Fig. 4. Somite or RA-loaded bead grafts. (A) Somite and RA bead grafts at the level of r2/r3, r3/r4 and r6/r7. (B-J) Flat mounts of E6 chick hindbrain preparations after somite or RA bead grafts, in situ hybridised with probes for Islet2, Hb9, Hoxa3 or Hoxd4 as indicated. Labels in white bar above each panel indicate the type of graft and embryonic stage at which the graft was undertaken. (D) Double in situ hybridisation with probes for Islet2 and Hoxd4. Asterisks indicate the grafted side and double-headed arrows indicate ectopic gene expression. (K-M,O-T) Double-immunostaining on a sagittal vibratome section (M) or transverse cryosections (K,L,O-T) of E6 chick embryos that have received a somite (M,O-T) or an RA bead graft (K,L) adjacent to the r3/r4 (O-S) or r6/r7 (T) level at stage 10. Antibodies are as indicated, abbreviations as in Figs 1, 2, 3. (M) Rhombomere boundaries are shown by a white line whereas the white asterisk highlights r4 ectopic SM axons. (O) QCPN stains the mesoderm, identifying the grafted somite (as in M). P and S are higher magnifications of O and R, respectively, showing the grafted side only. (N) Diagram of a transverse section of a chick embryo at the level of r4 summarising the normal Lim code on the control side (right) in comparison with the one on the somite/RA bead-grafted side (left). Although Islet1-positive motoneurones are present on either side, only the grafted side contains Islet2-positive/Lim3-negative motoneurones, consistent with an abducens identity for the r4 ectopic SM neurones. Note that when the somite was grafted at the level of r6/r7 the grafted side contains Islet2/Lim3-positive motoneurones consistent with a hypoglossal identity for the r7 ectopic SM neurones (see also Fig. 3P). Scale bar: 720 µm for B-D; 480 µm for E-H; 190 µm for I,J; 60 µm for L,P,S; 100 µm for M; 120 µm for K,Q,R,T.