Shi J et al. (2011), Snail2 controls mesodermal BMP/Wnt induction of...

XB-ART-43436

Development 2011 Aug 01;13815:3135-45. doi: 10.1242/dev.064394.

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Snail2 controls mesodermal BMP/Wnt induction of neural crest.

Shi J , Severson C , Yang J , Wedlich D , Klymkowsky MW .

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The neural crest is an induced tissue that is unique to vertebrates. In the clawed frog Xenopus laevis, neural crest induction depends on signals secreted from the prospective dorsolateral mesodermal zone during gastrulation. The transcription factors Snail2 (Slug), Snail1 and Twist1 are expressed in this region. It is known that Snail2 and Twist1 are required for both mesoderm formation and neural crest induction. Using targeted blastomere injection, morpholino-based loss of function and explant studies, we show that: (1) Snail1 is also required for mesoderm and neural crest formation; (2) loss of snail1, snail2 or twist1 function in the C2/C3 lineage of 32-cell embryos blocks mesoderm formation, but neural crest is lost only in the case of snail2 loss of function; (3) snail2 mutant loss of neural crest involves mesoderm-derived secreted factors and can be rescued synergistically by bmp4 and wnt8 RNAs; and (4) loss of snail2 activity leads to changes in the RNA levels of a number of BMP and Wnt agonists and antagonists. Taken together, these results identify Snail2 as a key regulator of the signals involved in mesodermal induction of neural crest.

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Species referenced: Xenopus laevis
Genes referenced: a2m bmp4 cer1 chd7 chrd eomes fgf8 frzb myc myf5 myod1 odc1 snai1 snai2 sox9 szl tbx2 tbx6 tbxt twist1 vegt wnt8a
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	Fig. 1. snail1 MO effects. Xenopus embryos were injected with RNAs encoding myc-tagged GFP (mt-GFP; 50 pg/embryo) and UTR-Snail1-GFP (which latter includes the target of the snail1 MO) RNAs (600 pg/embryo), either alone or together with the snail1 MO (7 ng/embryo). (A) Immunoblot analysis of stage 11 embryos using an anti-GFP antibody revealed a clear and specific reduction in the accumulation of Snail1-GFP as compared with GFP in the snail1 MO-injected sample. (B,C) UTR-Snail1-GFP RNA was injected into one cell of 2-cell embryos either alone (B) or together with the snail1 MO (C). At stage 11, the snail1 MO greatly reduced UTR-Snail1-GFP fluorescence. (D,E) RT-PCR (D) and qPCR (E) analyses indicate that the injection of the snail1 MO into both cells of 2-cell embryos led to a specific reduction in the levels of twist1 and snail2 RNAs at stage 11. Ornithine decarboxylase (ODC) RNA was used to control for non-specific effects. Error bars indicate s.d.
	Fig. 2. snail1 MO effects on germ layer markers. (A-B′) snail1 MO injection (into one cell of a 2-cell embryo) led to the loss of expression (as measured by in situ hybridization) of xbra (A′, versus control in A) and to an increase in endodermin (edd) (B′, versus control in B) RNA levels (at stage 11). (C,C′) Section analysis revealed that the edd expression domain, which is restricted to the superficial region in control embryos (C), extends deeper into the mesodermal region in snail1 MO-injected embryos (C′, black arrow indicates lacZ marker staining, and the red arrow indicates the extent of edd staining in deep mesodermal tissue). (D-F′) There was a loss of expression of the neural crest/placodal marker sox9 at stage 17/18 (D, severely affected; E, mildly affected; injected sides to the right), as well as a loss of expression of myoD at stage 25, as expressed in myotomal muscle, a mesodermal derivative (F′, versus control in F). (G-J) The effects on sox9 (G,H) and myoD (I,J) of snail1 MO injection (G,I) were rescued by the injection of the Snail1-GFP RNA (H,J), which lacks the snail1 MO target sequence. Injected sides are shown. (K) The percentage of normal embryos plotted with respect to xbra, edd, myoD and sox9 in situ expression. Blue, snail1 MO injected; green, snail1 MO together with snail1 RNA (600 pg/embryo); yellow, snail1 MO together with Snail2-GFP RNA (600 pg/embryo); red, snail1 MO together with Twist1-GFP RNA (600 pg/embryo). The number of embryos examined is indicated above each bar.
	Fig. 3. Loss of mesoderm leads to loss of neural crest. (A,B) Injection of the xbra MO has little effect on myoD (stage 25) or sox9 (stage 17/18) expression. Injection of the antipodean/vegT MO causes a decrease in myoD expression (Fukuda et al., 2010), but little effect on sox9 expression (data not shown). Together (XAP), the xbra and antipodean/vegT MOs block sox9 (stage 17/18) (B, versus control in A) and myoD (data not shown) expression. (C) The percentage of embryos with loss of myoD or sox9 staining, with the number of embryos examined shown at the end of each bar. Injection of snail1, snail2 or twist1 RNAs (600 pg/embryo) produced a modest rescue of sox9 expression in XAP morphant embryos. (D-G) Injection of 600 pg δNp63 RNA into one cell of a 2-cell embryo led to the loss of sox9 (D), xbra (not shown) and myoD (F, moderate effect; G, severe effect; versus control in E) expression in ∼50% of embryos. (H) Injection of RNA encoding the mutated and inactive δNp63R304W protein had no effect on sox9 or myoD (data not shown) expression. (I) Quantitative presentation of the data shown in D-H. (J-L) qPCR analyses of δNp63 RNA-injected embryos (both cells of 2-cell embryos injected, analyzed at stage 11) revealed a substantial decrease in the levels of the xbra, vegT/antipodean and myf5 mesodermal marker RNAs (J). In both δNp63 RNA (K) and XAP MO (L) injected embryos, there were similar decreases in the levels of snail2, snail1 and twist1 RNAs. Error bars indicate s.d.
	Fig. 4. Target blastomere injection effects. (A) Fate map of the 32-cell X. laevis embryo, lateral to front, with blastomeres marked with respect to tiers (A-D) and position (1, dorsal; 4, ventral). Blastomeres are marked that make a major (red) or moderate (green) contribution to neural crest (left) or to lateral somites and plate mesoderm (right). (B) At the 32-cell stage, this embryo was injected with RNA encoding GFP; the embryo was photographed under bright-field and epifluorescence illumination at stage 11. Ventral pole to front (dorsal and ventral are marked). (C-E) C2/C3 blastomeres were injected with snail2, snail1 or twist1 MOs; at stage 17/18 the embryos were fixed and stained in situ for sox9 RNA. sox9 expression was lost in snail2 morphant embryos (C), but was present in snail1 (D) and twist1 (E) morphants. (F) The phenotypes of C2/C3 morphant embryos with respect to xbra, myoD, c-myc and sox9 expression. Bars indicate percentage loss of expression; the number of embryos is indicated above each bar. (G-I) The effects of the snail2 MO could be partially rescued by injection of high levels (600 pg/embryo) of snail2 (G), snail1 (H) or twist1(I) RNAs. (J-L) snail2 RNA was more effective at rescuing the snail2 C2/C3 morphant sox9 phenotype than either snail1 or twist1 RNAs. snail2 C2/C3 morphant embryos were co-injected with 150 pg/embryo of snail2 (J), snail1 (K) or twist1 (L) RNA. (M) The rescue of the C2/C3 snail2 morphant sox9 phenotype by 150, 300 and 600 pg/embryo snail2, snail1 and twist1 RNAs. Bars indicate percentage loss of expression; the number of embryos is indicated above each bar. (N) The DLMZ of C2/C3 MO-injected embryos was dissected at stage 10.5 and subject to qPCR analysis. The effects on the mesodermal markers xbra, eomes, tbx6, myf5, as well as on wnt8 and fgf8 RNA levels were analyzed. The results shown are representative of studies carried out in triplicate. Error bars indicate s.d.
	Fig. 5. Dorsal mesoderm-animal cap explant studies. (A) When cultured alone, wild-type ectoderm (animal cap) contained little sox9 RNA, as visualized by in situ hybridization (analysis carried out when control embryos had reached stage 17/18). (B) When cultured together with DLMZ from wild-type Xenopus embryos, sox9 RNA accumulated (in the ectodermal region). (C) By contrast, DLMZ from snail2 C2/C3 morphant embryos failed to induce sox9 RNA. (D,E) snail1 morphant DLMZ appears to be intermediate in its ability to induce sox9 expression (D), whereas twist1 morphant DLMZ retained the ability of induce sox9 expression (E). (F) qPCR analysis of sox9 RNA levels supports this general trend. For each condition, 5-15 explants were analyzed when unmanipulated embryos had reached stage 17/18. ODC RNA was used as a standard. WE, whole embryo at stage 17/18 (level set to 1); AC, animal cap/ectodermal explant; ACM, animal cap plus DLMZ from uninjected control embryo; snail2, wild-type animal cap plus snail2 morphant DLMZ; snail1, wild-type animal cap plus snail1 morphant DLMZ; twist1, wild-type animal cap plus twist1 morphant DLMZ. The results shown are representative of more than three replications. Error bars indicate s.d.
	Fig. 6. Further characterization of the snail2 morphant phenotype. (A-E) Xenopus C2/C3 blastomeres were injected with snail2 MO. Compared with uninjected embryos (A), snail2 morphants displayed a reduction in sox9 expression at stage 17/18 (B). This reduction was rescued by the injection of 25 pg/embryo bmp4 RNA (C), wnt8 RNA (D), or the two RNAs together (E). In the case of wnt8 RNA injection, there was often evidence of the formation of a secondary axis (D, line to the right). (F) The percentage of snail2 MO C2/C3 injected embryos rescued (red bar) using 25 pg/embryo fgf8, wnt8, bmp4 or wnt8 and bmp4 (25 pg each) RNAs is shown. (G) In a similar study, lower amounts (10 pg/embryo) of bmp4 and wnt8 RNAs were used. Rescue was observed only when bmp4 and wnt8 RNAs were injected together. (H,I) In situ analysis indicated that the levels and extent of chordin expression increased in stage 11 C2/C3 snail2 morphant embryos (I, versus control in H). (J) At stage 10.5-11, DLMZ from snail2, snail1 or twist1 morphant C2/C3 dorsolateral zones were dissected, RNA was isolated and subjected to qPCR analysis. This revealed reproducible increases in the levels of sizzled, cerberus and chordin RNAs and decreases in the levels of wnt8, bmp4 and frzb1 RNAs; distinct patterns of change were observed in snail1 and twist1 morphant DLMZ. Error bars indicate s.d.
	Fig. S2. snail1 morphant effects on neural crest markers. (A-H) Control (A,D,G) and snail1 morphant (one cell of 2-cell embryos injected with 6 ng/embryo) (B,C,E,F,H) embryos were stained in situ at stage 17/18 for twist1 (A-C), chd7 (D-F) and snail2 (G,H). In the injected embryos, lacZ staining is particularly evident on the right-hand side of the embryos shown in C,E,F,H.
	Fig. S3. Later stage, Xbra, Antipodean and pδN63 myotomal phenotypes. (A-E) Injection (one cell of a 2-cell embryo) of an MO against xbra RNA had little effect on myoD expression (B, versus uninjected in A), whereas the antipodean/vegT MO caused a decrease in myoD expression (C), as expected (see Fukuda et al., 2010). Embryos were fixed and stained at stage 25. Together, the xbra and antipodean/vegT MOs had a similar effect on myoD expression as the antipodean/vegT MO alone (D, uninjected side; D′, injected side). A similar decrease in myoD RNA levels was observed in deltaNp63 RNA-injected embryos (E, injected side).
	Fig. S4. snail2, snail1 and twist1 morphant phenotype in Xenopus tropicalis. (A-E) The C2 and C3 blastomeres of 32-cell X. tropicalis embryos were injected with snail2 (B,C), snail1 (D) or twist1 (E) MOs (10 ng/embryo). At stage 18, embryos were fixed and stained in situ for sox9 (A, control uninjected embryo); 60% (n=20) of the snail2 morphant embryos showed a decrease in sox9 expression, whereas only 12% (n=25) of the snail1 and 11% (n=35) of the twist1 morphant embryos displayed a similar decrease in sox9 expression.

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