Shi J et al. (2012), sizzled function and secreted factor network dy...

XB-ART-46294

Biol Open 2012 Mar 15;13:286-94. doi: 10.1242/bio.2012019.

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sizzled function and secreted factor network dynamics.

Shi J , Zhang H , Dowell RD , Klymkowsky MW .

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Studies on the role of the E-box binding transcription factor Snail2 (Slug) in the induction of neural crest by mesoderm (Shi et al., 2011) revealed an unexpected increase in the level of sizzled RNA in the dorsolateral mesodermal zone (DMLZ) of morphant Xenopus embryos. sizzled encodes a secreted protein with both Wnt and BMP inhibitor activities. Morpholino-mediated down-regulation of sizzled expression in one cell of two cell embryos or the C2/C3 blastomeres of 32-cell embryos, which give rise to the DLMZ, revealed decreased expression of the mesodermal marker brachyury and subsequent defects in neural crest induction, pronephros formation, and muscle patterning. Loss of sizzled expression led to decreases in RNAs encoding the secreted Wnt inhibitor SFRP2 and the secreted BMP inhibitor Noggin; the sizzled morphant phenotype could be rescued by co-injection of RNAs encoding Noggin and either SFRP2 or Dickkopf (a mechanistically distinct Wnt inhibitor). Together, these observations reveal that sizzled, in addition to its established role in dorsal-ventral patterning, is also part of a dynamic BMP and Wnt signaling network involved in both mesodermal patterning and neural crest induction.

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Species referenced: Xenopus
Genes referenced: a2m bmp4 chd7 dkk1 myc myod1 nog sfrp2 snai1 snai2 sox9 szl tbx2 tbxt twist1 wnt8a
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	Fig 1. Expression analysis of sizzled. In situ hybridization of stage 12 (A,B) and stage 18 (C,D) embryos revealed high levels of expression in both rostral and caudal ventral regions, as well as lower, but detectable expression within the neural ectoderm and neural crest. In later stages, sizzled in situ hybridization staining (E) was readily detected in the ventral and cranial regions. Standard RT-PCR (F) (stage 11) revealed readily detectable levels of sizzled RNA in whole embryos (WE), the ventral axial marginal zone (VAMZ) and dorsal axial marginal zone (DAMZ). Quantitative RT-PCR of the dorsal and ventral regions of stage 25 embryos (G) revealed readily detectable levels of sizzled RNA in both regions. qPCR analysis (H) of control and morpholino-injected embryos (both blastomeres of 2 cell embryos injected, embryos analyzed at stage 11) reveal an increase in sizzled RNA snail2/slug, snail1, and twist1 morphant embryos.
	Fig. 2. Morpholino-based studies of sizzled function. (A) 1 cell embryos were injected with RNA encoding either Sizzled-HA or SizzledD92N-HA (200 pgs/embryo) together with 200 pgs/embryo GFP RNA either alone or together with 7 ngs/embryo sizzled morpholino; immunoblot analysis of stage 11 embryos indicated that GFP accumulation was unaffected by morpholino injection, while Sizzled-HA protein levels were reduced, but not completely eliminated. Injection of the sizzled morpholino into 1 cell of 2 cell embryos, together with RNA encoding lacZ as a lineage marker, revealed that compared to control embryos (B), there was a decrease in xbra in situ hybridization staining (C) that could be rescued by the co-injection of sizzled RNA (D). Similarly, the expression of the endodermal marker endodermin (E) was increased in sizzled morphant embryos (F), and this increase was reversed by sizzled RNA injection (G). These effects extended into later stages. Staining with the monoclonal antibody 4A6, which labels the pronephros, revealed a decrease in staining on the injected (H) compared to the uninjected sides (H) of stage 30 embryos. Similarly, the expression of myoD ((I)-control, (J)-sizzled morpholino) ( indicates injection artifact) and the organization of somatic myotomal muscle, visualized by whole-mount staining with an antibody against tropomyosin, ((K), (M)-control, (L)-sizzled morpholino-injected side, (N)-section of whole-mount stained, injected embryo) were disrupted in sizzled morpholino injected regions of the embryo (* indicates injected side in part N).
	Fig3. 32-cell sizzled morphant phenotypes and rescue. Sizzled morpholino was injected, together with GFP RNA, into the C2/C3 blastomeres of 32 cell stage embryos. Embryos were sorted based on fluorescence at stage 10. Stage 11 embryos were stained for xbra RNA ((A)-control, (B,C)-morphant); stage 18 embryos were stained for snail2 ((D)-control, (E,F)-morphant), twist1 ((G)-control, (H,I)-morphant), sox9 ((J)-control, (K,L)-morphant) RNAs. Effects on myoD RNA were examined at stage 25 ((M)-control, (N)-morphant). (O) sox9 expression in sizzled MO embryos was rescued by sizzled RNA injection (200 pgs/embryo) but not by sizzled D92N RNA (green bars indicate percentage of embryos with a wild type phenotype, red bars indicate loss of sox9 RNA staining).
	Fig 4. Sizzled-dependent induction in ectodermal-DLMZ explants. Ectodermal explants were isolated from stage 8/9 embryos and cultured either alone (A) or together with DLMZ explants, isolated from stage 10 wild type (B) or sizzled C2/C3 morphant (C) embryos. When control embryos reached stage 18, explants were stained in situ for sox9 RNA. In similar studies (D), explants were analyzed when control embryos reached stage 11 by qPCR for levels of sox9, snail2, or twist1 RNAs. Levels of these RNAs in whole embryos (WE), animal caps (AC), control animal cap-dorsal mesoderm (AC-DM) or control animal cap-sizzled morphant dorsal mesoderm (sizzled MO) were compared.
	Fig 5. Sizzled-dependent changes in DLMZ gene expression and antagonist rescue studies. (A) The DLMZ region (at stage 11) was isolated from C2/C3 sizzled morphant embryos and subjected to qPCR analysis. Compared to control DLMZs (green), sizzled morphant DLMZs (red) showed small but reproducible increases in wnt8 and bmp4 RNA levels, and reproducible decreases in noggin and and sfrp2 RNA levels. (B) We then examined the ability of injection of noggin and sfrp2 RNAs, either alone or together, to rescue the sizzled C2/C3 morphant sox9 phenotype: neither was effective alone, but in combination they produced a robust rescue. Green bars indicated rescued embryos, red bars indicate loss of sox9 expression. (C) Given that SFRP2 may have both anti-Wnt and anti-BMP activities, we examined the behavior of the ureWnt antagonist Dkk. As before, neither noggin or dkk RNAs alone rescued, but together they produced a greater than 50% rescue of sox9 expression.
	Fig 6. Effects of sizzled over-expression. The effects of sizzled over-expression were complex, as illustrated when the effects of injecting low (200 pgs/embryo) or high (600 pgs/embryo) amounts of sizzled-HA RNA into one blastomere of two cell embryos (control, uninjected side to the left in all images.) At stage 18, the effects on the expression of sox9 ((A)-control, (B)-low, (C)-high) and twist1 ((D)-control, (E)-low, (F)-high), snail2 ((G)-control, (H)-high), chd7 ((I)-high), and c-myc ((J)-control, (K,L)-high) were analyzed. In embryos injected with 200 pgs of Sizzled-HA RNA the sox9 expression domain was altered in 27 of 30 embryos (B); the twist1 expression domain was altered in 26 of 31 embryos (E); the snail2 expression domain was altered in 26 of 30 embryos (G); and the chd7 expression domain was altered in 19 of 24 embryos (data not shown). In embryos injected with 600 pgs of Sizzled-HA RNA, the sox9 expression domain was altered in 21 of 24 embryos (C); the twist1 expression domain was altered in 19 of 22 embryos (F); the snail2 expression domain was altered in 22 of 26 embryos (H); the chd7 expression domain was altered in 16 of 18 embryos (I); and the c-myc expression domain was altered in 24 of 28 embryos (K,L). Red-brown staining indicates lacZ staining due to co-injected LacZ RNA (used as a lineage marker).

References [+] :

Akkers, A hierarchy of H3K4me3 and H3K27me3 acquisition in spatial gene regulation in Xenopus embryos. 2009, Pubmed, Xenbase