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The skeletal structures of the face and throat are derived from cranial neural crest cells (NCCs) that migrate from the embryonic neural tube into a series of branchial arches (BAs). The first arch (BA1) gives rise to the upper and lower jaw cartilages, whereas hyoid structures are generated from the second arch (BA2). The Hox paralogue group 2 (PG2) genes, Hoxa2 and Hoxb2, show distinct roles for hyoid patterning in tetrapods and fishes. In the mouse, Hoxa2 acts as a selector of hyoid identity, while its paralogue Hoxb2 is not required. On the contrary, in zebrafish Hoxa2 and Hoxb2 are functionally redundant for hyoid arch patterning. Here, we show that in Xenopus embryos morpholino-induced functional knockdown of Hoxa2 is sufficient to induce homeotic changes of the second arch cartilage. Moreover, Hoxb2 is downregulated in the BA2 of Xenopus embryos, even though initially expressed in second arch NCCs, similar to mouse and unlike in zebrafish. Finally, Xbap, a gene involved in jaw joint formation, is selectively upregulated in the BA2 of Hoxa2 knocked-down frog embryos, supporting a hyoid to mandibular change of NCC identity. Thus, in Xenopus Hoxa2 does not act redundantly with Hoxb2 for BA2 patterning, similar to mouse and unlike in fish. These data bring novel insights into the regulation of Hox PG2 genes and hyoid patterning in vertebrate evolution and suggest that Hoxa2 function is required at late stages of BA2 development.
Figure 1. Inhibition of in vitro XHoxa2 translation by antisense morpholinos. A: Position of each morpholino with respect to the cDNA sequence of XHoxa2(b) (Pasqualetti et al.,2000). B: All three morpholinos, Mo1, Mo2, Mo3, significantly inhibit translation of XHoxa2 mRNA containing the 5′UTR sequence, with a higher efficacy for Mo2 (lane 4) than Mo1 and Mo3 (lanes 3 and 5, respectively). Lane 6 is a negative translation control in the presence of pBluescript. Mo2 (or Mo3, data not shown) did not affect translation of XHoxa2:GR mRNA lacking the target 5′UTR sequence (lane 9), whereas Mo1 is still effective in blocking translation (lane 8). Mo1 and Mo2 have no effect on the translation of the unrelated template luciferase mRNA (lanes 11,12). Lanes 1 to 6: Western blot of XHoxa2 in vitro translated products with a Hoxa2 mouse specific antibody recognizing both mouse (lane 1) and Xenopus (lane 2) Hoxa2 protein. Lanes 7 to 12: Autoradiography of 35S-labeled translated products.
Figure 2. Migration and contribution to dorsal and ventral cranial skeletal elements of individual neural crest segments. A–C: Modified from Sadaghiani and Thiebaud (1987) and Newman et al. (1997). The mandibular crest segment (1; yellow) contributes to Meckel's (M), palatoquadrate (Q), and subocular (So) cartilages, the hyoid crest segment (2; blue) contributes to the ceratohyal (C) cartilages, and the anterior (3; pink-purple) and posterior (4; red) branchial crest segments contribute to the ceratobranchial cartilages of the gills. D,E: Embryos were coinjected in one or two blastomeres at the two- or four-cell stage with morpholino oligos and RNA encoding lineage tracers (GFP and/or β-galactosidase). Embryos (not shown) or tadpoles were selected for asymmetric distribution of the lineage tracer in half of the head region before in situ hybridization or skeletal staining, respectively. In D is shown the ventral view of a living injected tadpole in which half of the head cartilages are fluorescent due to GFP expression. E shows the same specimen in bright field illumination. ET, ethmoid-trabecular plate.
Figure 3. Skeletal alterations of second arch structures in MoHoxa2-injected Xenopus embryos. A: In the drawing, the first arch (1; Hox-negative) and second arch (2; Hoxa2-expressing) streams of migrating NCCs are represented as yellow and blue arrows, respectively (see also Fig. 2). The white arrows indicate the NCC streams migrating in posterior branchial arches. B,D,E,G,H: Alcian Blue–stained skeletal preparations from injected tadpoles at stage 49. The injection side is always on the right. Representative examples of second arch-specific skeletal alterations in Mo2-injected tadpoles, with variable penetrance. B: Mild phenotype in which two ectopic skeletal extensions are present on the lateral margin of the ceratohyalcartilage, one extending anteriorly resembling a duplicated muscular process of the palatoquadratecartilage (Q2), the second one extending posteriorly (So2) evocative of a partial duplication of the caudal extension of the palatoquadrate, the subocular arc (So). Note that, on the injected side, the orthotopic So appears shorter than the So on the non-injected (control) side, possibly reflecting a slight decrease of developmental rate due to the morpholino injection and/or altered signaling between first and second arch territories following Hoxa2 functional knockdown. D,E: The ceratohyal is abnormally connected to the ethmoid-trabecular plate by a supernumerary (So2) subocular arc. D is the whole-mount preparation of the head of the embryo shown in E before dissection of the cartilaginous skeleton. G,H: Severe phenotype showing a mirror-image homeotic transformation in which the ceratohyalcartilage (C) is strongly reduced and replaced by elements resembling a duplicated palatoquadrate structure (Q2) connected posteriorly to a supernumerary subocular arc (So2) and conceivably to a duplicated lateral (proximal) part of the Meckel's cartilage (M2). The broken line represents the axis of symmetry of the mirror-image homeotic duplication. G represents an additional view of the transformation presented in H, after removal of the non-injected ceratohyal and flipping of M, Q, M2, and Q2 cartilages. Legends are as in I. C,F,I: Summary drawings of the malformations observed in B,E,H. First or second arch derivatives display the same color code as in A (yellow or blue, respectively). In F,I, it is assumed that the malformed structures in blue are of second arch origin.Legends are as in B,D,E,G,H.
Figure 4. Molecular changes in morpholino-injected Xenopus embryos. A,B: Embryos were injected with Mo2 and analyzed at stage 37. RNA encoding the lineage tracer β-galactosidase was co-injected to identify the injected side (red). Xbap is normally expressed in the first arch, as shown in B, and marks predominantly the precursors to the palatoquadratecartilage, which contributes to the structures of the mandibular arch (m). A shows the lateral view of the injected side of the same embryo, showing ectopic expression of Xbap (black arrowhead) selectively in the second, hyoid (h), arch.
Figure 5. XHoxb2 expression is precociously down-
regulated in second arch NCCs. A–D: Whole mount
in situ hybridizations with XHoxa2 (A,C) and XHoxb2
(B,D) antisense probes on stage-18 (A,B; dorsal views)
and stage-30 (C,D; lateral views) embryos. Anterior is
to the right. A,B: At stage 18, both XHoxa2 and
XHoxb2 are expressed in the neural plate and
premigratory NCCs of the hyoid (hcs) and branchial
crest (bcs) segments, albeit with distinct intensities.
C,D: At stage 30, the anterior limits of expression in the rhombencephalon are at the r1/r2 and r2/r3 borders for
XHoxa2 and XHoxb2, respectively (ov, otic vesicle). In
the NCCs, Xhoxa2 is strongly expressed in BA2 and at
lower levels in BA3-BA4, while Xhoxb2 expression is
no longer present in BA2 and BA3 and only persists in BA4.
