1. Volume 5, Issue 3, Pages 379-390 (September 2003)
Intrinsic, Hox-Dependent Cues Determine the Fate of Skeletal Muscle Precursors 
Lúcia E Alvares, Frank R Schubert, Colin Thorpe, Roy C Mootoosamy, Louise Cheng, Gary Parkyn, Andrew Lumsden, Susanne Dietrich 
Developmental Cell 
Volume 5, Issue 3, Pages (September 2003)
DOI: /S (03)

2. Figure 1
Neck- and Limb-Derived Lateral Mesoderm Interchangeably Induce MMPs from Neck- and Limb-Level Somites
(A) Dorsolateral view of a HH18 chick embryo stained for Lbx1 in dark blue and Pax3 in red. Arrows indicate MMPs (Lbx1+/Pax3++) at occipital, neck, and limb levels. Open arrowheads: nonmigratory (Lbx1−/Pax3++) hypaxial muscle precursors in the flank.
(B and E) Schemes of operation, (C, D, F, and G) operated embryos; dorsolateral views, anterior toward the top, lateral to the right; arrows indicate endogenous, arrowheads ectopic Lbx1 signals; grafts are marked by asterisks and dotted lines. Abbreviations: aer, apical ectodermal ridge; fl, forelimb; m, myotome; nt, neural tube; t, tongue muscle precursors.
(B–D) Exchange of neck and limb lateral mesoderm.
(B) A strip of host lateral mesoderm was excised and replaced by a strip of donor lateral mesoderm as indicated. (C) Limb lateral mesoderm grafted into the neck; (D) neck lateral mesoderm grafted into the limb. In both cases, MMP-specific Lbx1 expression (dark blue) and Pax3 (red) upregulation occurred in the normal fashion (arrowheads). Fgf8 (also blue) marks the aer of the host developing limbs and, more weakly, the somitic myotomes. Note that neither of the grafts participated in limb formation as evidenced by the absence of FGF8.
(E–G) Insertion of neck and limb lateral mesoderm medial to the somites.
(E) A slit was made between the axial midline tissues and the somites, and a strip of lateral mesoderm was inserted as indicated. (F) Medial grafting of limb lateral mesoderm into the neck, (G) neck lateral mesoderm grafting into the limb. Note that Lbx1 expression (blue) was induced in the medial wall of somites (arrowheads). However, the grafts lacked limb properties as judged by the absence of SF/HGF (stained in red).
Developmental Cell 2003 5, DOI: ( /S (03) )

3. Figure 2
All Lateral Mesoderm Can Induce MMPs from Normally MMP-Producing Somites, but Not from Flank Somites
(A–D) Exchange of lateral mesoderm as in Figures 1B–1D.
(B) Neck lateral mesoderm grafted into the flank cannot trigger Lbx1 expression (dark blue). Pax3 (red) is correctly upregulated, indicating that flank-specific, nonmigratory hypaxial muscle precursors were induced (open arrowheads). In contrast, flank lateral mesoderm into the neck (C) or the limb (D) induces Lbx1 expression (arrowheads).
(E–H) Medial insertion of lateral mesoderm similar to Figures 1E–1G.
(F) Neck-derived lateral mesoderm grafted medially to flank somites does not induce Lbx1 (open arrowheads). Flank-derived lateral mesoderm inserted medially to neck (G) or limb (H) somites induced ectopic Lbx1 signals (blue, arrowheads) medial to the endogenous Lbx1 signals (arrow). Note that SF/HGF (red) is expressed in the endogenous limb mesenchyme as shown in (H), but not in the grafts.
(I–L) Heterotopic somite grafting. Epithelial somites or anterior segmental plate of chick hosts were excised and replaced by epithelial somites/anterior segmental plate from quail donors as indicated in (I) (quail cells detected with QCPN antibody in brown). When flank somites were transplanted into the neck (J), they did not express Lbx1 (blue, open arrowheads). Neck- (K) or limb-level somites (L) grafted to the flank expressed Lbx1 at the correct maturation stage (arrowheads). Abbreviations: hl, hindlimb; others as in Figure 1. In (B–D), (F–H), and (J–L), embryos are orientated as in Figure 1; arrows indicate endogenous, arrowheads ectopic, and open arrowheads missing Lbx1 signals.
Developmental Cell 2003 5, DOI: ( /S (03) )

4. Figure 3
Shifting of Somite Axial Identities Somites Leads to a Corresponding Shift in Lbx1 Expression
(A–C) Dorsal view of HH18 wild-type embryos at hindlimb level stained in blue for HoxD9, HoxA10, and Lbx1. Note that the markers share the same anterior expression boundary.
(D–F) Shift of somitic boundaries with FGF8 beads.
(D) Scheme of FGF8 bead insertion. (E and F) Dorsal views of the operated area 36 hr post surgery, stained in blue for HoxA10 (E) and Lbx1 (F); anterior is to the top. Somites with shifted somitic boundaries are marked with red numerals. Normal expression boundaries in the control side (left) are indicated with arrows; ectopic signals in shifted somites on the right are marked with arrowheads. Note that somites with hindlimb-level identities (i.e., HoxA10 positive) faithfully express Lbx1 even when they encounter a flank environment. Abbreviations: som, somite; others as in Figures 1 and 2.
(G–K) Overexpression of Hox genes using in ovo electroporation.
(G) Bicistronic Hox-eGFP expression constructs were pressure injected into the somitocoels of flank somites at stages 0–III and electroporated as indicated. After 24 hr of reincubation, the presence of eGFP was examined under a fluorescent microscope (H). Fluorescing embryos were then analyzed for the expression of Lbx1 (blue) in regions that harbour the Hox-eGFP transcripts (red). (H and I) Lbx1 was found to be expressed in the lateral dermomyotomal lips of HoxA10/eGFP-positive somites in a cell-autonomous fashion (arrowheads). The same result was obtained, using a HoxD9/eGFP construct (J), while Lbx1 was absent in embryos electroporated with the HoxB1/eGFP control construct ([K], open arrowheads).
Developmental Cell 2003 5, DOI: ( /S (03) )

5. Figure 4 Extrinsic Cues to Trigger MMP Formation from Flank Somites
Flank somites were grafted to forelimb levels (A and D) or exposed to limb-derived lateral mesoderm grafted laterally (B and E) or medially (C and F), using the same grafting protocols as in Figures 1 and 2. When flank somites from quail were heterotopically transplanted to limb levels as indicated in (A), expression of Lbx1 (blue) was induced ([D], arrowheads). Likewise, when a strip of flank lateral mesoderm was replaced by forelimb-derived lateral mesoderm as indicated in (B), Lbx1 expression was strongly induced in the somites adjacent to the graft ([E], arrowheads). Note that the labeled cells began to migrate into the graft, which, judged by the Fgf8 signal in the aer, developed into an ectopic limb bud. (C and F) When the same, limb-derived strip of lateral mesoderm was inserted medially to the flank somites, Lbx1 expression failed ([F], open arrowheads). Note that the graft did not display any limb characteristics as monitored by the absence of SF/HGF (asterisk). Abbreviations as in Figures 1 and 2. Arrows in (D), (E), and (F) indicate endogenous, arrowheads ectopic, and open arrowheads missing Lbx1 signals.
Developmental Cell 2003 5, DOI: ( /S (03) )

6. Figure 5
Tissues from a Developing Limb and FGF4-Soaked Beads Can Stimulate MMP Production from Flank Somites
Operation schemes (A–F) and dorsal views (G–L) plus vibratome cross-sections (M–R) of the operated areas 24 hr post surgery; anterior is toward the top in (G–L), dorsal is toward the top in (M–R). In (G)–(J) and (M)–(P), the embryos were stained for Lbx1 expression in blue. The embryo in (J) and (P) was additionally labeled for Pax3 in red. In (K), (L), (Q), and (R), the embryos were stained in blue for HoxA10.
(A, G, and M) Following removal of the flank-level neural tube, limb-derived lateral mesoderm and overlying ectoderm were grafted medially into the gap. The graft developed into an ectopic limb bud with a morphologically discernible apical ectodermal ridge ([M], aer). In the surrounding flank somites, strong Lbx1 expression was induced, with the expressing cells invading the ectopic limb (arrowheads).
(B, H, and N) Strongly Lbx1-expressing, migratory cells were also induced from flank somites when upon neural tube ablation, only the aer from a HH22 limb was inserted (arrowheads).
(C, I, and O) In contrast, no Lbx1 expression was observed when the pzm was inserted (open arrowheads).
(D, I, and P) FGF4-loaded beads grafted medially between neural tube/notochord and flank somites also strongly induced Lbx1 expression (arrowheads). Note that the Lbx1-positive cells originate in the lateral dermomyotomal lips of the somite but move toward the bead.
(E, K, and Q) Operation as in (A), (G), and (M). Like endogenous limbs, the ectopic limb mesenchyme expresses HoxA10. Note that HoxA10 is also strongly expressed in the dermomyotomal lips facing the graft (arrowheads).
(F, L, and R) Insertion of FGF4 loaded bead as in (D), (J), and (P). Note the ectopic expression of HoxA10 (arrowheads). Labeling of grafts and of endogenous and ectopic marker gene expression was performed as before. Abbreviations: pzm, progress zone mesenchyme; others as in Figures 1 and 2.
Developmental Cell 2003 5, DOI: ( /S (03) )

7. Figure 6 Model for the Specification of Migratory Muscle Precursors
Developmental Cell 2003 5, DOI: ( /S (03) )