8.5 hpf (A,B) or sox17 at 8 hpf (C,D). The number and distribution of endoderm precursor cells is similar in wt (A,C) and ref mutant (B, n = 8/8; D, n = 14/14) embryos during gastrulation stages. (E–H) Dorsal views, anterior to the top, depict the anterior endoderm, visualized with the expression of axial at 24 hpf (E,F) or the endodermal reporter transgene Tg(sox17:egfp) at 18 s (G,H). The width and morphology of the anterior endoderm is similar in wt (E,G) and ref mutant (F, n = 13/13; H, n = 7/7) embryos during the stages when cardiac fusion takes place. Scale bars: 60 μm. DOI: http://dx.doi.org/10.7554/eLife.21172.005

• 

  Figure 1—figure supplement 3.. Endocardial cells move to the midline in ref mutant embryos. (A,B) Dorsal views, anterior to the top, depict the expression of the endothelial reporter transgene Tg(fli1a:negfp) in the endocardial precursor population at 18 s (n = 8). In both wt (A) and ref mutant (B) embryos, the endocardial precursors move to the midline during the process of cardiac fusion. Scale bar: 60 μm. DOI: http://dx.doi.org/10.7554/eLife.21172.006

• 

  Figure 2.. ref is a loss-of-function mutation in pdgfra. (A) Polymorphic markers (z21170, kdr_e28, 5’scfd2, z14614) were used to map meiotic recombination events, narrowing the region containing the ref mutation to <0.1 cM on linkage group (LG) 20. (See also Table 3). Fractions indicate frequencies of proximal (magenta) and distal (green) recombination between markers and ref. Six annotated genes are present in this region (GRCv10); sequence analysis of kdr, kita, gsx2, lnx1, and fip1l1a in ref mutants revealed only missense mutations that led to conserved amino acid changes. (B) RT-PCR spanning exons 13–19 of pdgfra generates a single, properly spliced product from homozygous wt embryos and multiple, smaller products from ref mutant embryos. Sequencing revealed that exon 14 was either omitted or truncated in these smaller products; in all cases, the observed missplicing would result in a frameshift followed by a premature stop codon. Although we did not detect any normally spliced pdgfra products in ref mutants, we cannot rule out the presence of low levels of wild-type mRNA. RT-PCR of gapdh demonstrates use of comparable amounts of template. (C,D) Sequencing the e14i15 exon-intron…

• 

  Figure 2—figure supplement 1.. ref mutants display craniofacial defects. (A–F) Lateral (A–C) and ventral (D–F) views of embryos at 6 days post-fertilization (dpf), stained with Alcian blue to show the dorsal and ventral cartilage in the head. Embryos homozygous for ref (B,E; n = 9/9) or transheterozgous for ref and b1059 (C,F; n = 5/6) display similar dorsal cartilage formation defects. Specifically, the dorsal ethmoid plate is diminished or absent in these mutants. Scale bars: 60 μm. (D'–F') Cartoons illustrate the dorsal cartilage structures shown in D–F. DOI: http://dx.doi.org/10.7554/eLife.21172.010

• 

  Figure 2—figure supplement 2.. Comparison of cardiac fusion phenotypes resulting from alteration of PDGF signaling. Bar graph compares severity of cardiac fusion phenotypes observed at 22 s in three sets of experiments: one set of experiments comparing ref homozygous mutant embryos, b1059 homozygous mutant embryos, ref/b1059 transheterozygous mutant embryos, pdgfra MO-injected embryos, and wt sibling embryos (as in Figure 2G–J); one set of experiments comparing embryos treated with DMSO and embryos treated with Pdgfr inhibitor V (as in Figure 5—figure supplement 1); and one set of experiments comparing wt embryos and Tg(hsp70l:pdgfaa-2A-mCherry) embryos, following heat shock at the tailbud stage (as in Figure 7Q–S). Phenotypes were classified as fused (blue; as in Figure 2G), almost fused (light blue; no representative image shown), only posterior fusion (yellow; as in Figure 2H), or no fusion (red; as in Figure 2J). The n for each data set is presented under its corresponding bar. The severity of the cardiac fusion defects appears similar in ref homozygous mutant embryos, b1059 homozygous mutant embryos, ref/b1059 transheterozygous mutant embryos, and embryos treated with Pdgfr inhibitor V. In contrast, the…

• 

  Figure 3.. Pdgfra mouse mutants display defects in heart tube assembly. (A–E') Ventral views (A,B,D,E), lateral views (A',B',C), and transverse sections (A'',E') depict Nkx2-5 expression in wt (A–A'') and Pdgfra homozygous mutant (B–E') mice on a C57BL/6J background at E9.5. Some Pdgfra mutants display relatively mild cardiac defects (B,B'), including defects in heart tube rotation. Other Pdgfra mutants display severe cardiac defects that could result from hindered cardiac fusion (C–E'). These defects include incomplete heart tube assembly (C,D) with two inflow/common atrial regions (red arrows) and a single ventricle (black arrowhead) or with cardia bifida (E,E'; arrows indicate unfused ventricles and atria). See Tables 4 and 5 for additional information on the prevalence of mutant phenotypes. A: atrium; AVC: atrioventricular canal; LA: left atrium; LV: left ventricle; OFT: outflow tract; RA: right atrium; RV: right ventricle; V: ventricle. Scale bars: 100 μm. (F–I) Ventral views depict localization of Pdgfra mRNA (F–H) and PDGFRα protein (I) in wt embryos from E7.5 to E8.5. Pdgfra expression is seen in anterior lateral plate mesoderm (ALPM) at E7.5 (F) and persists in the caudal region of…

• 

  Figure 4.. pdgfra is expressed within the ALPM while cardiac fusion is underway. (A–D) Dorsal views, anterior to the top, depict pdgfra expression in wt embryos at 10 s (A), 12 s (B), 14 s (C), and 18 s (D). Arrows (A) indicate pdgfra expression in the ALPM and the neural crest (NC). Asterisk (D) denotes position of the myocardium by 18 s; although pdgfra is expressed in the myocardial precursors within the ALPM at earlier stages, its expression in these cells appears to be gone by this time point. (E–J) Comparison of hand2 (green) and pdgfra (red) expression patterns demonstrates their overlap in the wt ALPM at 10 s. (E–G) Three-dimensional confocal reconstructions of dorsal views, anterior to the top, focused on the left side of the ALPM (area outlined by a dashed box in (A). Arrows (E) indicate pdgfra expression in the ALPM and the NC. (H–J) Single transverse (XZ) sections from (E–G), respectively. Yellow arrow (H) indicates overlap of hand2 and pdgfra expression in the ALPM. (K–P) Comparison of axial (green) and pdgfra (red) expression patterns demonstrates lack of pdgfra expression in the axial-expressing anterior endoderm in wt embryos at 12 s. (K–M) Three-dimensional confocal…

• 

  Figure 5.. pdgfra influences the movement of the ALPM after the 15 somite stage. (A–F) Dorsal views, anterior to the top, depict expression of hand2 in the wt (A–C) and ref mutant (D–F) ALPM from 12 to 20 s. The morphology and position of the ALPM are indistinguishable in wt (A) and ref mutant (D) embryos at 12 s. After 15 s (B,C,E,F), disrupted movement of the ALPM is evident in ref mutants. Scale bar: 60 μm. (G) Graph illustrates the average distance between the two sides of the ALPM in wt and ref mutant embryos from 8 to 20 s. In each embryo, the distance between the sides of the ALPM was calculated by measuring the distance between the medial edges of the hand2-expressing domains at three equidistant points (200 μm apart) along the anterior-posterior axis. The largest of those three measurements was selected as representative of the maximum distance between the bilateral ALPM domains for that embryo. Dots represent the selected measurements from individual embryos. The distance between the bilateral sheets in ref mutant embryos begins to diverge significantly from wt after 15 s. Error bars represent the standard error. Asterisks indicate p<0.05 (Student’s t-test): p=0.99 at 8 s; p=0.58…

• 

  Figure 5—figure supplement 1.. PDGF signaling is required after gastrulation for proper cardiac fusion. (A,B) Dorsal views, anterior to the top, display myl7 expression at 22 s in representative embryos treated with DMSO (A) or Pdgfr inhibitor V (B) from the tailbud stage until 22 s. DMSO-treated control embryos exhibited normal cardiac fusion, whereas treatment with Pdgfr inhibitor V disrupted cardiac fusion. See Figure 2—figure supplement 2 for additional information on the prevalence of these phenotypes. Scale bar: 60 μm. DOI: http://dx.doi.org/10.7554/eLife.21172.018

• 

  Figure 5—figure supplement 2.. Overexpression of pdgfaa influences ALPM movement by the 15 somite stage. (A–C) Dorsal views, anterior to the top, depict expression of hand2 in the ALPM of Tg(hsp70l:pdgfaa-2A-mCherry) embryos from 12 to 20 s, following heat shock at the tailbud stage. See Figure 5A–C for images of representative wt embryos at the same stages. Overexpression of pdgfaa at the tailbud stage does not seem to affect the morphology and position of the ALPM at 12 s (A), but it does disrupt ALPM movement by 16 s (B) and 20 s (C). Scale bar: 60 μm. (D) Graph illustrates the average distance between the two sides of the ALPM from 12 to 20 s in Tg(hsp70l:pdgfaa-2A-mCherry) embryos, following heat shock at the tailbud stage, shown together with the data presented in Figure 5G. The distance between the sides of the ALPM domains in transgenic embryos was measured as described for Figure 5G. Dots represent measurements from individual embryos. The distance between the bilateral sheets of ALPM in Tg(hsp70l:pdgfaa-2A-mCherry) embryos begins to diverge significantly from wt by 15 s. Error bars represent the standard error. Asterisks indicate p<0.05 in comparison to wt embryos (Student’s T-test): p=0.47 at 12 s;…