Candidate Heterotaxy Gene FGFR4 Is Essential for Patterning of the Left-Right Organizer in Xenopus

Abstract

Congenital heart disease (CHD) is the most common birth defect, yet its genetic causes continue to be obscure. Fibroblast growth factor receptor 4 (FGFR4) recently emerged in a large patient exome sequencing study as a candidate disease gene for CHD and specifically heterotaxy. In heterotaxy, patterning of the left-right (LR) body axis is compromised, frequently leading to defects in the heart's LR architecture and severe CHD. FGF ligands like FGF8 and FGF4 have been previously implicated in LR development with roles ranging from formation of the laterality organ [LR organizer (LRO)] to the transfer of asymmetry from the embryonic midline to the lateral plate mesoderm (LPM). However, much less is known about which FGF receptors (FGFRs) play a role in laterality. Here, we show that the candidate heterotaxy gene FGFR4 is essential for proper organ situs in Xenopus and that frogs depleted of fgfr4 display inverted cardiac and gut looping. Fgfr4 knockdown causes mispatterning of the LRO even before cilia on its surface initiate symmetry-breaking fluid flow, indicating a role in the earliest stages of LR development. Specifically, fgfr4 acts during gastrulation to pattern the paraxial mesoderm, which gives rise to the lateral pre-somitic portion of the LRO. Upon fgfr4 knockdown, the paraxial mesoderm is mispatterned in the gastrula and LRO, and crucial genes for symmetry breakage, like coco, xnr1, and gdf3 are subsequently absent from the lateral portions of the organizer. In summary, our data indicate that FGF signaling in mesodermal LRO progenitors defines cell fates essential for subsequent LR patterning.

Keywords: FGF signaling; Xenopus; congenital heart disease; gastrulation; heterotaxy; left-right patterning.

Figure 1

Fgfr4 F0 CRISPR editing disrupts LR development. (A,B) fgfr4 CRISPR tadpoles display organ laterality defects. Ventral view of a stage 45 live tadpole with a cardiac L-loop (B; outlined) and inverse gut coiling (B; dashed line and red arrowhead). (C) Percentages of tadpoles with laterality defects (L-loops and inverse gut coiling) for three different CRISPRs; tadpoles with L-loops and inverse gut coils were scored; only tadpoles with inverse but otherwise intact gut coiling were considered; animals with completely uncoiled guts were scored as normal, as this phenotype occurs in the control population as well. Tadpoles with both cardiac and gut looping defects were only counted once in this analysis. (D) Pitx2c expression in the LPM of tailbud stage animals (stage 28); red arrowhead indicates absent expression. (E) Percentages of stage 28 animals with different pitx2c phenotypes; ^****p < 0.0001, ^***p < 0.001.

Figure 2

GRP patterning is defective in fgfr4 CRISPR mutants. Most representative expression patterns of pre-somitic GRP markers coco, xnr1, and gdf3 in stage 17 (A,C,E) and stage 19 (B,D,F). GRPs of control and fgfr4 F0 CRISPR animals; ventral view of GRPs, anterior is to the top. Graphs show percentages of embryos with differential expression patterns of coco, xnr1, and gdf3. ^**p < 0.01, ^***p < 0.001, ^****p < 0.0001.

Figure 3

GRP morphology and identity are altered in fgfr4 CRISPR embryos. (A–D) GRPs of fgfr4 CRISPR animals are morphologically distinct, as shown by phalloidin (actin) and anti-acetylated tubulin (cilia) stain; phenotypes ranging from mild (B) to severe (C,D), depending on loss of small mesodermal ciliated cells. (E–H) Higher magnification of GRPs shows loss of ciliated GRP area in fgfr4 CRISPR embryos (G, H). (I–K) The pre-somitic, myoD positive portion of the GRP (outlined) is drastically reduced in fgfr4 CRISPR embryos, even in embryos in which the overall GRP morphology is preserved (J). (L) Quantification of total GRP area, defined morphologically by small, ciliated cells, is reduced in fgfr4 CRISPR embryos. (M) The myoD positive area of the GRP, normalized to total GRP area, is specifically reduced in fgfr4 CRISPR embryos. Scale bars in (A–D, I–K) = 40 μm, in (E–H) = 20 μm. ^**p < 0.01, ^***p < 0.001.

Figure 4

Fgfr4 expression during early X. tropicalis development, detected by in situ hybridization. (A,B) Whole stage 19 embryos; expression is detected in the head region (2: eyes), lateral plate mesoderm (3), anterior somites (1a) and posterior pre-somitic mesoderm (1b). (C,D) Transcripts were not detected in stage 16 and 19 GRPs (arrowheads). (E,F) Stage 10.5 embryos, whole (E, dorsal view) or bisected through the dorsal midline (F); fgfr4 is broadly expressed in the ectoderm (4) and dorsal marginal zone (5). (G,H) Stage 12 embryos, whole (G, dorso-vegetal view) or bisected (H); fgfr4 is absent from the marginal zone (5), but is expressed in the anterior migrating involuted mesoderm (6).

Figure 5

The paraxial myogenic mesoderm is mispatterned in fgfr4 CRISPR embryos. (A–F, G–J) Dorsal-vegetal views showing expression of an array of mesodermal markers in stage 10 (A–F) and stage 12 (G–J) embryos. Expression of paraxial mesoderm markers myf5 and myoD is perturbed in fgfr4 CRISPR embryos. (K, L) Xbra expression in embryos bisected through their dorsal midline at stages 10.5 and 12; red arrowheads point at xbra expression in the involuted mesoderm. Graphs show percentages of embryos with normal vs. abnormal expression of each marker; N = 32–40 embryos per Control or fgfr4 CRISPR; ^**p < 0.001, ^***p < 0.0001.