Sempou E , Lakhani OA , Amalraj S , Khokha MK .

             fibroblast growth factor receptor signaling pathway
             left/right pattern formation

                congenital heart disease

Xtr Wt + fgfr4 CRISPR (Fig. 1 B C)
Xtr Wt + fgfr4 CRISPR (Fig. 1 B C)
Xtr Wt + fgfr4 CRISPR (Fig. 1 D E)
Xtr Wt + fgfr4 CRISPR (Fig. 2 A)
Xtr Wt + fgfr4 CRISPR (Fig. 2 B)
Xtr Wt + fgfr4 CRISPR (Fig. 2 C)
Xtr Wt + fgfr4 CRISPR (Fig. 2 D)
Xtr Wt + fgfr4 CRISPR (Fig. 2 E)
Xtr Wt + fgfr4 CRISPR (Fig. 2 F)
Xtr Wt + fgfr4 CRISPR (Fig. 3 A-H, L, M)
Xtr Wt + fgfr4 CRISPR (Fig. 3 I-K)
Xtr Wt + fgfr4 CRISPR (Fig. 5 E)
Xtr Wt + fgfr4 CRISPR (Fig. 5 H)
Xtr Wt + fgfr4 CRISPR (Fig. 5 I)
Xtr Wt + fgfr4 CRISPR (Fig. 5 L)
Xtr Wt + fgfr4 CRISPR (Fig. S 2 A, B)

	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.
	Supplementary Figure 1. Injection of CRISPRs 1–3 results in edits in fgfr4 in the genome of F0 frogs. (A–C) ICE (Inference of CRSIPR Edits) analysis of Sanger sequencing data from a genomic 800 bp PCR-fragment that contains the target site for CRISPR-1. (A) Average editing and knock-out scores for CRISPR-1 as identified by the ICE/Synthego software (N = 8 for control/N = 8 for CRISPR). All eight CRISPR F0 tadpoles displayed edits at the fgfr4 target cut site, with overall editing efficiency within a single tadpole ranging from 75 to 95%, and knockout efficiency from 27 to 61%. In contrast, none of the Control tadpoles displayed mutations in the same genomic region. (B) Inferred distribution of Indels around the fgfr4 CRISPR-1 target site within a single F0 CRISPR animal. The x-axis indicates the size of the insertion/deletion and the y-axis shows the percentage of sequences that contained it. (C) Relative contributions of inferred sequences present in a single CRISPR-1 F0 animal. An example for one single animal is shown. The cut site is presented with a black vertical dotted line and the wildtype sequence is marked by a “+” symbol on the far left. (D) T7 Endonuclease assay for fgfr4 CRISPRs-2 (up) and−3 (down). Lanes show PCR products that contain the prospective cut sites, amplified from genomic DNA of different animals (numbered; N = 3–4 per CRISPR, N = 2 per Control), prior to digestion with T7 Endonuclease I (a) and after digestion (b). Red arrowheads point at fragments that are unique in CRISPR animals post-digestion and correspond to predicted fragment sizes: 300/550 bp for CRISPR-2 and 400/600 bp for CRISPR-3.
	Supplementary Figure 2. Cilia number in the GRP of fgfr4 CRISPR-1 F0 embryos. (A) An example of how GRPs were outlined in order to count cilia and measure GRP area. (B,C) The total number of cilia per GRP is reduced in fgfr4 CRISPR embryos (B), however when the cilia numbers are normalized to total GRP area, no difference is visible between control and CRISPR embryos (C). Scale bar = 40 μm.

Albertson, Roles for fgf8 signaling in left-right patterning of the visceral organs and craniofacial skeleton. 2005, Pubmed

Albertson, Roles for fgf8 signaling in left-right patterning of the visceral organs and craniofacial skeleton. 2005, Pubmed
Amaya, FGF signalling in the early specification of mesoderm in Xenopus. 1993, Pubmed , Xenbase
Amaya, Expression of a dominant negative mutant of the FGF receptor disrupts mesoderm formation in Xenopus embryos. 1991, Pubmed , Xenbase
Babu, Left-right asymmetry: cilia stir up new surprises in the node. 2013, Pubmed
Baker, Direct and indirect roles for Nodal signaling in two axis conversions during asymmetric morphogenesis of the zebrafish heart. 2008, Pubmed
Bhattacharya, CRISPR/Cas9: An inexpensive, efficient loss of function tool to screen human disease genes in Xenopus. 2015, Pubmed , Xenbase
Blum, The evolution and conservation of left-right patterning mechanisms. 2014, Pubmed , Xenbase
Boettger, FGF8 functions in the specification of the right body side of the chick. 1999, Pubmed
Campione, The homeobox gene Pitx2: mediator of asymmetric left-right signaling in vertebrate heart and gut looping. 1999, Pubmed , Xenbase
Caron, Wnt/β-catenin signaling directly regulates Foxj1 expression and ciliogenesis in zebrafish Kupffer's vesicle. 2012, Pubmed
Conant, Inference of CRISPR Edits from Sanger Trace Data. 2022, Pubmed
de Campos-Baptista, Nodal signaling promotes the speed and directional movement of cardiomyocytes in zebrafish. 2008, Pubmed
del Viso, Generating diploid embryos from Xenopus tropicalis. 2012, Pubmed , Xenbase
Essner, Conserved function for embryonic nodal cilia. 2002, Pubmed , Xenbase
Fisher, eFGF is required for activation of XmyoD expression in the myogenic cell lineage of Xenopus laevis. 2002, Pubmed , Xenbase
Griffin, RAPGEF5 Regulates Nuclear Translocation of β-Catenin. 2018, Pubmed , Xenbase
Guschin, A rapid and general assay for monitoring endogenous gene modification. 2010, Pubmed
Hardcastle, FGF-8 stimulates neuronal differentiation through FGFR-4a and interferes with mesoderm induction in Xenopus embryos. 2000, Pubmed , Xenbase
Hopwood, Xenopus Myf-5 marks early muscle cells and can activate muscle genes ectopically in early embryos. 1991, Pubmed , Xenbase
Hopwood, MyoD expression in the forming somites is an early response to mesoderm induction in Xenopus embryos. 1989, Pubmed , Xenbase
Isaacs, eFGF regulates Xbra expression during Xenopus gastrulation. 1994, Pubmed , Xenbase
Jin, Contribution of rare inherited and de novo variants in 2,871 congenital heart disease probands. 2017, Pubmed
Kawasumi, Left-right asymmetry in the level of active Nodal protein produced in the node is translated into left-right asymmetry in the lateral plate of mouse embryos. 2011, Pubmed
Khokha, Techniques and probes for the study of Xenopus tropicalis development. 2002, Pubmed , Xenbase
King, The role of the brachyury gene in heart development and left-right specification in the mouse. 1998, Pubmed
Kitaguchi, Xenopus Brachyury regulates mesodermal expression of Zic3, a gene controlling left-right asymmetry. 2002, Pubmed , Xenbase
Logan, The transcription factor Pitx2 mediates situs-specific morphogenesis in response to left-right asymmetric signals. 1998, Pubmed
Meyers, Differences in left-right axis pathways in mouse and chick: functions of FGF8 and SHH. 1999, Pubmed
Nakamura, Left-right patterning: conserved and divergent mechanisms. 2012, Pubmed
Neugebauer, FGF signalling during embryo development regulates cilia length in diverse epithelia. 2009, Pubmed , Xenbase
Piedra, Pitx2 participates in the late phase of the pathway controlling left-right asymmetry. 1998, Pubmed
Pownall, Myogenic regulatory factors and the specification of muscle progenitors in vertebrate embryos. 2002, Pubmed
Rohr, Asymmetric involution of the myocardial field drives heart tube formation in zebrafish. 2008, Pubmed
Rudnicki, MyoD or Myf-5 is required for the formation of skeletal muscle. 1993, Pubmed
Ryan, Pitx2 determines left-right asymmetry of internal organs in vertebrates. 1998, Pubmed , Xenbase
Schweickert, The nodal inhibitor Coco is a critical target of leftward flow in Xenopus. 2010, Pubmed , Xenbase
Schweickert, Cilia-driven leftward flow determines laterality in Xenopus. 2007, Pubmed , Xenbase
Shook, Pattern and morphogenesis of presumptive superficial mesoderm in two closely related species, Xenopus laevis and Xenopus tropicalis. 2004, Pubmed , Xenbase
Stark, FGFR-4, a new member of the fibroblast growth factor receptor family, expressed in the definitive endoderm and skeletal muscle lineages of the mouse. 1991, Pubmed
Stubbs, The forkhead protein Foxj1 specifies node-like cilia in Xenopus and zebrafish embryos. 2008, Pubmed , Xenbase
Teven, Fibroblast growth factor (FGF) signaling in development and skeletal diseases. 2014, Pubmed
Vonica, The left-right axis is regulated by the interplay of Coco, Xnr1 and derrière in Xenopus embryos. 2007, Pubmed , Xenbase
Walentek, ATP4a is required for Wnt-dependent Foxj1 expression and leftward flow in Xenopus left-right development. 2012, Pubmed , Xenbase
Walentek, Wnt11b is involved in cilia-mediated symmetry breakage during Xenopus left-right development. 2013, Pubmed , Xenbase
Yamauchi, Fgf4 is required for left-right patterning of visceral organs in zebrafish. 2009, Pubmed
Yoshiba, Roles of cilia, fluid flow, and Ca2+ signaling in breaking of left-right symmetry. 2014, Pubmed
Yoshioka, Pitx2, a bicoid-type homeobox gene, is involved in a lefty-signaling pathway in determination of left-right asymmetry. 1998, Pubmed
Zaidi, Genetics and Genomics of Congenital Heart Disease. 2017, Pubmed
Zaidi, De novo mutations in histone-modifying genes in congenital heart disease. 2013, Pubmed
Zetser, MAP kinase converts MyoD into an instructive muscle differentiation factor in Xenopus. 2001, Pubmed , Xenbase