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. 2020 Dec 1;34(23-24):1735–1752. doi: 10.1101/gad.342956.120

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© 2020 Ray et al.; Published by Cold Spring Harbor Laboratory Press

This article is distributed exclusively by Cold Spring Harbor Laboratory Press for the first six months after the full-issue publication date (see http://genesdev.cshlp.org/site/misc/terms.xhtml). After six months, it is available under a Creative Commons License (Attribution-NonCommercial 4.0 International), as described at http://creativecommons.org/licenses/by-nc/4.0/.

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Figure 3. — Coordinate effects Fgfr1 and Fgfr2 signaling mutation in development. (A) Schematic representation of the Fgfr2 allelic series. Critical effectors that bind to FGFR2 are listed at the left of the Fgfr2^WT allele (WT). Critical residues for this binding are annotated at the right. Amino acid substitutions for each allele are provided to the right of all mutant alleles generated. (B) Coimmunoprecipitation experiments confirmed the ability of F, C, and PG mutations to disrupt FRS2, CRKL, and PLCγ binding to FGFR2, respectively. Constructs expressing Fgfr2^WT, Fgfr2^PG, Fgfr2^CPG, Fgfr2^F, and Fgfr2^FCPG triple-FLAG tag cDNA were overexpressed in 3T3 cells. FLAG pull-downs were then immunoblotted to show interactions of FRS2, CRKL, and PLCγ to FGFR2^WT and mutant receptors. (C) Sagittal and ventral views of E16.5 alcian blue/alizarin red-stained skulls of Fgfr1 signaling mutants. In Fgfr1^F/cKO; Fgfr2^cKO/cKO mutants, the frontal bone (F), nasal cartilage (NC), squamosal bone (SQ), tympanic bulla (T), maxilla (MX), pterygoid (Pt), and mandible (MD) were affected. Defects in mandible, squamosal, and pterygoid bones were exacerbated in Fgfr1^FCPG/cKO;Fgfr2^cKO/cKO mutants. Most severe defects were observed in Fgfr1^cKO/cKO;Fgfr2^cKO/cKO-null mutants, where we observed a reduction in alizarin red staining, reduction of mandible, loss of nasal cartilage, and a wider mid-facial clefting (depicted by red bars). Midfacial clefting was quantified (in millimeters) across various genotypes for signaling mutants and is represented in the graph. Scale bar, 1 mm. (D) Sagittal and ventral views of E16.5 alcian blue/alizarin red-stained skulls of Fgfr1^cKO/cKO;Fgfr2^F/cKO signaling mutants compared with Fgfr1^cKO/cKO;Fgfr2^+/cKO mutants and Fgfr1^cKO/cKO;Fgfr2^cKO/cKO double mutants. Fgfr1^cKO/cKO;Fgfr2^F/cKO signaling mutants showed striking defects in the mandible along with reduced ossification of the premaxilla (PMX) and maxilla, and loss of the squamosal bone and tympanic bulla. Defects were more severe in Fgfr1^cKO/cKO;Fgfr2^cKO/cKO double mutants. Scale bar, 1mm. (E) DAPI stained frontal facial view for Fgfr1^F and Fgfr2^F compound mutant embryos at E15.5. Fgfr1^F/F;Fgfr2^+/F embryos showed a severe facial cleft compared with other genotypes. (F) Brachyury (T) and Fgf8 expression in Fgfr1^FCPG and Fgfr2^FCPG compound signaling mutants at E8.0. In contrast to Fgfr1^−/−;Fgfr2^−/− embryos, which fail to implant on the 129S4 coisogenic background, we could recover Fgfr1^FCPG/FCPG;Fgfr2^FCPG/FCPG compound mutants at E8.0 but not at E10.5. Fgfr1^FCPG/FCPG;Fgfr2^FCPG/FCPG mutants were growth-retarded and exhibit abnormal posterior development, but expressed mesodermal T and Fgf8.