First published online 5 October 2005
doi: 10.1242/dev.02065
Development 132, 4755-4764 (2005)
Published by The Company of Biologists 2005
FGFR1 function at the earliest stages of mouse limb development plays an indispensable role in subsequent autopod morphogenesis
Cuiling Li1,
Xiaoling Xu1,
Danielle K. Nelson2,
Trevor Williams2,
Michael R. Kuehn3 and
Chu-Xia Deng1,*
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
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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© The Company of Biologists Ltd 2005
