Koebernick K et al. (2003), A restrictive role for Hedgehog signalling duri...

XB-ART-4839

Dev Biol 2003 Aug 15;2602:325-38. doi: 10.1016/s0012-1606(03)00242-2.

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A restrictive role for Hedgehog signalling during otic specification in Xenopus.

Koebernick K , Hollemann T , Pieler T .

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Vertebrate inner ear development is initiated by the specification of the otic placode, an ectodermal structure induced by signals from neighboring tissue. Although several signaling molecules have been identified as candidate otic inducers, many details of the process of inner ear induction remain elusive. Here, we report that otic induction is responsive to the level of Hedgehog (Hh) signaling activity in Xenopus, making use of both gain- and loss-of-function approaches. Ectopic activation of Hedgehog signaling resulted in the development of ectopic vesicular structures expressing the otic marker genes XPax-2, Xdll-3, and Xwnt-3A, thus revealing otic identity. Induction of ectopic otic vesicles was also achieved by misexpression of two different inhibitors of Hh signaling: the putative Hh antagonist mHIP and XPtc1deltaLoop2, a dominant-negative form of the Hh receptor Patched. In addition, misexpression of XPtc1deltaLoop2 as well as treatment of Xenopus embryos with the specific Hh signaling antagonist cyclopamine resulted in the formation of enlarged otic vesicles. In summary, our observations suggest that a defined level of Hh signaling provides a restrictive environment for otic fate in Xenopus embryos.

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Species referenced: Xenopus
Genes referenced: dlx5 eya1 hhip neurod1 pax2 pax8 ptch1 rpe shh slc5a5 smo wnt3a

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	Fig. 1. Effect of XSmo on the proximodistal patterning of the visual system. Lateral view of the head region of embryos (st. 36) injected with mRNAs encoding XSmo (1.5 ng; A, B, D, E), XSmo-M2 (1.5 ng; F), or Shh (500 pg; G) into one of two blastomeres. (C) Transverse vibratome section of an XSmo-injected embryo at the level of the optic vesicles. (D-G) XPax-2 expression (purple) in manipulated embryos detected by whole-mount in situ hybridization. In (E) and (G), injected sides (is) were identified by beta-galactosidase staining (light blue). (H) In situ hybridization revealing ectopic XPtc1 expression in embryos injected unilaterally (one of two blastomeres) with mRNAs coding for XSmo (H, 1.5 ng, 86%, n 28 embryos). is, injected side; nis, noninjected side; ov, optic vesicle; rpe, retinal pigmented epithelium.
	Fig. 2. XSmo induces ectopic otic vesicles expressing XPax-2. Embryos injected with XSmo mRNA (1.5 ng) into one of two blastomeres were fixed at embryonic stage 36 and stained for XPax-2 expression (purple) by whole-mount in situ hybridization. Lateral (Aï¿½C, E) and dorsolateral (D) views of injected embryos showing ectopic vesicular structures expressing XPax-2 in -galactosidase-positive (light blue) manipulated sides (arrowheads, 12%, n 50). (F) Horizontal vibratome section of the embryo shown in (D) at the level of the otic vesicles revealing the vesicular morphology of the ectopic otic vesicle. The single otic vesicle of the noninjected side appeared in a section plane localized more ventrally (not shown). Fig.
	Fig. 3. Ectopic otic vesicles express the otic marker genes Xdll-3 and Xwnt-3A. Head region of embryos (st. 36) injected with XSmo (B, E, D, G, H) or XSmo-M2 (F) mRNA (1.5 ng/embryo each, one of two cells) and stained for Xdll-3 (A, B, E, F) or Xwnt-3A (C, D, G, H) expression. Ectopic vesicles in XSmo-injected embryos expressed Xdll-3 (29%, n 27) and Xwnt-3A (16%, n 31). Ectopic vesicles expressing Xdll-3 also appeared in XSmo-M2-injected embryos (F, 38%, n 26). is, injected side; nis, noninjected side.
	Fig. 4. Effects of mHIP on XPtc1 expression and otic specification. Following injection of mHIP mRNA (750 pg) into one of two blastomeres, embryos were fixed at stage 38 (A) or stage 36 (B-G) and stained for expression of XPtc1 (A), Xdll-3 (B-E), or XPax-2 (F, G). mHIP-injected embryos (n 24) exhibit strongly reduced XPtc1 expression (74%; A). In parallel, ectopic vesicular structures (depicted by triangles) expressing Xdll-3 (27%, n 22) and XPax-2 (10%, n 31) were also observed in mHIP-injected embryos. is, injected side; nis, noninjected side.
	Fig. 5. Misexpression of XSmo and mHIP alters the expression of the placodal marker genes XEya1 and XPax-8. Two-cell-stage embryos were injected into one blastomere with XSmo (A, C, D) mRNA (1.5 ng) or mHIP (B, E-G) mRNA (B, 750 pg; E-G, 500 pg), fixed at early neurula stages (13-14) and analyzed for XEya1 or XPax-8 expression (purple). Embryos are shown in dorsal view in (A) and (C-E) (anterior is down), anterior view in (B) and lateral view in (F) and (G) [anterior is right (F) or left (G)]. Embryos in which XEya1 or XPax-8 signals overlapped with -galactosidase-positive regions (light blue) were analyzed. Manipulated embryos exhibited enhanced and expanded XEya1 expression in the preplacodal region (XSmo: 59%, n 22; mHIP: 60%, n 25). Posterior expansion and enlargement of the XPax-8-positive preotic domain (located anteriorly) was observed in 57% (n 28) and 53% of XSmo-injected embryos, respectively (C), with a concomitant reduction of pronephric XPax-8 expression (C, 46%, n 28). The pronephric and the preotic domain merged in 18% (n 28) of XSmo-injected embryos (D) and in 60% (n 10) of mHIP-injected embryos (E, F). In a subset of mHIP-injected embryos, an expansion of the preotic domain (40%, n 10) and a reduction of the pronephric domain (20%, n 10) were also observed (not shown). Representative examples are shown for each injected construct. is, injected side; nis, noninjected side.
	Fig. 6. Lateral line placodes are disrupted in XSmo- and mHIP-injected embryos. Embryos were injected with XSmo mRNA (1.5 ng, C, D; or 2 ng, A, B) or mHIP mRNA (500 pg, E, F) into one blastomere of the two-cell stage, fixed at stage 28, and analyzed for NeuroD expression. High doses of injected XSmo mRNA resulted in a strong disruption and reduction of the NeuroD-expressing placodal region (B, 44%, n 18) Convergence of ganglia from the epibranchial placodes VII and IX (D, arrow) was observed in 33% (n 21) of embryos injected with low doses of XSmo mRNA. NeuroD expression was also disrupted in 64% (n 25) of mHIP-injected embryos (F). epVII, facial epibranchial placode; epIX, glossopharyngeal epibranchial placode; epX1, first vagal epibranchial placode; expX2/3, fused second and third vagal epibranchial placode; pAD, anterodorsal lateral line placode; pM, middle lateral line placode; pP, posterior lateral line placode; is, injected side; nis, noninjected side.
	Fig. 7. Effect of XPtc1 Loop2 and cyclopamine treatment on otic specification. (A-E) Embryos were injected with XPtc1 Loop2 mRNA (750 pg) into one of two cells, fixed at stage 36, and stained for Xdll-3 expression. Embryos injected with XPtc1 Loop2, a dominant-negative form of XPtc1, exhibit ectopic vesicular structures expressing Xdll-3 (31%, n 36) (indicated by arrows) and enlarged otic vesicles (E). (F) Normal-sized otic vesicle in control embryo treated with 0.38% ethanol (100%, n 10). (G) Enlarged otic vesicle developed upon treatment with 20 M cyclopamine (50%, n 8). Con, control; cyc, cyclopamine; is, injected side; nis, noninjected side.