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BMP controls proximodistal outgrowth, via induction of the apical ectodermal ridge, and dorsoventral patterning in the vertebrate limb

Sandrine Pizette^1,*, Cory Abate-Shen² and Lee Niswander^1,

¹ Molecular Biology Program and Howard Hughes Medical Institute, Memorial Sloan-Kettering Cancer Center, New York, NY 10021, USA
² Center for Advanced Biotechnology and Medicine, UMDNJ-Robert Wood Johnson Medical School, Piscataway, NJ 08854, USA
^* Present address: EMBL, Developmental Biology Programme, Meyerhofst 1, 69117 Heidelberg, Germany

View larger version (82K): [in a new window]   Fig. 1. Time course of Bmp and Msx RNA expression in ventral ectoderm of the chick early limb bud. RNA in situ hybridization of alternate sections from the hindlimb region of (A-F) an early stage 17 (29 somite) embryo and (G-L) a late stage 17 (32 somite) embryo; (M-Q) whole-mount of late stage 18 embryos which were subsequently cryosectioned (forelimbs). Hybridization with probes as indicated and immunolocalization of EN1 protein (I). Brackets outline the domains of expression in the ectoderm (except in F, where the bracket indicates mesenchymal domain), arrowhead in J indicates position of the AER. (A-F) Dorsal (medial) towards the left and ventral (lateral) to the right. (G-L and M-Q) Dorsal at top, ventral at bottom. In N-Q, expression on the dorsal side is in the mesenchyme, not the ectoderm.

View larger version (74K): [in a new window]   Fig. 2. Dorsoventral patterning is altered by loss or gain of BMP function. Alternate sections of (A-C) control uninfected stage 20 limb, (D-G) Noggin-infected stage 20 limb and (H-K) constitutively activated BmpRIB-infected stage 19 limb. Noggin misexpression results in dorsalized limbs due to loss of EN1 and ectopic Wnt7a and Lmx1 expression on the ventral side (marked by asterisks in D-F), whereas constitutively activated BmpRIB misexpression ventralizes the limb because of EN1 misexpression and downregulation of Wnt7a and Lmx1 on the dorsal side (marked by + in H-J). Note that in H-K, there is no morphological AER. (G,K) Anti-GAG antibody to reveal virus distribution. (L-M) Whole-mount double RNA in situ hybridization for Fgf8 in the AER and Shh in the mesenchyme. (L) Control, (M) Noggin-infected, (N) Lmx1-infected stage 24 limbs. The white line marks the midline of the AER and the DV interface. After dorsalization of the limbs due to misexpression of Noggin or Lmx1, Shh expression is ventrally expanded.

View larger version (95K): [in a new window]   Fig. 3. AER formation is altered by gain of BMP function. (A-I) Whole-mount RNA in situ hybridization for Fgf8 expression in the AER. (A) Uninfected stage 27 hindlimb. (B) Constitutively activated BmpRIB-infected stage 27 hindlimb. In B, the limbs are severely truncated, owing to the absence of the AER and Fgf8 expression. (C) Constitutively activated BmpRIB-infected stage 17 hindlimb. Fgf8 expression is normal in the left hindlimb but is almost absent in the right hindlimb. (D,E,H) Uninfected stage 18 embryo and (F,G,I) constitutively activated BmpRIB-infected stage 18 embryo; right and left forelimbs (RFL and LFL) viewed from the dorsal (D-G) and lateral (H,I) side. Note ectopic Fgf8 expression is limited to dorsal ectoderm (arrows in F,G) and not ventral ectoderm (I; same limb as shown in F). Alternate sections of constitutively activated BmpRIB-infected stage 19 forelimb (J-M) and stage 18 hindlimb (N,O). Fgf8 is ectopically expressed in the dorsal ectoderm (asterisks in J,N), where is correlates with viral GAG expression (K) and Msx2 (L). In another sample, ectopic Fgf8 expression is not observed in regions of high EN1 expression (line in N,O).

View larger version (84K): [in a new window]   Fig. 4. AER formation is altered by loss of BMP function. (A,B) Noggin-infected stage 27 hindlimbs; in B, arrowheads point to remnant AER marked by Fgf8, broken lines outline the notched regions. (C-E) Stage 17 embryos showing the hindlimb region, arrows point to somite 28. (C) Uninfected and (D,E) Noggin-infected embryos. In D, Fgf8 expression is not detected in either limb; in E, there is a small region of Fgf8 expression in the left limb and, in the right limb, the anterior region of Fgf8 expression is absent and the posterior region is disrupted. (F,G) Two examples of Noggin-infected hindlimbs where arrows mark ectopic AERs on the ventral (v) side; in G, the limb is also notched. Anterior is towards the top, posterior is towards the bottom; all are dorsal views, except F, where ventral is towards the bottom, and G, which is a ventral view.

View larger version (41K): [in a new window]   Fig. 5. Changes in BMP signaling alter Msx expression. (A-F) Constitutively activated BmpRIB misexpression induces ectopic Msx2 and Msx1 expression in dorsal ectoderm (B,D,F) compared with uninfected limbs (A,C,E). Asterisks in F highlight ectopic Msx1 expression in dorsal ectoderm. (G-J) Noggin misexpression represses Msx expression (H,J) compared with uninfected limbs (G,I). (H) Msx1 is not expressed in the hindlimb in either the ectoderm or mesenchyme, whereas, in this sample, it is still detected in the forelimb. (J) Msx2 is not expressed in the forelimb, hindlimb and flank ectoderm or mesenchyme. (A,B) Stage 24 forelimbs shown in whole-mount; (C,D) stage 19 and (E,F) late stage 20 forelimbs shown in section. (G,H) Stage 18 (37 somite) embryos; (I, J) stage 17 (32 somite) embryos.

View larger version (98K): [in a new window]   Fig. 6. Msx1 misexpression promotes AER formation. (A-D) Msx1 misexpression induces ectopic AER formation in dorsal ectoderm as detected by ectopic Fgf8 expression (arrows in A-C) and ectopic outgrowths (arrows in C, D). (A,B) Stage 24, dorsal view, (C) stage 27, dorsal towards the left, and (D) stage 30, dorsal view. Inset indicates outgrowth occurs from the dorsal aspect of the limb, the broken line highlights the AER. d and v indicate dorsal and ventral sides, respectively.

View larger version (76K): [in a new window]   Fig. 7. MSX and EN1 act in separate pathways to promote AER formation and DV patterning, respectively. (A-D) Msx1 misexpression does not disrupt DV patterning. Alternate sections from a stage 19 limb bud detected for (A) Fgf8 RNA, (B) Wnt7a RNA, (C) EN1 immunolocalization (bracket) and (D) virus with anti-GAG antibody. GAG staining in the mesenchyme reflects expression in blood vessels. Wnt7a is downregulated only in regions of ectopic Fgf8 expression (arrows in B and A, respectively) as Wnt7a is normally not detected in the AER. (E-H) En1 misexpression does not alter Msx expression, except in cases of AER loss, where Msx expression, which marks the AER, is lost. Msx1 expression in stage 23 uninfected (E) and En1-infected (F) limbs. Msx2 expression in stage 24 uninfected (G) and En1-infected (H) limbs. (I,J) Fgf8 expression in uninfected (I) and En1- infected (J) limbs. In J, arrows indicate faint Fgf8 staining, asterisk indicates dorsal displacement of the Fgf8-positive AER. Note that the dorsally displaced AER fragments are often still connected to the midline AER by a line of Fgf8 expression.

View larger version (18K): [in a new window]   Fig. 8 Model for BMP signaling in the ventral ectoderm in the control of both DV patterning and AER formation. See text for details.