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First published online May 30, 2007
doi: 10.1242/10.1242/dev.001677

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BMP signals control limb bud interdigital programmed cell death by regulating FGF signaling

¹ Laboratory of Cancer and Developmental Biology National Institutes of Health, Frederick, MD 21702, USA.
² SAIC, NCI-Frederick, National Institutes of Health, Frederick, MD 21702, USA.
³ Laboratory of Reproductive and Developmental Toxicology, National Institute of Environmental Health Sciences, Research Triangle Park, NC, USA.

View larger version (39K): [in this window] [in a new window]   Fig. 1. Skeletal abnormalities in Msx2-Cre; Bmpr1aflox/null limbs. (A) Mating scheme used to produce mutant (Msx2-Cre; Bmpr1aflox/null) and control (Bmpr1aflox/wt) mouse embryos. Inset, dorsal view of E10.5 normal (left) and mutant (right) forelimb buds stained via whole-mount ISH with a riboprobe specific for the Cre-deleted Bmpr1a exon. Note the lack of staining (arrowheads) in the mutant AER. (B,C) Lateral views of early mutant forelimb (B) and hindlimb (C) bud transgenic for the Cre reporter R26R and stained for ß-gal. Recombination (blue) in limb bud pre-AER (arrows) is extensive at the stages indicated. (D-I) Skeletal preparations of normal (D,F,H) and mutant (E,G,I) neonates. In mutants, the hindlimbs are absent (E) and the pelvic bones are reduced or absent (G). Mutant forelimbs contain all skeletal elements (E,I) with broader tips in distal phalanges (asterisks in I). AER, apical ectodermal ridge; IL, ilium; IS, ischium; PU, pubis; ss, somite stage.

View larger version (100K): [in this window] [in a new window]   Fig. 2. Lack of a Pre-AER/AER leads to loss of Fgf10 maintenance in mutant mouse hindimbs. Transverse sections through (A,B) E9.5 (31ss) and (C,D) E10.5 hindlimb buds. Note the lack of pre-AER (B) and AER (D) in mutants as compared with normal controls (A,C). (E-J) Dorsal views of whole-mount hindlimb bud region stained with the probe at the somite stage indicated. Mutant Fgf8 expression is not induced (F). Mutant Fgf10 expression is induced (H) but is not maintained (J).

View larger version (109K): [in this window] [in a new window]   Fig. 3. Reduced interdigital PCD in mutant forelimbs does not correlate with changes in interdigital BMP signaling. (A,B) Comparison of E13.0 normal (A) and mutant (B) mouse forelimbs stained with the fluorochrome Lysotracker Red to label regions of dying cells. (C-N) Whole-mount ISH of E12.5 normal (C,E,G,I,K,M) and mutant (D,F,H,J,L,N) forelimbs for the probes indicated. All are dorsal views, anterior to the left.

View larger version (104K): [in this window] [in a new window]   Fig. 4. AER-specific expression of Msx2 and Bambi are downregulated in mutant forelimb. Whole-mount ISH using probes specific for Msx2 (A,B) and Bambi (C-F) in normal (A,C,E) and mutant (B,D,F) mouse forelimb buds at the embryonic stages indicated. At E10.5, mutant Msx2 expression is absent (arrowheads in B), whereas mutant Bambi expression is reduced at E10.5 (arrowheads in D) and absent by E12.5 (arrowheads in F) specifically in the AER, although expression is extended in the subapical mesenchyme (F).

View larger version (101K): [in this window] [in a new window]   Fig. 5. Fgf4 and Fgf8 expression is upregulated in mutant forelimbs. (A-L) Fgf8 and (M-X) Fgf4 expression in normal and mutant mouse forelimbs at the somite or embryonic stage indicated. A,B,M,N are lateral views; C,D are distal views, dorsal toward the top; E,F are anterior views; in all other panels (i.e. G-L,O-X), dorsal views are shown with anterior to the left. *, aberrant expression. The boxed region in P is shown at high magnification in the inset; boxes in I,J,U,V indicate the regions shown at high magnification in K,L,W,X, respectively. The arrow in G indicates lack of Fgf8 expression over the interdigital region in normal forelimbs; the arrow in H indicates discontinuous Fgf8 expression in the AER in mutant forelimbs.

View larger version (86K): [in this window] [in a new window]   Fig. 6. Inactivation of Bmpr1a rescues hypodactyly in Msx2-Cre; Fgf8flox/null mouse forelimbs. (A-C) Skeletal preparation of forelimbs of the genotypes indicated. Individual digits are numbered. Note that hypodactyly in Msx2-Cre;Fgf8flox/null mutants (B) is rescued by also deleting Bmpr1a (C). (D-I) Dorsal views (anterior to left) of forelimbs of the embryonic age and genotype indicated, stained with an Fgf4 riboprobe for a short time period (2 hours, 4°C) to optimally visualize differences in Fgf4 expression levels. Fgf4 expression ceases in normal forelimbs by E11.5 (D), but continues in Msx2-Cre;Fgf8flox/null mutants through E12.5 (E,H). This enhanced expression is higher in Msx2-Cre; Bmpr1aflox/null;Fgf8flox/null mutants (F,I). Note also that Msx2-Cre Fgf8flox/null limb buds are smaller (E,H) than normal (D,G), prefiguring the hypodactyly phenotype (Lewandoski et al., 2000). This size difference is rescued in the double mutants (F,I). The boxed regions in H and I are shown at high magnification in the insets.

View larger version (79K): [in this window] [in a new window]   Fig. 7. Reduction of AER-FGF dose rescues syndactyly in Msx2-Cre; Bmpr1aflox/null mouse forelimbs. (A-F) Dorsal view of forelimbs (anterior to left) of the genotype shown. Interdigital webbing occurs owing to Msx2-Cre-mediated inactivation of Bmpr1a (B), even when Fgf8 is also inactivated (C). However, note that loss of Bmpr1a rescues the hypodactyly phenotype due to Msx2-Cre-mediated inactivation of Fgf8 (C) (also see Fig. 6). (D) Inactivation of one copy of Fgf8 partially rescues webbing in Msx2-Cre; Bmpr1Aflox/null forelimbs.(E,F) Inactivation of one copy of Fgf4 mostly rescues webbing in Msx2-Cre; Bmpr1Aflox/null;Fgf8flox/null forelimbs, as well as restoring the hypodactyly phenotype resulting from loss of Fgf8. The inset in F is a skeletal preparation showing hypodactyly and a missing phalange (*). All animals were approximately 8 weeks of age, except for that in E which was 3 months of age, which accounts for the longer nails of this limb. Note that owing to Msx2-Cre expression in the hair follicle (Pan et al., 2004), Msx2-Cre; Bmpr1aflox/null animals (B-F) have a hair phenotype.

View larger version (11K): [in this window] [in a new window]   Fig. 8. Model for BMP and FGF interaction during interdigital PCD in normal and mutant mouse limb development. During normal development (left), BMP signaling from the interdigit region signals to the BMPR1A in the AER, causing downregulation of Fgf8. FGF8 normally prevents apoptosis in the interdigit region; therefore its downregulation allows interdigit PCD to occur. In Msx2-Cre; BmpR1Aflox/null forelimb buds (right), BMPR1A is absent, Fgf4 and Fgf8 expression persists and abnormal cell survival occurs.

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