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First published online 18 January 2006
doi: 10.1242/dev.02242

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Context-specific requirements for Fgfr1 signaling through Frs2 and Frs3 during mouse development

Program in Developmental Biology, Division of Basic Sciences, Fred Hutchinson Cancer Research Center, 1100 Fairview Avenue North, Seattle, WA 98109, USA.

View larger version (31K): [in a new window]   Fig. 1. Fgfr1 targeting. (A) Alleles introduced by gene targeting. (B) Partial cDNA knock-in approach: Fgfr1 exons 8-17, encoding the transmembrane and cytoplasmic domains, were replaced with a pseudoexon encoding the corresponding region of Fgfr1wtKI or Fgfr1Frs. Partial cDNAs were spliced at the 5' end into exon 8 and at the 3' end into exon 17, upstream of the endogenous polyadenylation sequence. (C) To generate the null allele, the first exon common to all reported Fgfr1 splice variants (exon 4) was flanked with loxP sites and excised in vivo by Meox2-Cre (Tallquist and Soriano, 2000). RT-PCR from embryonic RNA confirmed the generation of a stop codon soon after the exon3-exon5 splice junction (data not shown). (D) Southern blots verifying correct targeting of all alleles in ES cell clones. Abbreviations: A, ApaI; H, HindIII; KI, knock-in; Nd, NdeI; Nh, NheI; P, PstI; Rv, EcoRV; T, Tth111I; X, XbaI; ext, external; int, internal. (E) Relative Fgfr1 mRNA levels in homozygous embryos, assessed by semi-quantitative RT-PCR. Each point is the mean of triplicate reactions for a single embryo (±s.d.).

View larger version (72K): [in a new window]   Fig. 2. Rescue of early Fgfr1 functions by Fgfr1Frs. (A) Viability (percent of expected) of homozygotes recovered from Fgfr1Frs/+ and Fgfr1ex4/+ intercrosses. (B-G) Sonic hedgehog (Shh, E9.5) and Meox1 (E10.5) in situ hybridization. Arrows indicate width of the Shh expression domain (more than two cell diameters in Fgfr1ex4/ex4 compared with one cell diameter in control embryos). (C) The paraxial mesoderm population is reduced and disorganized in Fgfr1ex4/ex4 embryos, so Meox1 staining is faint and diffuse. (Color development time required to visualize any positive signal was notably longer in this embryo than in F,G.)

View larger version (40K): [in a new window]   Fig. 3. Neural tube defects. (A-C) Hoxb1 whole-mount in situ hybridization, labeling dorsal neural tube in the spinal region. Edges of open neuroepithelium are outlined in B. In C, the open cranial region (left embryo) is a procedural artifact. (D-F) Hematoxylin and Eosin stained sections through spinal neural tubes of E10.5 littermates. Embryo in E completed closure and embryo in F exhibited the intermittent neural tube closure phenotype. (G) Mitotic and apoptotic indices of neural tube sections (E10.5), assessed at similar spinal levels by phosphohistone H3 immunostaining and TUNEL. Each data point is the mean of positive/total cells on 12-16 sections per embryo, two embryos/genotype. Error bars indicate ±1 s.d.; P values were determined by Student's t-test.

View larger version (52K): [in a new window]   Fig. 4. Posterior truncations and tail bud development. (A-C) Bright-field photographs of E13.5 littermates (caudal region). (D,E) Hematoxylin and Eosin stained sections through kidneys of wild-type and severely truncated Fgfr1Frs/Frs embryos; bar indicates relative magnification. (F) T (Brachyury) and (G,H) Wnt3a whole-mount in situ hybridization. (I) Quantitation of proliferation (pH3, P=0.33) and apoptosis (TUNEL, P=0.09) in E10.5 tail bud sections. Each data point represents the mean of three to eight sections through each of at least three embryos. Error bars indicate ±1 s.d.; P values were determined by Student's t-test.

View larger version (93K): [in a new window]   Fig. 5. Pharyngeal arch (PA) development. (A-C) Phosphohistone H3 immunostained PA sections (E10.5). PA1 is labeled, arrowheads indicate PA2. (D,E) Fgf8 whole-mount in situ hybridization (PA region); arrows indicate PA2 Fgf8 expression domain lost in mutant embryos. (F-H) Migrating NCCs in the pharyngeal region of stage-matched embryos, visualized by Sox10 in situ hybridization. Arrows indicate limits of NCC migration into PA1, 2. (I-L) Nile Blue staining of cell death in the pharyngeal region (E10.5). I/J and K/L are stage-matched control/mutant embryo pairs.

View larger version (82K): [in a new window]   Fig. 6. PA2 neural crest derivatives. Skeleton preparations of E15.5 middle ears (A,B,D) and hyoid cartilages (E,F). PA1, 2 derivatives are traced in C,G,H. PA1 neural crest derivatives, traced in blue: i, incus, m, malleus, ty, tympanic ring, mc, Meckel's cartilage. PA2 derivatives, traced in red, are hypoplastic or missing in mutants: (C) s, stapes, sp, styloid process; (G,H) lesser horns of hyoid. Ectopic cartilages were observed in mutant ears (arrows in B,D; yellow in C).

View larger version (69K): [in a new window]   Fig. 7. p2 MEC responses to Fgf. (A) pMAPK and pAkt responses of Fgfr1Frs/Frs and control MECs stimulated with Fgf, assayed over a dose titration (Ai, 5 minute stimulations) and a 1 hour timecourse (Aii). RasGAP, loading control. (B,C) Crk and Frs2 activation responses to Fgf (50 ng/ml aFgf, 5 µg/ml heparin, 5'), assayed by IP-phosphotyrosine (4G10) western blot. Blots were stripped and reprobed with anti-Crk or anti-Frs2 (lower panels). *No antibody IP control. (D) Fgfr2 responses to Fgf (50 ng/ml aFgf, 5 µg/ml heparin, 5'), assayed by 4G10 IP-Fgfr2 western blot (upper panel). Total cell lysate Fgfr2 (middle panel) and actin (loading control, lower panel) western blots show relative protein levels. Matching arrowheads indicate bands of the same size recognized by different antibodies within B-D. (E) Relative Fgfr2 mRNA levels in homozygous embryos, determined by semi-quantitative RT-PCR. (F) Growth curves of p2 MECs derived from four mutant and two wild-type embryos cultured in DMEM, 4% FBS, 50 ng/ml aFgf.

View larger version (85K): [in a new window]   Fig. 8. Phospho-MAPK immunohistochemistry. Whole-mount phospho-MAPK immunohistochemistry of stage-matched embryos: (A-C) E8.5, (D-F) E9.5. High magnification views of (B,E) caudal gastrulating regions and (C,F) more rostral areas undergoing somitogenesis and neurulation. Arrowheads and arrows indicate phospho-MAPK-positive somites and neural folds, respectively. (G-L) G/J, H/K and I/L are stage-matched embryo pairs (E10.5). (G,H,J,K) Pharyngeal arch 1, 2 region; (I) dorsal and (L) lateral views of premigratory neural crest cells (arrows). Lateral view is shown in L because this embryo has an open neural tube.