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EphA4/ephrin-A5 interactions in muscle precursor cell migration in the avian forelimb

¹ Division of Biological Sciences, University of Missouri-Columbia, Columbia, MO 65211, USA
² The Burnham Institute, 10901 N. Torrey Pines Rd., La Jolla, CA 92037, USA

View larger version (125K): [in a new window]   Fig. 1. EphA4 RTK is expressed during muscle precursor migration. (A) Cross-section through stage 15 embryo at forelimb levels, hybridized with probe for EphA4 mRNA. EphA4 mRNA is present in lateral dermomyotomal cells (arrow), in the lateral plate mesoderm (*) and the developing nephric system (n). dm, dermomyotome; nt, neural tube. (B-F) Confocal z-series stacks (8 µm) from vibratome sections stained with EphA4 and Pax7 antibodies. (B) At stage 15, EphA4 appears diffusely distributed in the dermomyotome, localizing primarily to its lateral (arrow) and ventral edges, and at low levels in the lateral plate mesoderm. (C) At early stage 16, EphA4 is restricted to the lateral edge of the dermomyotome (arrow). (D) At stage 17, EphA4 is prominent in the lateral dermomyotome and encircles migrating muscle precursors in the proximal limb. Boxed area is high magnification view in inset. (E) EphA4 protein appears absent from muscle precursor cells at stage 19 but is strongly expressed in the progress zone (pz) and dorsoproximal limb mesenchyme (arrow). (F) High-magnification view of E, the lateral edge of the dermomyotome and adjacent dorsoproximal mesenchyme at stage 19.

View larger version (73K): [in a new window]   Fig. 2. EphA4 protein marks the surfaces of muscle precursors. High magnification views of proximal limb mesoderm from early stage 17 embryo, stained with annexin, EphA4 (green) and Pax7 (red) antibodies. (A) Many muscle precursors possess EphA4 protein on their surfaces (arrows in A-C). (B) Annexin labeling reveals cell surfaces of all cells in forelimb. (C) Merged image of A,B, with annexin labeling in blue.

View larger version (91K): [in a new window]   Fig. 3. Ephrin-A5 is primarily localized to forelimb mesoderm. (A) Cross-section through stage 15 embryo at forelimb levels, hybridized with probe for ephrin-A5 mRNA. Ephrin-A5 mRNA is present at apparently low levels in the dermomyotome (arrow) and in the limb mesoderm (*). nt, neural tube. (B-F) Confocal z-series stacks (8 µm) from vibratome sections stained with ephrin-A5 and Pax7 antibodies. (B) At stage 15, ephrin-A5 protein is found in the ventral dermomyotome (dm, arrow), in the dorsal neural tube (nt) and at very low levels in the limb mesoderm (l). (C) At stage 16, ephrin-A5 is found at low levels in the lateral dermomyotome (dm) and in the limb (l). (D) At stage 17, ephrin-A5 localizes to the surfaces of many limb mesoderm cells (b, boxed area) but is expressed at very low levels in proximal territory containing Pax7 muscle precursor cells (a, boxed area). Boxed areas are shown at higher magnification in Da and Db. Prominent ephrin-A5 expression is present in the developing nephric system (n). (E,F) Ephrin-A5 is strictly associated with the ventral portion of the limb at stage 19 and 23. v, ventral; d, dorsal.

View larger version (37K): [in a new window]   Fig. 4. In ovo electroporation targets gene expression to the forelimb mesoderm. (A) The microinjection of plasmid DNA into the coelom (co) and electrode placement, dorsal (+) and ventral (–) to the embryo. (B) Confocal z-series stack (16 µm) from a vibratome section through a stage 23 embryo that was electroporated at forelimb levels with plasmid DNA encoding EGFP. Red, Pax7-positive muscle precursor cells; green, EGFP expression in limb mesodermal cells. ao, aorta; dm, dermomyotome; ec, ectoderm; no, notochord; nt, neural tube; som, somite; sop, somatopleure; spl, splanchnic mesoderm.

View larger version (103K): [in a new window]   Fig. 5. Ectopic expression of ephrin-A5 results in a reduction of muscle precursor cells in the proximal forelimb. All images are confocal z-series (21 µm) from vibratome sections at forelimb levels. (A-D,E-H,I-L) Forelimbs from three different stage 17 embryos in which ephrin-A5/EGFP was targeted to the lateral plate mesoderm via in ovo electroporation at stage 13. For the top three rows, the contralateral, unelectroporated (control) limb is on the left (A,E,I). (B,F,J) Distribution of Pax7 muscle precursor cells in limbs ectopically expressing ephrin-A5. (C,G,K) Same field, EGFP images. (D,H,L) Merged Pax7 and EGFP images. Note that Pax7-positive cells cluster near the lateral dermomyotome in B,F, compared with controls (A,E). (M-P) Forelimbs from control embryos electroporated with pMES-EGFP. (M) Distribution of Pax7 cells in the contralateral, unelectroporated limb. (N-P) The distribution of Pax7 cells in the presence of EGFP alone is similar to the contralateral limb (M).

View larger version (8K): [in a new window]   Fig. 6. Quantitative analysis of the effects of ectopic ephrin-A5 on muscle precursor cell numbers. Pax7-positive cells were counted, as described in the Materials and Methods. Ratios of cell numbers in ectopic ephrin-A5 limbs versus the contralateral limbs (pMES ephrin-A5) were compared with limbs expressing EGFP alone versus the contralateral limbs (pMES). Ratios were analyzed with the Statistical Analysis System general linear models procedure. The mean ratio for pMES ephrin-A5 is 0.53, whereas the mean ratio for pMES is 0.86. Ectopic expression of ephrin-A5 using pMES results in significant reductions in the mean numbers of muscle precursor cells in the forelimb (P=0.0008).

View larger version (138K): [in a new window]   Fig. 7. The effects of ephrin-A5 on muscle precursor cells are direct and specific. Surgically isolated muscle precursor cells from somites at forelimb levels were applied to tissue culture dishes upon which alternating lanes of proteins had been applied. Twenty-four hours later, muscle precursor cells and the striped substrates were visualized and photographed. (A) EphA4 protein (arrow) is distributed in a fine manner on the surfaces of migrating dermomyotomal cells; phase contrast (top) and fluorescence (bottom) microscopy. (B) EphA4 protein is absent on a presumed non-migratory subset of muscle precursors; phase contrast (top) and fluorescence (bottom) microscopy. (C) Dermomyotomal cells on ephrin-A5/fibronectin (fn) versus fn lanes express Pax7, indicating they are muscle precursors. (D) Muscle precursors avoid substrate-bound ephrin-A5/fn (light lanes) and migrate instead on fn alone (dark lanes). (E) Addition of soluble ephrin-A5 blocks the repulsive effects of substrate-bound ephrin-A5 on muscle precursor cells. (F) Muscle precursors grow uniformly on substrates coated with Fc/fn versus fn.