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Transcriptional regulation of mesoderm genes by MEF2D during early Xenopus development.

Kolpakova A, Katz S, Keren A, Rojtblat A, Bengal E - PLoS ONE (2013)

Bottom Line: At the molecular level, MEF2D knockdown reduced the expression of genes involved in mesoderm formation and patterning.The same promoter region was necessary but not sufficient to mediate MEF2D activity in a reporter gene assay.In sum, our results indicate that the MEF2D protein is a key transcription factor in the marginal zone acting in a positive feedback loop with FGF signaling that promotes mesoderm specification at late blastula stages.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Faculty of Medicine, Technion-Israel Institute of Technology, Haifa, Israel.

ABSTRACT
In Xenopus, specification of the three germ layers is one of the earliest developmental decisions occurring prior to gastrulation. The maternally-expressed vegetally-localized transcription factor VegT has a central role in cell autonomous specification of endoderm and in the generation of mesoderm-inducing signals. Yet, marginally-expressed transcription factors that cooperate with mesoderm-inducing signals are less investigated. Here we report that the transcription factors MEF2A and MEF2D are expressed in the animal hemisphere before mid-blastula transition. At the initiation of zygotic transcription, expression of MEF2D expands into the marginal region that gives rise to mesoderm. Knockdown of MEF2D delayed gastrulation movements, prevented embryo elongation at the subsequent tailbud stage and caused severe defects in axial tissues. At the molecular level, MEF2D knockdown reduced the expression of genes involved in mesoderm formation and patterning. We also report that MEF2D functions with FGF signaling in a positive feedback loop; each augments the expression of the other in the marginal region and both are necessary for mesodermal gene expression. One target of MEF2D is the Nodal-related 1 gene (Xnr1) that mediates some of MEF2D mesodermal activities. Chromatin immunoprecipitation analysis revealed that MEF2D associates with transcriptional regulatory sequences of the Xnr1 gene. Several MEF2 binding sites within the proximal promoter region of Xnr1 were identified by their in vitro association with MEF2D protein. The same promoter region was necessary but not sufficient to mediate MEF2D activity in a reporter gene assay. In sum, our results indicate that the MEF2D protein is a key transcription factor in the marginal zone acting in a positive feedback loop with FGF signaling that promotes mesoderm specification at late blastula stages.

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Axial abnormalities in MEF2D-knockdown embryos.(A) Upper panel: Western blot analysis of MEF2D protein extracted from control embryos and MEF2D AMO-injected embryos at different stages from fertilization to late gastrula stages. Lower panel: Embryos were injected with different concentrations of MEF2D-Flag mRNA, without or with AMO to MEF2D. MEF2D-Flag protein was detected by Western blot analysis using anti Flag antibodies (M2, Sigma). (B) Control uninjected embryos, MEF2D AMO-injected embryos and mismatch-AMO injected embryos. Left panel: Stage 12 embryos; MEF2D AMO delays blastopore closure. Right panel; Western blot of endogenous MEF2D from uninjected, MEF2D AMO and mismatch AMO-injected embryos. (C) Stage 26 embryos that were injected with different amounts of MEF2D AMO and mismatch AMO. (D) Transverse sections in the trunk region of stage 26 control uninjected embryo (left panel) and MEF2D AMO-injected embryo (right panel). The scheme at the right shows the position where sections were performed. Abbreviations: Sm-Somite; Nt-Notochord; S.c-Spinal cord.
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pone-0069693-g002: Axial abnormalities in MEF2D-knockdown embryos.(A) Upper panel: Western blot analysis of MEF2D protein extracted from control embryos and MEF2D AMO-injected embryos at different stages from fertilization to late gastrula stages. Lower panel: Embryos were injected with different concentrations of MEF2D-Flag mRNA, without or with AMO to MEF2D. MEF2D-Flag protein was detected by Western blot analysis using anti Flag antibodies (M2, Sigma). (B) Control uninjected embryos, MEF2D AMO-injected embryos and mismatch-AMO injected embryos. Left panel: Stage 12 embryos; MEF2D AMO delays blastopore closure. Right panel; Western blot of endogenous MEF2D from uninjected, MEF2D AMO and mismatch AMO-injected embryos. (C) Stage 26 embryos that were injected with different amounts of MEF2D AMO and mismatch AMO. (D) Transverse sections in the trunk region of stage 26 control uninjected embryo (left panel) and MEF2D AMO-injected embryo (right panel). The scheme at the right shows the position where sections were performed. Abbreviations: Sm-Somite; Nt-Notochord; S.c-Spinal cord.

Mentions: The expression levels of the mef2a and mef2d gene transcripts from fertilization until gastrula stages were analyzed by quantitative PCR (qPCR). In agreement with previous studies [17], [20], pre-MBT maternal transcripts of both isoforms were detected (Figure 1A). These transcripts were localized animally at the 4 cell stage (Figure 1B). While mef2a expression levels declined after MBT, zygotic mef2d transcripts progressively accumulated due to the onset of zygotic transcription (Figure 1A). Mef2c was not detected by qPCR at these stages (data not shown) [20]. Localization of the mef2d mRNA and protein was analyzed in embryos at stages 8 and 9. Reverse transcription-PCR and Western analyses indicated that MEF2D was confined to the animal hemisphere at stage 8 (Figure 1C, left panels). Likewise, sections that were immunostained with anti MEF2 antibody also indicated animal expression of MEF2D (Figure 1C, right panel). This antibody detected a single band of the MEF2D expected size that was diminished in embryos that were injected with specific antisense morpholino to mef2d (see Figure 2A). In addition, the same anti MEF2 antibody specifically and robustly stained cell nuclei of early neurula stage paraxial mesoderm as was expected for MEF2D (Figure S1). At stage 9, both in situ hybridization (ISH) and immunostaining analyses indicated that the expression of zygotic MEF2D was expanded into the marginal region (Figure 1D). At stage 10.5, mef2d transcript was localized to the dorsal marginal zone (DMZ) (Figure 1E, left panel). At that stage, the expression of injected Mef2-reporter gene (x3 Mef2 Luc) indicated that MEF2 activity was enriched in the DMZ relative to the ventral marginal zone (VMZ) (Figure 1E, right panel). In situ hybridization at later stages revealed zygotic expression of mef2d in the paraxial mesoderm, while expression of zygotic mef2a was detected first at stage 15 (Figure S2). In conclusion, expression of MEF2D before MBT is confined to the animal half of embryos whereas at late blastula it expands to the marginal zone. At early gastrula stage MEF2D expression is reduced to the DMZ and in the latter neurula stages it takes place in the paraxial mesoderm.


Transcriptional regulation of mesoderm genes by MEF2D during early Xenopus development.

Kolpakova A, Katz S, Keren A, Rojtblat A, Bengal E - PLoS ONE (2013)

Axial abnormalities in MEF2D-knockdown embryos.(A) Upper panel: Western blot analysis of MEF2D protein extracted from control embryos and MEF2D AMO-injected embryos at different stages from fertilization to late gastrula stages. Lower panel: Embryos were injected with different concentrations of MEF2D-Flag mRNA, without or with AMO to MEF2D. MEF2D-Flag protein was detected by Western blot analysis using anti Flag antibodies (M2, Sigma). (B) Control uninjected embryos, MEF2D AMO-injected embryos and mismatch-AMO injected embryos. Left panel: Stage 12 embryos; MEF2D AMO delays blastopore closure. Right panel; Western blot of endogenous MEF2D from uninjected, MEF2D AMO and mismatch AMO-injected embryos. (C) Stage 26 embryos that were injected with different amounts of MEF2D AMO and mismatch AMO. (D) Transverse sections in the trunk region of stage 26 control uninjected embryo (left panel) and MEF2D AMO-injected embryo (right panel). The scheme at the right shows the position where sections were performed. Abbreviations: Sm-Somite; Nt-Notochord; S.c-Spinal cord.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3716644&req=5

pone-0069693-g002: Axial abnormalities in MEF2D-knockdown embryos.(A) Upper panel: Western blot analysis of MEF2D protein extracted from control embryos and MEF2D AMO-injected embryos at different stages from fertilization to late gastrula stages. Lower panel: Embryos were injected with different concentrations of MEF2D-Flag mRNA, without or with AMO to MEF2D. MEF2D-Flag protein was detected by Western blot analysis using anti Flag antibodies (M2, Sigma). (B) Control uninjected embryos, MEF2D AMO-injected embryos and mismatch-AMO injected embryos. Left panel: Stage 12 embryos; MEF2D AMO delays blastopore closure. Right panel; Western blot of endogenous MEF2D from uninjected, MEF2D AMO and mismatch AMO-injected embryos. (C) Stage 26 embryos that were injected with different amounts of MEF2D AMO and mismatch AMO. (D) Transverse sections in the trunk region of stage 26 control uninjected embryo (left panel) and MEF2D AMO-injected embryo (right panel). The scheme at the right shows the position where sections were performed. Abbreviations: Sm-Somite; Nt-Notochord; S.c-Spinal cord.
Mentions: The expression levels of the mef2a and mef2d gene transcripts from fertilization until gastrula stages were analyzed by quantitative PCR (qPCR). In agreement with previous studies [17], [20], pre-MBT maternal transcripts of both isoforms were detected (Figure 1A). These transcripts were localized animally at the 4 cell stage (Figure 1B). While mef2a expression levels declined after MBT, zygotic mef2d transcripts progressively accumulated due to the onset of zygotic transcription (Figure 1A). Mef2c was not detected by qPCR at these stages (data not shown) [20]. Localization of the mef2d mRNA and protein was analyzed in embryos at stages 8 and 9. Reverse transcription-PCR and Western analyses indicated that MEF2D was confined to the animal hemisphere at stage 8 (Figure 1C, left panels). Likewise, sections that were immunostained with anti MEF2 antibody also indicated animal expression of MEF2D (Figure 1C, right panel). This antibody detected a single band of the MEF2D expected size that was diminished in embryos that were injected with specific antisense morpholino to mef2d (see Figure 2A). In addition, the same anti MEF2 antibody specifically and robustly stained cell nuclei of early neurula stage paraxial mesoderm as was expected for MEF2D (Figure S1). At stage 9, both in situ hybridization (ISH) and immunostaining analyses indicated that the expression of zygotic MEF2D was expanded into the marginal region (Figure 1D). At stage 10.5, mef2d transcript was localized to the dorsal marginal zone (DMZ) (Figure 1E, left panel). At that stage, the expression of injected Mef2-reporter gene (x3 Mef2 Luc) indicated that MEF2 activity was enriched in the DMZ relative to the ventral marginal zone (VMZ) (Figure 1E, right panel). In situ hybridization at later stages revealed zygotic expression of mef2d in the paraxial mesoderm, while expression of zygotic mef2a was detected first at stage 15 (Figure S2). In conclusion, expression of MEF2D before MBT is confined to the animal half of embryos whereas at late blastula it expands to the marginal zone. At early gastrula stage MEF2D expression is reduced to the DMZ and in the latter neurula stages it takes place in the paraxial mesoderm.

Bottom Line: At the molecular level, MEF2D knockdown reduced the expression of genes involved in mesoderm formation and patterning.The same promoter region was necessary but not sufficient to mediate MEF2D activity in a reporter gene assay.In sum, our results indicate that the MEF2D protein is a key transcription factor in the marginal zone acting in a positive feedback loop with FGF signaling that promotes mesoderm specification at late blastula stages.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Faculty of Medicine, Technion-Israel Institute of Technology, Haifa, Israel.

ABSTRACT
In Xenopus, specification of the three germ layers is one of the earliest developmental decisions occurring prior to gastrulation. The maternally-expressed vegetally-localized transcription factor VegT has a central role in cell autonomous specification of endoderm and in the generation of mesoderm-inducing signals. Yet, marginally-expressed transcription factors that cooperate with mesoderm-inducing signals are less investigated. Here we report that the transcription factors MEF2A and MEF2D are expressed in the animal hemisphere before mid-blastula transition. At the initiation of zygotic transcription, expression of MEF2D expands into the marginal region that gives rise to mesoderm. Knockdown of MEF2D delayed gastrulation movements, prevented embryo elongation at the subsequent tailbud stage and caused severe defects in axial tissues. At the molecular level, MEF2D knockdown reduced the expression of genes involved in mesoderm formation and patterning. We also report that MEF2D functions with FGF signaling in a positive feedback loop; each augments the expression of the other in the marginal region and both are necessary for mesodermal gene expression. One target of MEF2D is the Nodal-related 1 gene (Xnr1) that mediates some of MEF2D mesodermal activities. Chromatin immunoprecipitation analysis revealed that MEF2D associates with transcriptional regulatory sequences of the Xnr1 gene. Several MEF2 binding sites within the proximal promoter region of Xnr1 were identified by their in vitro association with MEF2D protein. The same promoter region was necessary but not sufficient to mediate MEF2D activity in a reporter gene assay. In sum, our results indicate that the MEF2D protein is a key transcription factor in the marginal zone acting in a positive feedback loop with FGF signaling that promotes mesoderm specification at late blastula stages.

Show MeSH
Related in: MedlinePlus