Genetic interaction between Wnt/beta-catenin and BMP receptor signaling during formation of the AER and the dorsal-ventral axis in the limb

Abstract

By conditional gene ablation in mice, we found that beta-catenin, an essential downstream effector of canonical Wnt signaling, is a key regulator of formation of the apical ectodermal ridge (AER) and of the dorsal-ventral axis of the limbs. By generation of compound mutants, we also show that beta-catenin acts downstream of the BMP receptor IA in AER induction, but upstream or parallel in dorsal-ventral patterning. Thus, AER formation and dorsal-ventral patterning of limbs are tightly controlled by an intricate interplay between Wnt/beta-catenin and BMP receptor signaling.

Figure 1.

Conditional ablation of β-catenin inhibits hindlimb development. (A) Section along the dorsal–ventral axis of hindlimb of Brn4Cre;lacZ embryos. Cre activity is restricted to limb ectoderm. (B,C) Analysis of β-catenin expression in wild-type and severely affected Brn4Cre;β-catenin^flox/flox hindlimbs. β-Catenin is absent in the ventral ectoderm of mutants, as detected by immunofluorescence (β-catenin is indicated in green, DAPI nuclei staining in blue). Arrows mark the surface ectoderm. (D,E) Loss-of-function mutation of β-catenin in the limb ectoderm results in variable hindlimb deformations. (F–H) Skeletal preparations of hindlimbs of wild-type and mutant mice. The malformation of mutant hindlimbs (70 animals were examined) consists of truncation at the proximal femur (G, 50%), disgenesis of tibia and fibula, and loss of digits 1–4 (H, 11%). The distal element of digit V is often bifurcated (inset in H, 83%). (I,J) Gross morphology of wild-type and Brn4Cre;β-catenin^flox/flox autopods (ventral view). Autopods of newborn mutant mice are dorsalized, as dermal pads (arrows) are absent on the ventral surface of mutant paws, and nails are circumferential (arrowhead). (K,L) Sagittal sections of wild-type and mutant digits, demonstrating nail plates on dorsal and ventral surfaces in the mutants (arrows), as indicated by in situ hybridization for Msx1. (A–C,K,L) Dorsal is to the top. (fe) Femur; (fi) fibula; (ti) tibia. Bars: A, 40 μm; B,C, 50 μm; D,E, 3 mm; F–H, 1 mm; I,J, 0.5mm; K,L, 100 μm.

Figure 2.

β-Catenin is essential for AER formation. (A,B) Reduction of Fgf8 expression in hindlimbs of mutants carrying loss-of-function mutation of β-catenin, in comparison with wild type, at the 38–42-somite stage (arrowheads). (C) Strong ventrally expanded expression domain of Fgf8 in mutants harboring the gain-of-function mutation of β-catenin. Note ectopic expression of Fgf8 in dorsal limb ectoderm (arrowheads). (D,E) Expression of β-catenin in spots, but not in the surrounding ectoderm of mildly affected mutants carrying loss-of-function mutation of β-catenin, as detected by immunofluorescence at the 38–42-somite stage (β-catenin is indicated in green, DAPI nuclei staining in blue; the surface of the ectoderm is marked by a broken line). Note that the exposition time was reduced in F. Bars: A–C, 150 μm; D–F, 15μm.

Figure 3.

β-Catenin acts downstream of the BMP receptor IA during AER formation (see scheme at right). (A–D) AER is absent or only few groups of cells resembling AER are present in β-catenin loss-of-function mutant limbs at the 42-somite stage. (E,F) AER and overall size of limb are strongly enlarged in β-catenin gain-of-function mutants at the 42-somite stage. (G,H) AER is not formed in Bmp receptor IA loss-of-function mutants at the 42-somite stage. (I,J) Limbs are strongly enlarged and the AER is expanded to ventral side in compound mutants at the 42-somite stage (cf. E,F). Dorsal ventral is as indicated. Bar, 100 μm.

Figure 4.

β-Catenin signaling depends on BMP receptor signaling during AER formation. (A–D) Expression of Wnt3 and Fzd6 are not changed in the limb ectoderm of Bmp receptor IA loss-of-function mutants at the 30-somite stage. (E–H) Fzd1 and conductin are not expressed in ventral ectoderm of Bmp receptor IA loss-of-function mutants at the 30-somite stage. Note that conductin expression was analyzed in compound mice expressing lacZ under the control of the conductin promoter. Dorsal ventral is as indicated. Bar, 50 μm.

Figure 5.

β-Catenin acts upstream of, or in parallel to, the BMP receptor IA during dorsal–ventral patterning of limbs (see scheme at right). (A,C) En-1 is not expressed in ventral limb ectoderm of mutants carrying loss-of-function mutation of β-catenin at the 30-somite stage. (B,D) Wnt-7a is expressed ectopically in ventral ectoderm of β-catenin loss-of-function mutant limbs at the 30-somite stage. (E,F) En-1 and Wnt-7a expression domains in β-catenin gain-of-function mutants resemble the wild type. (G) En-1 is not expressed in ventral ectoderm of mutants carrying the loss-of-function mutations of Bmp receptor IA. (H) Wnt-7a is expressed in both dorsal and ventral ectoderm in mutants carrying loss-of-function mutation of Bmp receptor IA. (I) En-1 is not expressed in the ventral ectoderm of compound mutants at the 30-somite stage. (J) Wnt-7a is expressed in ventral limb ectoderm of compound mutants at the 30-somite stage (cf. H). Dorsal ventral is as indicated. Bar, 50 μm.