An Allelic/Nonallelic Series of BMP-Deficient Limbs
Mice were generated with limbs deficient in BMP2, BMP4, or BMP7, both BMP2 and BMP4, or both BMP2 and BMP7. To obtain an initial indication of the range of phenotypes produced in these animals, we first examined the limb skeletons of newborn animals. BMP2-deficient limbs appeared remarkably normal, both in skeletal pattern and in gross aspects of chondrogenesis and osteogenesis indicated by Alcian blue/Alizarin red staining. The one notable defect in the appendicular skeleton of these animals is a characteristic malformation of the scapula (Figure 1, red arrow). BMP2-deficient limbs also exhibit 3/4 soft-tissue syndactyly with variable penetrance (Figure 2).
Figure 2  Depletion of BMP Signaling Causes Interdigital Syndactyly
(A–D) Forelimb of adult wild-type (A) and Bmp2C/C; Prx1::cre mouse (B) and hindlimbs of newborn wild-type (C) mouse and newborn Bmp2C/C; Bmp4C/C; Prx1::cre (D) mouse. The black arrow in (B) shows soft tissue syndactyly in Bmp2C/C; Prx1::cre mouse.
(E and F) Wild-type and Bmp2C/C; Bmp4C/C; Prx1::cre, respectively, showing acridine orange–stained hindlimbs of E15.5 mouse embryos. Acridine orange stain is in yellow.
(G and H) Enlarged views of selected regions from (E) and (F), respectively. Black arrow in (G) and (H) show acridine orange–stained apoptotic cells in the interdigital mesenchyme, and asterisk in (H) shows the remnant of the AER.
(I and J) Fgf8 mRNA expression in the hindlimbs of E13.5 wild-type (I) and Bmp2C/C; Bmp4C/C; Prx1::cre (J) embryos. The thick black arrows in (J) show Fgf8 mRNA expression. Mice with limbs deficient in BMP4 activity and the limbs of Bmp7 mutant mice have both been previously described. As reported [16], in the absence of BMP4 activity, limbs display a variable penetrance of preaxial and postaxial polydactyly, but otherwise, normal digit patterns and apparently normal skeletal differentiation (Figure 1G and 1O) take place. As previously described, mice homozygous for a null mutation in Bmp7 [12] have no defects in the formation of the normal appendicular skeletal elements (Figure 1H and 1P). We do occasionally observe preaxial polydactyly in these mutants.
Compound heterozygous mice, with one functional copy each of Bmp2 and Bmp4 in the limb (Bmp2+/C; Bmp4+/C; Prx1::cre), show no effect on either limb patterning or skeletogenesis (unpublished data). Similarly, limb skeletons of mice in which both copies of the Bmp4 gene and one copy of the Bmp2 gene have been conditionally removed in the limb (Bmp2+/C; Bmp4C/C; Prx1::cre) are phenotypically normal other than exhibiting the variable penetrance preaxial and postaxial polydactyly seen in mice deficient in BMP4 activity alone (Figure 1J and 1R). In contrast, mice in which both copies of Bmp2 and one copy of Bmp4 had been removed (Bmp2C/C; Bmp4+/C; Prx1::cre) showed more severe skeletal defects, including significantly thinner skeletal elements. However, the digit patterns of those animals are completely normal (Figure 1I and 1Q). Animals in which both copies of Bmp2 and Bmp4 were removed (Bmp2C/C; Bmp4C/C; Prx1::cre) had extremely malformed limbs (Figure 1K and 1S). They displayed severely short and malformed stylopods; one of the zeugopod elements was almost always missing, and the remaining one was so deformed that it was difficult to identify the element correctly. Moreover, the joint articulations are defective such that zeugopod and stylopod elements are fused (see below and Figure S1A–S1C). Interestingly, the autopods are less affected than the proximal elements. Nonetheless, the autopod elements are significantly reduced in size, and strikingly the two posterior-most digits are missing in the forelimbs of these animals.
Simultaneous removal of BMP2 and BMP7 had far less of an effect than did removal of BMP2 and BMP4 activity. Compound heterozygous removal of BMP2 and BMP7 (Bmp2+/C, Bmp7+/ −; Prx1::cre) and heterozygous removal of BMP2 with complete removal of BMP7 (Bmp2+/C, Bmp7 −/−; Prx1::cre) were both completely wild-type in skeletal pattern and differentiation (unpublished data). Mice homozygous for removal of Bmp2 and heterozygous for Bmp7 (Bmp2C/C, Bmp7+/ −; Prx1::cre) displayed the same subtle scapular defect seen in Bmp2C/C; Prx1::cre mutants alone (unpublished data). In limbs developing in the complete absence of both BMP7 and BMP2 activity (Bmp2C/C, Bmp7 −/−; Prx1::cre), the last phalanx was missing from digit III in the forelimb, and these limbs displayed the same phenotype in the hindlimb with variable penetrance (Figure 1L and 1T, black arrow). Additionally, the fibulae of these hindlimbs are malformed and do not articulate with the femur at the knee (Figure 1T, thick red arrow). They also display the same scapular defects seen in Bmp2C/C; Prx1::cre mice, and the overall size of the appendicular skeleton is slightly diminished. However, skeletal differentiation appears normal in the limbs of these animals.