Zhang C et al. (2005), SOX7 and SOX18 are essential for cardiogenesis ...

XB-ART-1304

Dev Dyn 2005 Dec 01;2344:878-91. doi: 10.1002/dvdy.20565.

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SOX7 and SOX18 are essential for cardiogenesis in Xenopus.

Zhang C , Basta T , Klymkowsky MW .

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Early in vertebrate development, endodermal signals act on mesoderm to induce cardiogenesis. The F-type SOXs SOX7 and SOX18beta are expressed in the cardiogenic region of the early Xenopus embryo. Injection of RNAs encoding SOX7 or SOX18beta, but not the related F-type SOX, SOX17, leads to the nodal-dependent expression of markers of cardiogenesis in animal cap explants. Injection of morpholinos directed against either SOX7 or SOX18mRNAs lead to a partial inhibition of cardiogenesis in vivo, while co-injection of SOX7 and SOX18 morpholinos strongly inhibited cardiogenesis. SOX7 RNA rescued the effects of the SOX18 morpholino and visa versa, indicating that the proteins have redundant functions. In animal cap explants, it appears that SOX7 and SOX18 act indirectly through Xnr2 to induce mesodermal (Eomesodermin, Snail, Wnt11), organizer (Cerberus) and endodermal (endodermin, Hex) tissues, which then interact to initiate cardiogenesis. Versions of SOX7 and SOX18 with their C-terminal, beta-catenin interaction domains replaced by a transcriptional activator domain failed to antagonize beta-catenin activation of Siamois, but still induced cardiogenesis. These observations identify SOX7 and SOX18 as important, and previously unsuspected, regulators of cardiogenesis in Xenopus.

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Species referenced: Xenopus laevis
Genes referenced: a2m cer1 eomes hhex mixer myc myh4 myh6 ncam1 nkx2-5 nodal nodal1 nodal2 nodal5 nodal5.2 nodal5.4 nodal6 odc1 ptk2b sia1 snai1 sox17a sox17b.1 sox17b.2 sox18 sox7 tbx5 tnni3 wnt11
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	Figure 1. Expression and activity of SOX18 beta . A: Fertilized eggs were analyzed by RT-PCR for SOX18 RNA at various stages. A weak signal was observed in pre-MBT embryos, but strong expression was detected by stage 10. When animal caps and vegetal masses were analyzed at stage 9.5, SOX18 beta mRNA appeared to be present at similar levels in both regions. Ornithine decarboxylase (ODC) was used as a control for RNA quality throughout. B: In situ hybridization staining with an anti-sense SOX18 beta probe revealed expression primarily in the anterior region of stage 30 embryos; at earlier stages no consistent, region-specific staining was observed and no staining was observed using a sense probe (data not shown). C: To define the regional distribution of SOX7 and SOX18 in the gastrula (stage 10/11) embryos, such embryos were dissected into four regions: Spemann Organizer/Dorsal marginal zone ( ldquo SO rdquo ), lateral marginal zones ( ldquo MZ rdquo ), and ventral marginal zone ( ldquo VZ rdquo ). The schematic shows these regions. D: RNA was isolated from the various regions and used for RT-PCR analysis (30 cycles); SOX7 and SOX18 RNAs were found in the SO and MZ, but not in the VZ region; Hex RNA was restricted to the SO region. Ornithine decarboxylase ( ldquo ODC rdquo ) was found to be uniformly distributed in all four regions. E: A similar analysis was carried out with neurula stage embryos (stage 16/17); these embryos were dissected into anterior dorsal ( ldquo AD rdquo ), anterior ventral ( ldquo AV rdquo ), posterior dorsal ( ldquo PD rdquo ), and posterior ventral ( ldquo PV rdquo ) regions and RT-PCR analysis was performed. F: Using 27 cycles of amplification, we found SOX7 and SOX18 RNA in both anterior regions, but little if any in the posterior regions. Hex and Nkx2.5 RNAs were found in the AV region only, while NCAM RNA (a marker of neural specification) was found in the two dorsal regions (AD and PD) but not in ventral regions. G: When 30 cycles of PCR were used, Tbx5 and MHC alpha , but not TnIc RNAs, could be detected in the anterior ventral region of the embryo. Images in parts C and E are modified from Nieuwkoop and Faber ([1967]).
	Figure 4. Inhibition of cardiogenesis by SOX7 and SOX18 morpholinos: Fertilized eggs were injected at animal and vegetal sites with a total of 30 ng of morpholino and examined at stage âˆ¼30. A: A schematic of a stage-30 embryo showing the location of the cardiogenic region (rrow (image from Nieuwkoop and Faber, 1967). In situ hybridization using antisense RNAs directed against either MHCÎ± (B) or Nkx2.5 (E,F). Both the SOX7 morpholino (MO7) (B,E) and the SOX18 morpholino (MO18) (C,E) reduced the size and intensity of the MHCÎ± and Nkx2.5 staining domains. A combination of both morpholinos (15+15 ng/embryo) (D,F) produced a more complete suppression of MHCÎ± and Nkx2.5 staining (see also Table 1). Rescue studies: To confirm the specificity of the morpholino effects, MO7+MO18-injected embryos were injected with RNAs (0.5 ng/embryo) encoding either altSOX7-GFP (G,K), SOX7GGG-GFP (H,L), SOX18Î²-GFP (J,M), or mtSOX18Î² Î´C-VP16 (18VP16) (J); at stage 30 the embryos were stained in situ for MHCÎ± (G) or Nkx2.5 (K). SOX7, SOX18, and mtSOX18Î² Î´C-VP16 rescued the effects of the combined morpholinos, whereas SOX7GGG-GFP did not.
	Figure 5. Induction of cardiogenesis in animal caps: To determine whether SOX7 induced markers of cardiogenesis, fertilized eggs were injected with RNA encoding SOX7-GFP (0.5 ng/embryo); animal caps were prepared at stage 8 and analyzed by in situ hybridization when control embryo had reached stage 30. Both Nkx2.5 (A) or MHCÎ± (B) were expressed in discrete domains, one per animal cap; no expression of either gene was observed in caps derived from uninjected embryos (data not shown). C: A similar analysis was carried using RT-PCR. Fertilized eggs were injected with RNA (0.5 ng/embryo) encoding either SOX7-GFP (SOX7), mtSOX7Î´C-VP16 (7Î´CVP16), SOX18Î²-GFP (SOX18), mtSOX18Î´C-VP16 (18Î´CVP16), or mtSOX17Î²-GFP (SOX17). Animal caps were prepared and analyzed when controls reached stage 25. Each of the injected RNAs induced the expression of the endodermal marker Edd, but only SOX7 or SOX18 constructs induced markers of cardiogenesis, Nkx2.5, MHCÎ±, Tbx5, and TnIc. D: In a similar study, SOX7-GFP and SOX18Î²-GFP induced expression of the mesodermal markers Snail and Eomesodermin (Eomes). E: The SOX7-GFP and SOX18Î²-GFP induction of Eomes, Hex, and Nkx2.5 expression was blocked by the co-injection of RNA encoding the nodal inhibitor CerS (0.2 ng/embryo).
	sox18 (SRY-box 18) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 28, lateral view, anterior left, dorsal up.
	Figure 2. SOX18 induction of Nodal-related genes in animal caps: To compare the activity of SOX18 to SOX7 and SOX17 in the animal cap system, fertilized eggs were injected with RNAs encoding SOX7-GFP, mtSOX17Î²-GFP, or SOX18Î²-GFP (0.5 ng/embryo). At stage 8, animal caps were prepared, cultured for approximately 4 hr (when control embryos had reached stage 10/11), and then analyzed by RT-PCR (unless otherwise noted, this is the â€œstandardâ€ animal cap assay used throughout this work). As reported previously, SOX7-GFP induced the expression of the five mesoderm-inducing nodal-related genes Xnr1, Xnr2, Xnr4, Xnr5, and Xnr6, as well as Mixer and Endodermin (Edd), while SOX17Î²-GFP induced the expression of Xnr4 and Edd, but not Mixer. SOX18Î²-GFP induced weak expression of Xnr1, and stronger expression of Xnr2, Xnr4, Mixer, and Edd.
	Figure 3. SOX7 and SOX18 morpholinos: A: The sequence of the morpholinos against SOX7 and SOX18, their alignment with their target sequences, and the analogous regions of the other F-type SOX mRNAs are shown. The ability of the SOX7 morpholino to inhibit SOX7 RNA translation has been established previously (Zhang et al., 2005). B: To test the specificity of the SOX18 morpholino, fertilized eggs where injected with 0.5 ng/embryo utr-SOX18Î²-V5 RNA either alone or in the presence of control (CMO) or SOX18 (18MO) morpholinos (15 ng/embryo). At stage 8/9, embryonic lysates were generated and analyzed by SDS-PAGE/immunoblot using a monoclonal antiV5 antibody. The accumulation of SOX18-V5 polypeptides (marked with an asterisk) was blocked by the SOX18 morpholino, but unaffected by the control morpholino. C: Rabbit reticulocyte extracts (25 Î¼L final volume) were programmed with 2 Î¼g each of utr-SOX18Î²-V5 andSOX3-V5 RNAs either alone or together with 80 ng SOX18 morpholino. The reactions were then analyzed by SDS-PAGE and immunoblot using the monoclonal antiV5 antibody. The SOX18 morpholino completely inhibited the accumulation of SOX18-V5 polypeptide, but had no effect on SOX3V5 polypeptide accumulation.
	Figure 6. Direct versus indirect targets of SOX7 regulation: Fertilized eggs were injected with RNA encoding GR-SOX7-GFP (0.5 ng/embryo). Animal caps were treated in four different ways: either left in standard media (AC) with 1% ethanol (the carrier for the dexamethasone), incubated with 20 Î¼M dexamethasone (+DEX), pretreated with 100 Î¼g/ml emetine for 30 min and then incubated with 20 Î¼M dexamethasone and 100 Î¼g/ml emetine (+DEX +Eme), or incubated with 100 Î¼g/ml emetine alone (+Eme). The presence of emetine had no apparent effect on the expression of the genes examined. In the presence of dexamethasone, the Xnrs were induced, along with Eomes, Hex, Wnt11, and Cerberus; of these genes, Xnr4, Xnr5, and Xnr6 were expressed in the presence of both dexamethasone and emetine, indicating that they are direct targets of SOX7 regulation.
	Figure 7. SOX7 antagonism of Î²-catenin signaling and cardiogenic ability. A: Fertilized eggs were injected with RNA encoding either SOX7-GFP (Sx7GFP) or SOX7Î´C-GFP (Sx7Î´C-GFP) (0.5 ng/embryo); at stage 9/10 embryo lysates were prepared and immunoprecipitated with a rabbit antiGFP antibody. Immunoprecipitates where analyzed by SDS-PAGE and immunoblot using an rabbit anti-Î²-catenin antibody. In addition to the immunoglobulin heavy chain (â€œHCâ€), we found that Î²-catenin was co-precipitated with SOX7-GFP (arrow), but that little or no Î²-catenin associated with the SOX7Î´C-GFP immunoprecipitation. Immunoblot with antiGFP antibody of embryonic lysates indicated that both SOX7-GFP and SOX7Î´C-GFP polypeptides accumulated to similar extents (data not shown). B: In an animal cap assay, injection of RNA (0.5 ng/embryo) encoding SOX7Î´C-GFP failed to induce Nkx2.5, Tbx5, or MHCÎ± expression (animal caps analyzed when control embryos reached stage 25). C: Injection of RNA encoding a mutationally stabilized form of Î²-catenin (mt-Î´G-Î²-catenin) induces the expression of Siamois in animal caps. Fertilized eggs were injected with RNA encoding mt-Î´G-Î²-catenin (50 pg/embryo) alone or together with RNAs encoding SOX7-GFP, mt-SOX7Î´C-VP16, or SOX18Î²-GFP (500 pg/embryo); animal caps were prepared at stage 8 and analyzed when control embryos reached stage 10/11. Î²-catenin induced expression of Siamois; both SOX7-GFP and SOX18Î²-GFP suppressed Î²-catenin-induced Siamois expression, and induced expression of Xnr2, Xnr4, Cerberus, and Nkx2.5; SOX7-GFP also induced Xnr5 expression. SOX7Î´C-VP16 failed to suppress Î²-catenin-induced Siamois expression, but induced Xnr2, Xnr4, Xnr5, Cerberus, and Nkx2.5 expression. D: The SOX7GGG-GFP construct is analogous to the SOX17G3 construct generated and characterized by Sinner et al (2004). In animal caps assayed at stage 25, SOX7GGG-GFP (0.5 ng/embryo) induced the expression of Edd, but not Nkx2.5, Tbx5, MHCÎ±, or TnIc. E: To examine the ability of SOX7-GFP, SOX7GGG-GFP, and SOX18Î²-GFP to inhibit Î²-catenin activation of the OT reporter, fertilized eggs were injected with OT and pTK-Renilla DNAs (20 pg each/embryo) together with RNAs encoding mt-Î´G-Î²-catenin (50 pg/embryo) and varying amounts of SOX RNAs; animal caps were analyzed when control embryos reached stage 10/11. SOX7-GFP and SOX18Î²-GFP RNAs inhibited of Î²catenin activation of OT; SOX7GGG-GFP was a much less active antagonist of Î²-catenin in this assay.
	Figure 8. SOX7, SOX18, and the cardiogenic pathway: In this diagram, direct interactions are indicated by solid lines, indirect interactions by dotted lines. SOX17 induces Xnr4, but fails to induce cardiogenesis in animal caps, whereas SOX7 and SOX18 induce both Xnr2 and Xnr4 and induce cardiogenesis. The presence of the nodal inhibitor CerS inhibits SOX7 and SOX18-induced cardiogenesis. How other direct and indirect targets of SOX7 and SOX18 interact with â€œdownstreamâ€ targets of Xnr-regulation (e.g., Eomes, Wnt11, Cerberus, and Hex) remains unclear; nevertheless, the end result in the activation of Nkx2.5 and other cardiac markers by SOX7 and SOX18.
	Suppl Fig 1. Fertilized eggs were injected with either SOX18(delta)C-VP16myc (B), SOX7(delta)C-VP16myc (C), SOX18-GFP (E), or SOX7-GFP (F) RNAs (0.5 ng/embryo). At stages between 25 (A-C) and 30 (D-F) the embryos were stained in situ for Nkx2.5. Compared to uninjected controls (A,D), both GFP and DC-VP16 myc forms of SOX7 and SOX18 induced an apparent increase in the extent and intensity of the Nkx2.5 expression domain.

References [+] :

Agius, Endodermal Nodal-related signals and mesoderm induction in Xenopus. 2000, Pubmed, Xenbase