Hoxa4/a5/a6/a7 genes are necessary for wing bud formation.

Schemes showing the electroporation in transverse section (a) and in the dorsal view (b). The somite 16 is marked (b). Successful transfection of plasmids as verified by EGFP expression(c). The dn Hox genes downregulated the expression of Tbx5 (B-E), Fgf10 (G-J), and Fgf8 (L-O) and inhibited wing bud formation at the ipsilateral (right) side (Q-T). A-E: HH14; F-O: HH18-19; P-T: HH22; scale bars in c (for c, A-O) and in P (for P-T): 500μm. The proximodistal (P-D) distance (left in U) of wing buds is significantly reduced in Hox dn-expressing wing buds compared to EGFP electroporated wing buds (right in U). The scheme on the left-hand side shows how measurements were made. Red dotted line: baseline of the wing bud; CTRL: normal control wing buds without any operation; Elect.: wing buds after electroporation without constructs; GFP: wing buds after electroporation with EGFP-expressing constructs; A4dn, A5dn, A6dn, A7dn: wing buds after electroporation with dn Hoxa4/5/6/7 expressing constructs, respectively. Each dot represents one embryo; error bars represent mean ±SEM. **p < 0.01.

Dominant negative expression of Hoxa4/a5/a6/a7 down-regulated gene expression.

The numbers of embryos with an unambiguous effect and the total number of embryos analysed are given (effect/total number analysed).

Hoxa6/a7 but not Hoxa4/a5 are sufficient to induce a neck wing bud.

Scheme showing electroporation of the neck region in the dorsal view (a). The somite 16 is marked. The expression domain of electroporated constructs is marked by a green bar. Expression of Hoxa4(b), Hoxa5(c), Hoxa6(d), Hoxa7(e) in the LPM anterior to the wing field after electroporation with the respective plasmids as documented by in situ hybridization. Whereas ectopic cervical expression of Hoxa6/a7 induced the anterior expression (indicated by arrows) of Tbx5 (D, E, I, J), overexpression of Hoxa4/a5 did not induce anterior expression of it (B, C, G, H). Also, only Hoxa6 and Hoxa7, but not a4 or a5 resulted in the anterior extension of the wing bud (arrows in I-J). The ectopic wing buds (fused with or separated from the endogenous one) induced by HoxPG6-7 are indicated by GFP fluorescence (K-O) and in situ hybridization for Tbx5 (arrows in P-T). b-e and A-E: HH14; F-T: HH22; scale bars in b (for b-e and A-E), in F (for F-J) and in K (for K-T): 500μm. Arrows indicate induced wing buds (P-T).

HoxPG6/7 upregulated wing bud formation in the neck region.

Number indicate the numbers of embryos in which a cervical extension of the wing bud, or a cervical wing bud separated from the normal wing bud could be observed.

The neck wing bud is smaller than the natural wing bud.

The scheme (A) indicates how tissue samples were collected for RNA sequencing. Venn diagram (B) showing the overlap between up-regulated genes expressed in normal wing bud (Wing 948), HoxA6-induced wing bud (A6-Bud 347) and neck tissue (Neck 2202) in the cervical LPM; the heatmap (B’) showing the expression profiles of genes in neck tissue, normal wing bud and HoxA6-induced wing bud. FC: fold change. Gene Ontology (C) analyses showing top 15 terms in biological process for 221 genes of A6-Bud. The heatmap (D) shown the expression levels of genes related to outgrowth, patterning. The expression of Fgf10 (E, F), Fgf8 (G, H), Shh (I) and Lmx1 (J) in transfected embryos is rechecked by ISH. E and G: HH18-19; F, H, I and J: HH22; scale bars in E (for E, G) and in F (for F, H-J): 500μm. Arrows indicate induced wing buds.

Gene Ontology analyses showing top ten terms in Biological Process.

Permissive, instructive, and inhibitory Hox codes regulate the forelimb positioning.

The phylogenetic tree of gnathostomes (A, redrawn from Hirasawa et al., 2016) show that despite the variation in the number of cervical vertebrae (C.V.), the pectoral fin and forelimb (dark blue) are always located at the cervical–thoracic boundary. However, their axial positions with respect to somite number vary widely across species (B and C modified from Burke, 1995). B, C: Bright circles: numbered somites; grey shaded circles: thoracic somites; black bars: spinal nerves of the brachial plexus; curved lines: limb bud. In lamprey embryos (D), expression of Tbx5 homologue is restricted to the heart region (Adachi et al., 2016). In skate embryos (E), Tbx5 expression (blue) extended slightly caudally from the heart anlage (Adachi et al. 2016). In avian embryos (F), it extends from the heart over the neck to the wing field. During wing bud formation, Tbx5 expression is restricted to only the heart and the wing bud. In lateral plate mesoderm, Hox4/5 expression (yellow) extends into the neck region, whereas the anterior expression domain of Hox6/7 (green) is at the wing level. Hox9 expression (magenta) starts posteriorly to the wing. The yellow, green, and magenta colours represent permissive, instructive, and inhibitory functions, respectively. The blue curved line outlines the wing bud.

Primer sequences used for generating in situ hybridization probes by PCR.