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First published online 5 October 2005
doi: 10.1242/dev.02065

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FGFR1 function at the earliest stages of mouse limb development plays an indispensable role in subsequent autopod morphogenesis

¹ Genetics of Development and Disease Branch, NIDDK, NIH, 10/9N105, 10 Center Drive, Bethesda, MD 20892, USA
² Department of Craniofacial Biology and Cellular and Developmental Biology, University of Colorado Health Sciences Center, 4200 East Ninth Avenue, Denver, CO 80262, USA
³ Laboratory of Protein Dynamics and Signaling, Center for Cancer Research, National Cancer Institute, NCI-Frederick, Frederick, MD 21702, USA

View larger version (98K): [in a new window]   Fig. 1. Activity of Ap-2Cre revealed using Rosa-26 reporter mice. Ages of embryos are as indicated. (A) Whole embryo at the 20-somite stage. (B) Forelimbs at the 20 (left) and 28 (right)-somite stage. The embryo in B was slightly bigger than embryo in A. (C) Forelimbs of embryos at the 32 (left) and 36 (right)-somite stage. (D) Hindlimb of an E10.5 embryo (32 somites). (F) Section of E11.5 forelimbs. (G) Whole-mount in situ hybridization of E11.5 forelimbs using a probe for Fgfr1. Genotypes of the embryos were: mutant (Mt) Fgfr1Co/Co;Ap-2Cre and controls (Wt) Fgfr1Co/Co. FL, forelimb; HL, hindlimb; AER, apical ectodermal ridge; a, anterior; p, posterior.

View larger version (78K): [in a new window]   Fig. 2. Abnormal digit formation and limb development in Fgfr1Co/Co;Ap-2Cre mice. (A) Forelimb of E11.5 Fgfr1Co/Co;Ap-2Cre (Mt) and Fgfr1Co/Cocontrol (Wt) mice. (B) Whole-mount in situ hybridization for Sox9 in E12 forelimbs. (C,D) Hoxd12 stained E13.5 forelimbs (C) and hindlimbs (D). The Hoxd12-negative digit is the first digit (1). Arrowheads in C indicate fused digits. (E-I) Alizarin Red and Alcian Blue stained newborn forelimbs (E-G), and P10 hindlimbs (H,I). The arrows in F-H indicate fused digits. The boxed area in E is enlarged in F. Ra, radius; Ul, ulna; In, intermedium; Fib, fibulare; Ti, tibia; Fi, fibula; Sc, scapula; Hu, humerus.

View larger version (114K): [in a new window]   Fig. 3. Anterior-distal abnormalities in Fgfr1Co/Co;Ap-2Cre limbs. Whole-mount in situ hybridization probes were as indicated. Ages of limb buds were E11.5 (A,B,D-G), E10.5 (C) and E12.5 (H). Arrow in A indicates the enlarged anterior portion of the AER. Frozen sections of limb buds in A are shown in B. Note, samples shown in C were hybridized with probes for Fgf8 and Shh simultaneously. The red lines in D-F mark the corresponding position of truncation in mutant limbs. Wt, wild type; Mt, mutant.

View larger version (106K): [in a new window]   Fig. 4. Activity of Hoxb6-Cre revealed by analysis of Rosa-26 reporter mice. Ages of embryos are as indicated; the number of pairs of somites are: 8 (A), 25 (B,D,F), 20 (C) and 22 (E) pairs of somites. The arrow in A indicates the presumptive hindlimb territory. Arrows in C,D,G indicate anterior areas lacking Cre activity. In E and F the extent of the forelimbs is marked with lines. The arrowheads indicate a boundary between solid blue and diffuse ß-gal staining, indicating that Cre activity gradually extends anteriorly. FL, forelimb; HL, hindlimb.

View larger version (90K): [in a new window]   Fig. 5. Malformation of hindlimbs of Fgfr1Co/Co;Hoxb6-Cre mice. Images of E10.5 (A,C,D), E11.5 (B), E12.25 (E,F), E12.5 (G) and E14.5 (H,I) limbs. Probes used for whole-mount in situ hybridization are as indicated. Anterior is to the left and posterior to the right in all the panels. Arrows indicate the abnormal AER (B), the significantly reduced Shh expression (C), and the expanded Alx4 expression domain (D). Wt, wild type; Mt, mutant.

View larger version (106K): [in a new window]   Fig. 6. Abnormal autopod formation and patterning in Fgfr1Co/Co;Hoxb6-Cre mice. (A,B) Alizarin Red and Alcian Blue stained P1 (A) and P10 (B) hindlimbs. (C,D) Higher magnification of the fifth digit of a mutant (C), and a wild type (D). The boxed area in B is enlarged in E. Cl, claw; Fib, fibulare; In, intermedium; Ph, phalanges; Wt, wild type; Mt, mutant.

View larger version (82K): [in a new window]   Fig. 7. Phenotypes of forelimbs of Fgfr1Co/Co;Hoxb6-Cre mice. Whole-mount in situ hybridization using probes as indicated. Ages of limb buds were E11.5 (A,B), E12.25 (C), E12.5 (D,E), E14.5 (F) and P21 (G). Arrowheads in A-E indicate the border between the relatively normal anterior and the abnormal posterior portions of the limbs. The mutant limb bud shown in C is placed at two different angles to show a reduced height for the PD axis (middle) and an increased length of the DV axis (right). Wt, wild type; Mt, mutant.

View larger version (104K): [in a new window]   Fig. 8. Increased apoptosis in the hindlimbs of Fgfr1Co/Co;Hoxb6-Cre mice detected by staining with LysoTracker Red. Whole-mount images of E10.5 (A,B), E11.5 (C,D) and E12.5 (E,F) limbs. Arrows and arrowheads in mutant limbs indicate apoptotic cells. The arrowhead marked with an asterisk in F indicates an apoptotic area that is out of focus, but it was confirmed under the microscope. Wt, wild type; Mt, mutant.

View larger version (95K): [in a new window]   Fig. 9. Increased apoptosis and altered gene expression in FGFR1-deficient limbs. (A-D) TUNEL assay on E11.5 wild type (A,C) and mutant (B,D) limbs. C and D are cross sections of hindlimbs of wild-type (C) and Fgfr1Co/Co;AP-2Cre (D) mice. Note that the mutant limb shown in D is wider because of an increased length of the DV axis, which can only been seen in the cross sections. All other images are hindlimbs of wild type and Fgfr1Co/Co;Hoxb6-Cre mice. (E-H) Whole-mount in situ hybridization of E10.5 (G,H, arrowheads indicate areas of Dkk1 expression), and E11.5 (E,F) limbs. Genotypes and probes are as indicated.