Conditional inactivation of Fgfr1 in mouse defines its role in limb bud establishment, outgrowth and digit patterning

Abstract

Previous studies have implicated fibroblast growth factor receptor 1 (FGFR1) in limb development. However, the precise nature and complexity of its role have not been defined. Here, we dissect Fgfr1 function in mouse limb by conditional inactivation of Fgfr1 using two different Cre recombinase-expressing lines. Use of the T (brachyury)-cre line led to Fgfr1 inactivation in all limb bud mesenchyme (LBM) cells during limb initiation. This mutant reveals FGFR1 function in two phases of limb development. In a nascent limb bud, FGFR1 promotes the length of the proximodistal (PD) axis while restricting the dimensions of the other two axes. It also serves an unexpected role in limiting LBM cell number in this early phase. Later on during limb outgrowth, FGFR1 is essential for the expansion of skeletal precursor population by maintaining cell survival. Use of mice carrying the sonic hedgehog(cre) (Shh(cre)) allele led to Fgfr1 inactivation in posterior LBM cells. This mutant allows us to test the role of Fgfr1 in gene expression regulation without disturbing limb bud growth. Our data show that during autopod patterning, FGFR1 influences digit number and identity, probably through cell-autonomous regulation of Shh expression. Our study of these two Fgfr1 conditional mutants has elucidated the multiple roles of FGFR1 in limb bud establishment, growth and patterning.

Fig. 1.

Inactivation of Fgfr1 by Tcre and Shh^cre. (A-D) β-Gal staining of embryos at E8.5 (A) and E10.0 (B), an E10.0 forelimb bud in transverse section (C) and an E10.5 intact limb bud (D). The arrowheads in A and B delineate the rostral boundary of robust staining. Arrow in B indicates the forelimb bud. Inset in C is a magnified view of the boxed domain to illustrate that the staining is robust in the mesenchyme, while absent in the AER (arrowhead). (E-L) Gene expression analysis using whole-mount in situ hybridization probes indicated on the left. (E,F) E10.0 embryos. Arrows in E,F indicate forelimb buds. Arrowhead in F delineates the rostral boundary of Fgfr1 inactivation. (G,H) E10.5 forelimb buds. Broken line and arrowhead in H indicate Fgfr1 inactivation in posterior mesenchyme. (I,J) E10.0 forelimb buds. Expression of Spry4 in the mutant is reduced to the posterior mesenchyme and a thin line subjacent to the AER. (K,L) Oblique dorsoposterior view of E10.5 forelimb buds. Arrowhead in L indicates reduced Spry4 expression in the dorsoposterior mesenchyme. (M) RT-PCR of normal (n) and Tcre;Fgfr1 mutant (m) limb buds to illustrate that Fgfr1 is inactivated in mutant LBM at E10.0 and E10.5.

Fig. 2.

Tcre;Fgfr1 skeletal phenotypes and gene expression. (A-H) Skeletal preparations of E17.5 limbs, stained for cartilage (Alcian Blue) and bone (Alizarin Red). (A-D) All segments of the limb, the stylopod (S), zeugopod (Z) and autopod (A) are reduced in the mutant, while the structures outside of the limb, the scapula (sc) and pelvic girdle (pg), remain normal. (E-H) Magnified views of autopod skeletons. The positions of metacarpals (mc) and metatarsals (mt) are indicated. Dots in F and H indicate individual phalange. (I-P) Forelimb buds at E11.5 (I,J) and E12.5 (K-P). Arrowheads in K-P indicate the position of the most anterior digit.

Fig. 3.

Tcre;Fgfr1 limb bud morphology and cell survival. (A-L) Forelimb buds at the indicated stages with the AER labeled by Fgf8 whole-mount in situ hybridization. Each normal versus mutant pair is shown at the same magnification. (A-D) Dorsal view with the remaining axes indicated in A. Corresponding solid and broken lines in each pair are of the same length to assist comparisons of limb bud dimensions. (E-H) Transverse sections of limb buds. The mutant is thicker along the DV axis at E10.0 and E10.5. (I-L) Distal view with the remaining axes indicated in I. Arrowheads indicate that the mutant AER is wider than normal along the DV axis at E10.0 and E10.5. (M,N,Q-T) Gene expression, as indicated, in E10.5 forelimb buds as assayed by whole-mount in situ hybridization. Arrowheads in Q and R indicate expression in the mesenchyme. (O,P) Cell death in E10.5 forelimb buds assayed by LysoTracker Red staining. Limb buds are outlined by broken white lines. Abbreviations: An, anterior; D, dorsal; Di, distal; Po, posterior; Pr, proximal; V, ventral.

Fig. 4.

Whole-mount in situ hybridization analysis of gene expression in Tcre;Fgfr1 E10.5 forelimb buds. The mesenchymal expression of Shh, gremlin (Grem), Bmp4, Hoxa13 and Hoxd13 is reduced in the mutant, while the mesenchymal expression of Alx4 and Bmp2 and the AER expression of Bmp4 and Bmp2 is increased in the mutant.

Fig. 5.

Gene expression in Shhcre;Fgfr1 limb buds. (A,B) β-Gal staining in (A) a Shh^cre/+;Fgfr1^co/+;R26R/+ and (B) a Shh^cre/+;Fgfr1^co/co;R26R/+ littermate E10.75 forelimb bud to label cells in which CRE has acted. (C-L) Gene expression in E10.75 (C-J) and (K,L) E11.5 forelimb buds. Shh^cre/+;Fgfr1^co/+ limb buds are used as normal control. Expression of Shh, Mkp3, Bmp4 and Hoxd13 is reduced within the CRE-active domain, while Ptch1 is reduced outside of the domain. Arrowheads indicate the posterior end of the AER. Asterisks indicate signal in the mesenchyme proximal to the end of the AER. Arrows indicate that the anterior boundaries of Bmp4 and Hoxd13 reduction corresponds to that of Fgfr1 inactivation as reported by Mkp3 reduction.

Fig. 6.

Shhcre;Fgfr1 autopod skeletal phenotype. Autopod skeletons of E18.5 embryos demonstrate consistent patterns that are observed in wild-type (n=4) and mutant (n=6) embryos. Shown within each box are a skeletal preparation of an E18.5 autopod (top left panel), a diagram outlining all elements (top right panel) and a magnified view of the wrist region (lower panel). Brackets indicate regions magnified in lower panels. In a normal autopod, individual digits are numbered and colored differentially. In the mutant, each identifiable digit is assigned the number and color of the corresponding normal digit, while the non-identifiable digits are colored grey. In the magnified views, carpal elements relevant to the identification of digits are labeled with letters: a, trapezium; b, trapezoid; c, central carpal; d, capitate; e, hamate; f, intermediate cuneiform; g, lateral cuneiform; h, cuboid; i, navicular. The metacarpals/metatarsals and carpals/tarsals are outlined with broken lines to delineate the connections between them. In both mutant forelimb and hindlimb, digits 1 and 5 are easily identifiable based on their phalanx number, digit length and their metacarpal/metatarsal articulation with the carpals/tarsals. Digit 2 is identified in the mutant forelimb based on its length (shorter than digits 3, 4 and longer than 1, 5), and more importantly on its metacarpal articulation with both the trapezoid (b) and central carpal (c). The grey digit in the mutant forelimb is either a digit 3, 4 or a chimeric 3/4 based on its length (longest of all) and its articulation with both the capitate (d) and the hamate (e). Digit 4 is identified in the mutant hindlimb based on its articulation with the Cuboid (h). The grey digit in the mutant hindlimb is either a digit 2, 3 or a chimeric 2/3 as it articulates with the fused intermediate/lateral cuneiform (f/g) atop the navicular (i). FL, forelimb; HL hindlimb.

Fig. 7.

Shhcre;Fgfr1 digit condensation and Shh-expressing cell lineage. In all panels, anterior is towards the left and posterior towards the right. (A-D) Digit condensations outlined by Sox9 expression. Dots in A and B indicate individual digit condensations that are beginning to separate. All curved lines in C and D are the same length. All lines are the same length. The lines in C span the entire distance between each pair of condensations, while the line in D does not, indicating that the middle two condensations in D are farther apart than normal. Pattern in C indicates that each condensation has its unique morphology. In particular, digit 2 and 4 condensations have a slight curve towards the straight digit 3 condensation at the midline of the AP axis. In D, the middle two condensations exhibit a curve towards the AP midline, resembling digit 2 and 4 condensations in a normal limb bud. (E-H) β-Gal staining to label Shh-expressing cell lineage in normal (Shh^cre/+;Fgfr1^co/+;R26R/+) and Shhcre;Fgfr1 (Shh^cre/+;Fgfr1^co/co;R26R/+) forelimb buds. Dots in E and F indicate the position of condensations as deduced from Sox9 expression in an equivalent staged limb bud. Broken lines in E-H indicate anterior boundary of the Shh-expressing lineage.

Fig. 8.

Mechanism of FGFR1 function. Based on data presented here, we propose that in limb development, FGFR1 serves as a principle receptor for AER-FGF signaling. Activation of FGFR1 then impacts limb skeletal formation through three distinct cellular mechanisms. As elaborated in the Discussion, FGFR1 regulation of Shh and 5′-Hox gene expression probably contributes to the molecular mechanism underlying FGF function during limb bud development.