1. Volume 4, Issue 5, Pages 839-849 (November 1999)
Control of Vertebrate Limb Outgrowth by the Proximal Factor Meis2 and Distal Antagonism of BMPs by Gremlin 
Javier Capdevila, Tohru Tsukui, Concepción Rodríguez Esteban, Vincenzo Zappavigna, Juan Carlos Izpisúa Belmonte 
Molecular Cell 
Volume 4, Issue 5, Pages (November 1999)
DOI: /S (00)

2. Figure 1
Expression of Meis2, BMPs, Gremlin, and Spalt in the Chick Limb Bud
Whole-mount in situ hybridizations (probes indicated to the left). Meis2 is detected in the lateral mesoderm of the trunk (data not shown) and in the whole limb bud mesenchyme at the initial stages of limb bud development (stage 17 [A]). Later, Meis2 becomes restricted to a proximal domain of the limb bud mesenchyme, shown at stages 20 (B), 21 (C), 23 (D), and 25 (E). BMP-2, -4, and -7 are all expressed in the AER ([F–H], stage 22). BMP-2 is also expressed in a posterior mesenchymal domain (F), and BMP-4 and BMP-7 show anterior and posterior mesenchymal domains of expression (G and H). Gremlin is detected in dorsal and ventral aspects of the mesenchyme of stage 17 limb buds, in a pattern that is maintained during the fist stages of limb budding ([I] shows a stage 19 limb), being characteristically excluded from the posterior margin. Later, it is expressed in a wide domain that surrounds the posterior margin of the limb bud, being stronger distally ([J], stage 22; [K], stage 23; [L], stage 25), until expression is detected in the interdigital spaces ([M], stage 27). Spalt is always observed in the AER and the distal-most part of the mesenchyme ([N], stage 17; [O], stage 21; [P], stage 23), until it starts fading distally at stages 24/25 (Q). (A–E) and (I–M) show hindlimb buds, and (F–H) and (N–Q) show forelimb buds. All are dorsal views, and limbs are oriented so that anterior is up and posterior is down in these and all subsequent panels, unless otherwise indicated.
Molecular Cell 1999 4, DOI: ( /S (00) )

3. Figure 2
Limb Outgrowth Requires Proximal Restriction of Meis2 Expression
Phenotypes caused by infection of right-side stage 10 presumptive limb regions with an adenovirus expressing Meis2 (Ad-Meis2). (A) Contralateral (uninfected) control hindlimb bud photographed at stage 25, and (B) Ad-Meis2-infected hindlimb bud showing a mild phenotype consisting of slight widening of the limb (arrowheads, compare with [A]). (C) Contralateral (uninfected) control forelimb bud (stage 24), and (D) Ad-Meis2-infected forelimb bud showing a stronger phenotype of severe disruption of the AER (arrowhead). (E) Day 10 legs that developed from Ad-Meis2-infected hindlimb buds, showing distal truncations of toes (asterisks) and shortening of tibia and fibula (black arrow); compare with the normal length of the control (white arrow). (F) Alcian blue staining of a control day 10 wing that shows the normal cartilage pattern. (G and H) Same staining of day 10 wings that developed from Ad-Meis2-infected forelimb buds, showing truncations of cartilage elements, predominantly distal (arrowheads). H, humerus; R, radius; U, ulna; II, III, and IV, digits (asterisks indicate pattern alterations in skeletal elements).
Molecular Cell 1999 4, DOI: ( /S (00) )

4. Figure 3
Ectopic Meis2 Causes Generalized Repression of Distal Limb Bud Gene Expression
Ad-Meis2 infection of stage 10 presumptive limb regions. The gene detected in each in situ hybridization is indicated at the bottom of each panel. (A and B) Fgf-4 ([A], mild phenotype, with only a small gap in the Fgf-4 pattern, indicated by an arrowhead; [B], severe phenotype, with a big gap in the AER and almost complete disappearance of Fgf-4). (C and D) Fgf-8 ([C], mild, with a gap in the AER; [D], severe, with multiple gaps). (E) Shh is almost completely absent, even in limbs with no detectable morphological alterations in the AER. Note the altered shape (shorter and wider) of the infected limb bud when compared to the contralateral noninjected limb bud. Similar downregulation is observed for Tbx2 (F), Bmp-2 (G), and Bmp-7 (H). (I) Hoxa-13 is severely repressed (the picture shows an extreme phenotype), as are Hoxd-13 (J), Msx-1 (K), and Spalt ([L], extreme phenotype). Infected limbs are to the right, and the arrowheads indicate sites of changes in gene expression in all panels. The limb buds shown constitute a representative example of the various phenotypes obtained that range from mild (e.g., [A]) to extreme (e.g., [I]).
Molecular Cell 1999 4, DOI: ( /S (00) )

5. Figure 4 Chick Meis2 Promotes Nuclear Translocation of Pbx1
The coexpression of chick Meis2 induces nuclear localization of mouse Pbx1 protein in Drosophila Schneider cells, which were transiently transfected with expression constructs for Pbx1 and Meis2 and processed for indirect immunofluorescence using anti-Pbx1 polyclonal antibodies. Pbx1 protein (in red [A]) is localized to the cytoplasm of expressing Schneider cells, and coexpression of Meis2 with Pbx1 causes nuclear localization of Pbx1 (in red [B]). The arrowhead indicates nuclear accumulation of Pbx1 in one of the doubly transfected cells.
Molecular Cell 1999 4, DOI: ( /S (00) )

6. Figure 5
Modulation of BMP Activity by the Secreted Antagonist Gremlin Regulates Distal Limb Outgrowth
(A–E) Infection of stage 10 presumptive limb regions with RCAS–Gremlin results in a number of phenotypical alterations that include repression of PCD in the anterior margin of the limb ([A], revealed by Nile blue staining, arrowheads) and in the interdigital spaces ([B], note the presence of interdigital membranes in the infected leg, which are already absent from the contralateral noninjected leg, arrowheads). Also, in the day 10 specimen shown in (B), the toes are truncated. (C) RCAS-Gremlin-injected limb buds are initially wider (arrowheads), but, later on, distal outgrowth is inhibited and the limbs end up being shorter, displaying distal truncations. (D and E) Overexpression of Gremlin in the forelimb bud results in distal truncations. (D) Alcian blue staining of the contralateral control wing, and (E) same staining of the wing that developed from a RCAS-Gremlin-injected forelimb bud. Note the truncation of the digits (asterisks) and the shortening of radius (R*) and ulna (U*). (F and G) In RCAS-Gremlin-injected limbs Fgf-4 (F) and Fgf-8 (G) expression is reinforced, and Fgf-4 expands into the anterior AER (asterisk in [F]). Expression of Shh and Hoxd-13 is also initially reinforced (H and I), but, later on, transcripts for both genes begin to fade in the injected limbs (K and L), along with other distal markers such as Msx-1 (M). Injected limbs display severe hyperplasia of the AER (arrowheads in [J], stage 28), and they do not develop distal structures (C) or, in the case of mildly affected limbs, they show distal truncations (E). (N–Q) Gremlin expression is regulated by Shh and BMPs. (N) Mouse Gremlin fails to be maintained in Shh−/− limbs. (O) RCAS-Shh induces Gremlin in the chick limb (arrowhead points to anterior expansion of the domain). (P) RCAS–Noggin downregulates Gremlin (arrowhead). (Q) A bead soaked in BMP-2 protein implanted in the anterior margin of the limb bud induces Gremlin expression along the whole P/D axis of the limb (arrowhead). Infected limbs are to the right when not indicated, and the arrowheads point to sites of changes in gene expression in all panels.
Molecular Cell 1999 4, DOI: ( /S (00) )

7. Figure 6
Regulatory Interactions between Proximal and Distal Factors in the Vertebrate Limb
(A) A bead soaked in BMP-2 protein represses proximal expression of Meis2.
(B) A BMP-2 bead, when applied to previously injected RCAS-Wnt3a limb buds, induces in proximal limb bud cells the distal marker, Spalt.
(C) Similar induction is achieved by BMP-2 plus RCAS-Fgf-8.
(D) BMP-2 plus RCAS-Fgf-8 induces Hoxd-13 proximally.
(E and F) Hoxd-11 and Hoxd-13 both repress Meis2 when ectopically expressed using an RCAS retrovirus. Arrowheads point to sites of changes in gene expression, and the injected limbs in (E) and (F) are to the right.
Molecular Cell 1999 4, DOI: ( /S (00) )

8. Figure 7 Model of P/D Determination during Vertebrate Limb Outgrowth
(A) Meis2 is expressed in the flank region of the embryo prior to limb induction (stage 15) and initially in the mesenchyme of the whole nascent limb bud (stages 17/18). Concomitantly with the initiation of Shh expression in the posterior margin of the limb bud and the induction of the AER Meis2 begins to be restricted proximally (stage 19). Shh expression is depicted in pink and the AER in yellow.
(B) Proximal confinement of Meis2 is absolutely required for distal gene expression in the limb bud, since Meis2 is able to repress AER genes (such as Fgf-8, Bmps, and presumably Wnt3a, all indicated in yellow) and mesenchymal genes (such as Shh, Tbx2, Bmps, Hoxa-13, Hoxd-13, Msx-1, and Spalt) (only Bmps are indicated in the figure for clarity and to stress their roles in distal development). Gremlin (in red) is a target of Bmp-2 (and possibly of other Bmps) and encodes an extracellular antagonist of BMPs that acts by preventing AER repression by BMPs, as shown by the anterior expansion of Fgf-4 and the prolonged maintenance of the AER caused by ectopic Gremlin. Red lines indicate the types of antagonistic regulatory relationships between Meis2 and distal genes described in the main text. Green arrows depict the maintenance of Fgf-4 (in green) in the posterior AER by the antagonistic interaction of Gremlin with BMPs. These schemes are not intended to depict all the interactions involved in P/D determination, and the domains of gene expression are simplified for clarity. See main text for details.
Molecular Cell 1999 4, DOI: ( /S (00) )