Wei S et al. (2012), Roles of ADAM13-regulated Wnt activity in early...

XB-ART-44671

Dev Biol 2012 Mar 01;3631:147-54. doi: 10.1016/j.ydbio.2011.12.031.

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Roles of ADAM13-regulated Wnt activity in early Xenopus eye development.

Wei S , Xu G , Bridges LC , Williams P , Nakayama T , Shah A , Grainger RM , White JM , DeSimone DW .

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Pericellular proteolysis by ADAM family metalloproteinases has been widely implicated in cell signaling and development. We recently found that Xenopus ADAM13, an ADAM metalloproteinase, is required for activation of canonical Wnt signaling during cranial neural crest (CNC) induction by regulating a novel crosstalk between Wnt and ephrin B (EfnB) signaling pathways (Wei et al., 2010b). In the present study we show that the metalloproteinase activity of ADAM13 also plays important roles in eye development in Xenopus tropicalis. Knockdown of ADAM13 results in reduced expression of eye field markers pax6 and rx1, as well as that of the pan-neural marker sox2. Activation of canonical Wnt signaling or inhibition of forward EfnB signaling rescues the eye defects caused by loss of ADAM13, suggesting that ADAM13 functions through regulation of the EfnB-Wnt pathway interaction. Downstream of Wnt, the head inducer Cerberus was identified as an effector that mediates ADAM13 function in early eye field formation. Furthermore, ectopic expression of the Wnt target gene snail2 restores cerberus expression and rescues the eye defects caused by ADAM13 knockdown. Together these data suggest an important role of ADAM13-regulated Wnt activity in eye development in Xenopus.

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Species referenced: Xenopus tropicalis
Genes referenced: adam13 cat.2 cer1 chrd dvl1 efnb1 efnb2 otx2 pax6 rax snai2 sox2 tbx2 tbxt
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	Fig. 1. Knockdown of ADAM13 affects eye development in Xenopus. Eight-cell stage embryos were injected in one dorsal-animal blastomere with the indicated MO (1.5 ng), and cultured to desired stages. (A) Embryos were scored at stage ~35 for eye defects. One example of each phenotype is shown in the upper panels, and results of multiple experiments are graphed in the lower panels. **, p < 0.001 for comparison between control (CT) and 13-1 morphants, and p = 0.002 for comparison between CT and 13-3 morphants; NS, not significant (p = 0.91). (B and C) Embryos were cultured to stage ~12.5, and in situ hybridization was carried out for pax6 (B) or rx1 (C). The injected side is denoted with a red asterisk. e, eye field; l, lateral stripes. (D and E) Wild-type (D) or γ1-crys/GFP3 transgenic (E) embryos were injected with MO CT (upper panels) or 13-1 (lower panels). Embryos were cultured to stages 21–24 and processed for in situ hybridization for pax6 (D), or to stage ~45 (E). Representative embryos were photographed on both the uninjected (left panels) and injected (right panels) sides. Images taken in red (for co-injected red dextran) and green (for GFP) channels and bright field were merged in E. Note the lack of eye structures and GFP expression on the injected side of the 13-1 morphant. N = number of independent experiments; n = number of embryos scored (same below).
	Fig. 2. ADAM13 metalloproteinase activity is required for eye field formation and eye morphology. One dorsal-animal blastomere of 8-cell stage embryos was injected with the indicated MO (1.5 ng) and, as indicated, rescue mRNA encoding wild-type or the E/A mutant of ADAM13 (25 pg). (A) Embryos were allowed to develop to stage ~12.5 and then processed by in situ hybridization for pax6. A representative embryo of each injection is shown in the upper panels (the injected side is denoted with a red asterisk), and combined results summarized in graphs. (B) Embryos were scored for eye phenotype at stage ~35. **, pb0.001; NS, not significant (p=0.28). See Fig. 1A and Materials and methods for phenotype scoring.
	ig. 3. Effects of ADAM13 MO on eye development can be rescued by blocking EfnB sig- naling or by restoring canonical Wnt signaling. One dorsal-animal blastomere of 8-cell stage embryos was injected with the indicated MO (1.5 ng) and, as indicated, with 100 pg mRNA encoding EphB1δC, or full-length (FL) or deleted forms of Xdsh. (A) Embryos were cultured to stage ~ 12.5 and then processed by in situ hybridization for pax6. The injected side is denoted with a red asterisk. (B) Embryos were scored for eye phe- notype at stage ~ 35. **, p b 0.001 in each case; NS, not significant (p = 0.17). See Fig. 1A and Materials and methods for phenotype scoring.
	Fig. 4. Snail2 rescues the eye phenotypes caused by ADAM13 MO. One dorsal-animal blastomere of 8-cell stage embryos was injected with the indicated MO (1.5 ng) to- gether with or without Snail2 transcript (200 pg). Embryos were processed for in situ hybridization for pax6 at stage ~12.5 (A), or scored for eye defects at stage ~35 (B). The injected side is denoted with a red asterisk. See Fig. 1A and Materials and methods for phenotype scoring. **, p=0.008.
	Fig. 5. ADAM13 controls cerberus expression through Snail2. One-cell stage embryos were injected with 12 ng of MO CT (A and B) or 13-1 (C and D), or MO 13-1 with 1 ng mRNA encoding Snail2 (E and F), and cultured to stage ~11. In situ hybridization was carried out for cerberus, and embryos were cleared with 2:1 benzyl benzoate/ benzyl alcohol before photographed. One representative embryo of each injected group is shown in animal pole view (with dorsal at the top) in the left panels, and all embryos of each injected group are shown in the right panels.
	Fig. 6. The effects of ADAM13 knockdown on pax6 expression and eye morphology can be rescued by Cerberus. One dorsal-animal blastomere of 8-cell stage embryos was injected with the indicated MO (1.5 ng) with or without an mRNA encoding Cerberus (50 pg). Embryos were allowed to develop and then processed by in situ hybridization for pax6 (stage ~12.5; A), or scored for eye phenotype (stage ~35; B). See Fig. 1A and Materials and methods for phenotype scoring. *, p=0.02
	Supplementary Fig. 1. Effects of ADAM13 knockdown on sox2 expression. One-cell stage embryos were injected with the indicated amount of control (CT) MO or MO 13-3, and cultured to stage 13/14. In situ hybridization was carried out for sox2. A representative embryo of each group is shown in the left panels (anterior views with dorsal at the top), and multiple embryos of the same groups are shown in the right panels.
	Supplementary Fig. 2. Effects of ADAM13 knockdown on otx2 expression. Two-cell stage embryos were injected in one blastomere with the indicated MO (6 ng each), cultured to stage 18/19, and processed for in situ hybridization for otx2. Red asterisks denote the injected side. Of the 43 embryos injected with MO 13-3, 35 (81%) displayed a phenotype similar to that shown in B. Fb, forebrain; mb, midbrain.
	Supplementary Fig. 3. Knockdown of ADAM13 has no apparent influence on xbra expression. One-cell stage embryos were injected with the indicated MO (12 ng each), and cultured to stage ~11. In situ hybridization was carried out for xbra, and multiple embryos of the same injection groups are shown in A and C. Embryos were also sectioned as illustrated in E (with animal cap removed to reveal the staining of xbra in mesoderm), and one representative example of each group is shown in B and D (animal pole views with dorsal at the top).
	Supplementary Fig. 4. Knockdown of ADAM13 does not alter chordin expression. One-cell stage embryos were injected with 12 ng control MO (A and B) or MO 13-3 (C and D), and cultured to stage 12/12.5. In situ hybridization was carried out for chordin, and multiple embryos of the same injection groups are shown in A and C (dorsal views with vegetal pole at the top). Embryos were also sectioned as illustrated in Supplementary Fig. 3E, and one representative example of each group is shown in B and D (animal pole views with dorsal at the top).
	Supplementary Fig. 5. Developmental expression of adam13 and related genes. (A-E) The expression pattern of adam13 (A) overlaps with those of efnB1 (B), efnB2 (C), snail2 (D) and cerberus (cer; E) in dorsal mesoderm during early gastrulation (stage ~10.5). Embryos were cleared with 2:1 benzyl benzoate/benzyl alcohol before photographed. All embryos are shown in vegetal pole views, and arrows point to dorsal lip of blastopore. (F-H) Expression of adam13 (F), efnB1 (G) and efnB2 (H) in the presumptive eye field during early neurulation (stage 13-15). All embryos are shown in dorsal views, and brackets indicate the anterior neural plate that corresponds to the eye field.
	Supplementary Fig. 6. The effects of ADAM13 knockdown on pax6 expression and eye morphology can be rescued by exogenous β-catenin (β-cat). One dorsal-animal blastomere of 8-cell stage embryos was injected with the indicated MO (1.5 ng) together with or without β-cat transcript (50 pg). Embryos were processed for in situ hybridization for pax6 at stage ~12.5 (A), or scored for eye defects at stage ~35 (B). The injected side is denoted with a red asterisk. See Fig. 1A and Materials and Methods for phenotype scoring. **, p < 0.001.
	Supplementary Fig. 7. Knockdown of ADAM13 does not affect the contribution of D1.1 blastomere to the eye field. The D1.1 blastomere of 16-cell stage embryos was injected with the indicated MO (0.75 ng) together with red Dextran, as described in Material and Methods. Embryos were cultured to stage 37/38 and analyzed for the presence of red fluorescence in the eyes. (A and B) Injected side of whole embryos showing fluorescence in the eye field. Note the lack of eye pigments in 13-3 morphants. (C and D) Cross sections of the eyes of control (CT; C) and 13-3 (D) morphants showing fluorescence on the injected side. Red dashed circles indicate the eye field, and green dotted lines demarcate the midline of the embryos.

References [+] :

Alfandari, ADAM 13: a novel ADAM expressed in somitic mesoderm and neural crest cells during Xenopus laevis development. 1997, Pubmed, Xenbase