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Fig. 2. XHoxa2 developmental expression pattern. (A,B) Lateral and dorsal view of a stage 26 embryo showing XHoxa2 expression in branchial arches and hindbrain up to the r1/r2 boundary (arrow). (C,D) Lateral view and horizontal section, respectively (broken line in C represents level of section in D), of stage 28 embryos showing higher XHoxa2 transcript levels in PA2 (arrows) than more posterior arches (C), as well as a specific distribution in NC cells (D).
(E,G) Coronal and horizontal sections, respectively, of stage 41 embryos showing XHoxa2 expression in the procartilaginous condensation of the ceratohyal (arrow), which is identified at stage 44 by Alcian Blue/Sefranine O staining (F,H). BH, basihyal; C, ceratohyal; CG, cement gland; E, eye; Et, ethmoid; G, gill; Ih, interhyoideus; NT, neural tube; Oh, orbitohyoideus; PA, pharyngeal arch; Q, palatoquadrate; r, rhombomere.
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Fig. 3. Skeletal alterations in injected tadpoles. (A) Percent distribution of normal and abnormal skeletal phenotypes observed in stage 49 tadpoles co-injected with XHoxa2 and GFP RNAs, showing low (GFP B) or high (GFP+, C) GFP expression in the branchial region. (D,E) The migration pathways (D, modified from Sadaghiani and Thiebaud, 1987) and contribution to the visceral skeleton (coupled to cartilage staining of the actual structures, E) of individual cranial NC segments in Xenopus. Infrarostral (I) and basihyal (BH) are mesodermal derivatives. (F-L) Flat-mounts of Alcian Blue-stained skeletal preparations from stage 49 tadpoles. Uninjected side always on the left. (F,G,K) Segmentation phenotype of XHoxa2 injected tadpoles showing fusion of mandibular-hyoid arch (F,G), and 3rd-hyoid arch
(K) elements. In K, branchial rays (arrow) characteristic of the 3rd (first gill; G1) arch are fused on the posterior surface of the ceratohyal cartilage. (H,L) Homeotic phenotype showing serial duplication of the ceratohyal cartilage (C2) replacing 1st (H) and 3rd (L) arch elements, respectively. (I,J) Mirror-image homeotic phenotype of palatoquadrate (Q) and proximal portion of Meckel (M) cartilage in embryos injected with mouse Hoxa2 and XHoxa2, respectively. The broken line represents the axis of symmetry. aBCS, anterior branchial crest segment;
C, ceratohyal; Et, ethmoid; G, gill; HCS, hyoid crest segment; Mes and Mes MCS, mesencephalic and metencephalic mandibular crest segment, respectively; pBCS, posterior branchial crest segment.
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Fig. 5. Neural crest segregation and migration in XHoxa2- injected embryos. (A,B) Lateral views of a whole-mount stage 18 embryo hybridised with XSlug. (A) Pre-migratory NC blocks are clearly segregated on the uninjected side (arrows), though not on the injected side (B, asterisks). (C-E) Lateral views of stage 28 embryos hybridized with XAP-2 showing mild (D) and severe (E) alterations in NC stream segregation and migration, as compared with wild type (C). BCS, branchial crest segment; CG, cement gland; E, eye; HCS, hyoid crest segment; MCS, mandibular crest segment; PA, pharyngeal arch.
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Fig. 5. Neural crest segregation and migration in XHoxa2- injected embryos. (A,B) Lateral views of a whole-mount stage 18 embryo hybridised with XSlug. (A) Pre-migratory NC blocks are clearly segregated on the uninjected side (arrows), though not on the injected side (B, asterisks). (C-E) Lateral views of stage 28 embryos hybridized with XAP-2 showing mild (D) and severe (E) alterations in NC stream segregation and migration, as compared with wild type (C). BCS, branchial crest segment; CG, cement gland; E, eye; HCS, hyoid crest segment; MCS, mandibular crest segment; PA, pharyngeal arch.
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Fig. 6. XHoxa2 induction after NC migration results in homeosis of mandibular arch skeletal elements. (A) Luciferase activity from COS cell extracts transiently transfected with the pAdML ARE reporter, β-gal, and Pbx1 together with either Xenopus (XHoxa2), mouse (mHoxa2), zebrafish (zHoxa2) or DEX inducible (XHoxa2:GR) cDNA constructs. Bars represent the mean luciferase activity.e.m. of six independent experiments after normalising to β-gal activity. (B) Distribution of normal (N), segmentation (S) and homeotic (H) phenotypes in XHoxa2:GR-injected tadpoles after DEX treatment at stages (st) 15, 20, 26, 28 and 30. In C, embryos have been co-injected with GFP and selected at stage 49 for high levels of GFP expression in the branchial region. Note the significant enrichment of homeotic phenotypes in stage 26 and 28, but not stage 30, in DEX-treated GFP-selected embryos, when compared with (B). (D) Dorsal view of a whole-mount Alcian Blue-stained skeleton of a stage 49 injected tadpole, DEX-treated at stage 28, showing the loss (vertical arrow) of the muscular process (MP) of the palatoquadrate (Q) on the injected side (right); the broken red line marks the palatoquadrate edge. (E) Skeletal flat-mount of a stage 49 tadpole, treated with DEX at stage 28, showing a mirror-image homeotic transformation of the palatoquadrate and the proximal portion of the Meckel
(M) cartilage into ceratohyal (C2) on the injected side. The red and black broken lines indicate the distal part of the Meckel and the axis of symmetry, respectively. (F,G) Lateral views of the uninjected (F) and injected (G) sides of a stage 37 embryo, treated with DEX at stage 28 and hybridised with Xbap. In F, expression is in the precursors of palatoquadrate and proximal part of the Meckel cartilage (arrow, Q+M). In G, a selective loss of Xbap expression (arrow) at the level of these precursors is observed. (H,I) Lateral views of uninjected (H) and injected (I) sides of a stage 37 embryo, treated with DEX at stage 28, and hybridised with Xgsc. Fusion of 1st and 2nd arch expression domains is indicated in I and J (arrows). (J) Ventral view of the embryo in H,I after dissection of the cement gland. Spatial reorganisation of Xgsc transcripts is evident in 1st arch (PA1, arrowhead). C, ceratoyal; E, eye; Et, ethmoid; I, infrarostral; PA2, 2nd pharyngeal arch.
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