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First published online 19 December 2007
doi: 10.1242/dev.013268

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FGF signaling regulates mesenchymal differentiation and skeletal patterning along the limb bud proximodistal axis

Department of Developmental Biology, Washington University School of Medicine, St Louis, MO 63110 USA.

View larger version (84K): [in this window] [in a new window]   Fig. 1. Limb development phenotypes in Fgfr2Msx2-Cre mouse embryos. (A-F) Comparison of limb phenotypes in wild-type control (WT) and Fgfr2Msx2-Cre mouse embryos. (A,D) Skeletal preparations of E15.5 embryos. (B,E) Skeletal preparations of P0 forelimbs. (C,F) Gross appearance of limb buds at E12.5. Note that the hindlimb bud is never formed and only appears as a small bulge (arrow and dotted line) in Fgfr2Msx2-Cre embryos. (G-J) Histology of the distal hindlimb buds at E10 (G,H) and E10.5 (I,J) showing the failure to form a multi-layered AER (arrow) in Fgfr2Msx2-Cre embryos. (K-N) Histology of the distal forelimb buds at E10 (K,L) and E10.5 (M,N) showing initial formation of an AER (arrow) in Fgfr2Msx2-Cre embryos at E10 and regression to a single-layered epithelium by E10.5. (O-R) Whole-mount in situ hybridization showing Fgf8 expression in the AER at E10.5 (O,P) and E11.5 (Q,R). Note that Fgf8 is never expressed in Fgfr2Msx2-Cre hindlimbs (arrow) and is prematurely lost in Fgfr2Msx2-Cre forelimbs, accompanied by distal deformities (dotted line). Data are representative of at least three embryos. Scale bar: in G for G-N, 20 µm.

View larger version (132K): [in this window] [in a new window]   Fig. 2. Reduced mesenchymal proliferation and increased cell death after loss of the AER in Fgfr2Msx2-Cre mouse embryos. Phosphohistone H3 (pHH3) immunohistochemistry was used to assess mesenchymal proliferation. Caspase 3 immunohistochemistry and TUNEL assay were used to assess cell death. Hindlimb (A-H) and forelimb (I-T) sections are shown at the developmental times indicated. Insets in G and H show TUNEL labeling on adjacent sections. Insets in S and T show the overall size of the limb buds at E11.5. Data are representative of at least three embryos. Scale bar: 50 µm.

View larger version (114K): [in this window] [in a new window]   Fig. 3. Limb development phenotypes resulting from FGF receptor inactivation in limb mesenchyme with Prx1-Cre. (A) β-galactosidase staining of Prx1-Cre, R26R forelimb at E9.5. (B-D) Skeletal preparations of P0 wild-type control (WT), Fgfr1Prx1-Cre and Fgfr1/2Prx1-Cre mice. (E-L) Appearance (E-H) and histology (I-L) of E10 forelimb buds of WT, Fgfr1Prx1-Cre, Fgfr2Prx1-Cre and Fgfr1/2Prx1-Cre embryos showing a reduced proximodistal axis in Fgfr1/2Prx1-Cre forelimb buds. (M-P) Phosphohistone H3 (pHH3) immunohistochemistry to assess proliferation at E10 (M,N) and E10.5 (O,P). (Q-T) Caspase 3 immunohistochemistry used to assess cell death at E10 (Q,R) and E10.5 (S,T). Note that the region of reduced density of pHH3-positive cells in proximal mesenchyme of Fgfr1/2Prx1-Cre embryos at E10 (N) corresponds to the region with increased apoptosis (R). Data are representative of at least three embryos. Scale bars: in A, I for I-L and M for M-T, 50 µm.

View larger version (29K): [in this window] [in a new window]   Fig. 4. Comparison of forelimb development and phenotypes derived from genetic studies in the mouse. (A) Models for AER-FGF functions in mesenchymal differentiation and chondrogenic primordia formation along the PD axis during normal limb development. Fgf8 expression in limb field ectoderm at early stages of development stimulates the FGFR-dependent MAP kinase signaling pathway in all mesenchymal cells of the nascent limb bud (red shading) (Corson et al., 2003). Initial Sox9 expression demarcates the stylopod primordia. Having received AER-FGF signals, these Sox9-expressing cells commit to osteochondroprogenitors and will form a mesenchymal condensation. Distal mesenchymal cells, which remain undifferentiated, continue to proliferate under the influence of the AER and receive AER-FGF signals. As Sox9 expression expands with limb outgrowth, the zeugopod and autopod primordia are sequentially established. (B-E) Phenotypes resulting from genetic manipulations of AER-FGF signaling. Loss of AER-FGF signaling does not prevent mesenchymal cells from expressing Sox9, but insufficient AER-FGF signaling triggers mesenchymal cell death that leads to skeletal hypoplasia. (B) Attenuated mesenchymal FGF signal transduction achieved by inactivation of Fgfr1 and Fgfr2 with the Prx1-Cre transgene (see Fig. 3). With progressive Fgfr inactivation during early limb bud development, AER-FGF signals are attenuated in limb mesenchyme. Although reduced in size, chondrogenic primordia still form along the PD axis, which leads to a normally segmented but small and dysmorphic skeleton. (C) The RAR-Cre transgene results in complete inactivation of Fgf8 before forelimb bud initiation (Moon and Capecchi, 2000). Without Fgf8, mesenchymal cells in the nascent limb bud fail to receive FGF signaling. Sox9-expressing cells that are derived from these mesenchymal cells cannot commit to osteochondroprogenitors and fail to form the stylopod primordia. Increased (and precocious) Fgf4 expression in the AER restores FGF signaling in distal undifferentiated mesenchyme allowing the zeugopod and autopod primordia to sequentially form following Sox9 expression. (D) The Msx2-Cre transgene inactivates Fgf4 and Fgf8 after forelimb bud initiation, allowing transient AER-FGF signaling (Sun et al., 2002). Initial FGF signaling in nascent limb mesenchyme ensures stylopod primordia formation. Subsequent loss of Fgf4 and Fgf8 impedes continual commitment of distal mesenchymal cells to osteochondroprogenitors, which is required for formation of normally sized skeletal segments. The severely hypoplastic zeugopod and autopod are formed from small numbers of committed mesenchymal cells that are either derived from nascent limb mesenchyme or result from partial rescue of distal limb mesenchyme by Fgf9 and Fgf17. (E) The RAR-Cre transgene results in complete inactivation of Fgf4 and Fgf8 before forelimb bud initiation (Boulet et al., 2004). Without AER-FGF signaling, Sox9-expressing cells cannot commit to osteochondroprogenitors and fail to form any chondrogenic primordia. Owing to distal mesenchymal defects, the AER or AER functions are not maintained at later stages and distal mesenchymal proliferation is inevitably reduced, which further reduces limb bud size. (F,G) Phenotypes resulting from genetic ablation of the AER at different times of limb development. Proliferation in distal mesenchyme or in mesenchyme adjacent to the AER is reduced after loss of the AER, but mesenchymal cell death is manifested only when the AER is disrupted at early stages. (F) Inactivation of Fgfr2 in the AER after forelimb bud initiation (see Figs 1, 2). AER degeneration results in an arrest of development in distal mesenchyme and autopod primordia fail to form owing to decreased mesenchymal proliferation and loss of the differentiation function of AER-FGFs. Further skeletal development of stylopod and zeugopod primordia, which are established before AER degeneration, is not affected by loss of AER functions at later developmental stages. (G) Inactivation of Fgfr2b (Revest et al., 2001) or conditional inactivation of Fgfr2 in limb field ectoderm before limb bud initiation (in the hindlimb, Figs 1, 2). The absence of any AER function results in decreased mesenchymal proliferation, massive mesenchymal cell death, and subsequent limb bud agenesis. S, stylopod; Z, zeugopod; A, autopod.

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