Gray RS et al. (2009), Diversification of the expression patterns and ...

XB-ART-40065

Dev Dyn 2009 Aug 01;2388:2044-57. doi: 10.1002/dvdy.22028.

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Diversification of the expression patterns and developmental functions of the dishevelled gene family during chordate evolution.

Gray RS , Bayly RD , Green SA , Agarwala S , Lowe CJ , Wallingford JB .

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Dishevelled (Dvl) proteins are key transducers of Wnt signaling encoded by members of a multi-gene family in vertebrates. We report here the divergent, tissue-specific expression patterns for all three Dvl genes in Xenopus embryos, which contrast dramatically with their expression patterns in mice. Moreover, we find that the expression patterns of Dvl genes in the chick diverge significantly from those of Xenopus. In addition, in hemichordates, an outgroup to chordates, we find that the one Dvl gene is dynamically expressed in a tissue-specific manner. Using knockdowns, we find that Dvl1 and Dvl2 are required for early neural crest specification and for somite segmentation in Xenopus. Most strikingly, we report a novel role for Dvl3 in the maintenance of gene expression in muscle and in the development of the Xenopus sclerotome. These data demonstrate that the expression patterns and developmental functions of specific Dvl genes have diverged significantly during chordate evolution.

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Species referenced: Xenopus laevis
Genes referenced: dvl1 dvl2 dvl3 fn1 myf5 myod1 pax1 snai2 tcf15 twist1
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	Figure 2. Divergent expression patterns of Dvl1, Dvl2, and Dvl3 in Xenopus embryos. A: Dorsal view of a stage-20 embryo exhibits Dvl1 expression in neural crest, optic placodes, and neural folds. B: Lateral view of a stage-23 embryo shows continued Dvl1 expression in the streaming cranial neural crest and the developing eye, as well as the otic placodes and the segmenting somites. C: Lateral view of a stage-29/30 embryo expressing Dvl1 in the somites and in multiple cranial placodes (see Supp. Fig. 2). D: Dorsal view of a stage-22 embryo exhibiting Dvl2 expression in the streaming cranial neural crest, developing eyes, and neural folds. E: Lateral view of a stage-23 embryo expressing Dvl2 in the otic placodes, neural crest, and eyes. F: Lateral view of a stage-29/30 embryo exhibits Dvl2 staining in the somites, developing eye, and branchial arches. G: Lateral view of a stage-18 embryo expressing Dvl3 in paraxial mesoderm. H: Lateral view of a stage-23 embryo exhibits Dvl3 expression in the presomitic mesoderm and developing somitomeres. I: Lateral view of a stage-35/36 embryo expressing Dvl3 in mature somites, multiple head placodes, and the developing heart.
	Figure 5. Neural crest induction in Xenopus requires Dvl1 and Dvl2, but not Dvl3. A-A rdquo : Unilateral injection of MOs + GFP mRNA targeted to one dorsal lateral blastomere of a 16-cell embryo. Slug expression is lost on the injected side due to Dvl1 and Dvl2 morpholinos. B-B rdquo : Dvl1 and Dvl2 MOs eliminate expression of Twist. C,C': Graphs of combined morpholino data for Slug in situ hybridizations. Data shown are percent of embryos showing normal or abnormal expression of Slug or Twist. Uninjected Control N=16; Dvl-1MO 72 ng, N=26; Dvl-2MO 72 ng, N=10; Dvl-3MO 72 ng, N=12; Dvl-1MO + Dvl-2MNO 36 ng each, N=15; Dvl-1MMMO 72 ng, N=13; Dvl-1MO + Dvl-1MMMO 36 ng each, N=25; Dvl-2MO + Dvl-1MMMO 36 ng each, N=11. D,D': Graphs of combined data for Twist in situ hybridizations. Uninjected Control N=36; Dvl-1MO 72 ng N=84; Dvl-2MO 72 ng, N=28; Dvl-3MO 72 ng, N=20; Dvl-1MO + Dvl-2MNO 36 ng each, N=31; Dvl-1MMMO 72 ng, N=54; Dvl-1MO + Dvl-1MMMO 36ng each, N=18; Dvl-2MO + Dvl-1MMMO 36 ng each, N=24.
	Figure 7. Knock down of distinct Dishevelled genes generates distinct somite phenotypes. A: In situ hybridization to MyoD at control stage 29/30. B: Injection of Dvl1+2MO cocktail yields shorter embryos that exhibit a disruption of the normal MyoD pattern. C: Dvl3 morphants are normal in length, but embryos exhibit diminished anterior MyoD expression. D: Stage 32, normal MyoD (purple) in situ hybridization and (D') 12/101 (green) antibody staining. E: In Dvl3 morphants, MyoD expression is reduced anteriorly but 12/101 (E') is maintained. 12/101 staining is increased at the somite boundaries (red arrows). F: Normal Myf5 expression (purple) within somites and along both epaxial and hypaxial somite edges and (F') normal 12/101 staining. G: Myf5 staining of the hypaxial and epaxial somite edges appears unaffected in Dvl3 morphants, but expression is lost within the somites, though 12/101 is maintained (G'). H: Control stage-13 in situ hybridization for MyoD. I: Sibling stage-13 Dvl3 morphant embryo in situ hybridized to MyoD (purple). J, L: Normal expression patterns of bHLH myogenic transcription factors; MyoD and Myf5 at stage 22. K, M: Reduced anterior expression of assayed myogenic markers in Dvl3 morphants at stage 22.
	Figure 8. Knock down of Dvl3 in presomitic mesodermal tissues disrupts normal expression of both paraxis and Pax-1 genes. A,B: Single-stage 29/30 tailbud stage embryo. A: Control uninjected side displays normal paraxis expression in the posterior presomitic mesoderm and within individual somites. a': A magnified image of posterior half of the (A) side of the embryo. B: Morpholino-injected side displays a loss of paraxis expression within individual somites as well as diminished expression in the unsegmented mesoderm. b': A magnified image of the posterior half of the (B) side of the embryo. C: Normal expression of Pax-1 in a wild type stage-37 embryo in the branchial arches and perinotochordal region. D: Dvl3 morphants lack any appreciable Pax-1 staining surrounding the notochord (targeted by injection) while the brachial arches (not targeted by injection) retain Pax-1 expression. Red arrows point to paraxis expression within individual somites. Red bracket highlights defects in both expression and patterning of paraxis transcript expression.
	Figure 6. Divergent, gene-specific somite defects in Dvl morphants Lateral confocal projections displaying somite morphology of Dvl morphants with a muscle specific mouse anti-12/101 antibody (green) and the somite boundaries with a mouse anti- fibronectin antibody (red). (A): Normal morphology of the medial flank of a stage 29/30 embryo (B): Normal fibronectin deposition in somite boundaries stage 29/30 embryo. (C): Dvl1 morphant stage 29/30 embryo displaying segmentation defect (white arrow) (D): Dvl1 morphant stage 29/30 embryo displays defects in the deposition of fibronectin (red) while hevronshape is conserved. (E): Dvl3 morphant stage 29/30 embryo displaying un- hevronedsomites and multiple cells are disrespecting the established somite boundaries. (F): Dvl3 morphant stage 29/30 embryo displaying normal deposition of fibronectin between un-hevronedboundaries. Somite defects were observed in 50-90% of morphants, depending upon the experiment.
	fn1 (fibronectin 1) gene expression in Xenopus laevis embryo, assayed via immunohistochemistry, NF stage 28, lateral view, anterior left, dorsal up.

References [+] :

Afonin, Cell behaviors associated with somite segmentation and rotation in Xenopus laevis. 2006, Pubmed, Xenbase