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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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Animal cap- MEF2D-depleted explants do not express mesoderm markers in Nieuwkoop recombinants.(A) Embryos were injected with vegt mRNA without or with MEF2D AMO. AC explants (10 explants per treatment) were dissected at stage 8 and were allowed to grow to stage 9 (left) or to stage 14 (right). RNA was extracted and semi quantitative RT-PCR was performed. (B) Left panel: Scheme of the experiment. Animal cap explants from control or AMO-injected embryos were dissected at stage 9 and combined with stage 9 vegetal explants for 3 hours (n = 20). Following a co-culture period, explants were separated and AC explants grown to stage 12. RNA was extracted and analyzed by semi quantitative RT-PCR. Right panel: Expression of mesoderm genes was induced in control AC explants previously combined with vegetal explants (left lane) but was barely induced in AC explants from AMO-injected embryos (middle lane).
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pone-0069693-g006: Animal cap- MEF2D-depleted explants do not express mesoderm markers in Nieuwkoop recombinants.(A) Embryos were injected with vegt mRNA without or with MEF2D AMO. AC explants (10 explants per treatment) were dissected at stage 8 and were allowed to grow to stage 9 (left) or to stage 14 (right). RNA was extracted and semi quantitative RT-PCR was performed. (B) Left panel: Scheme of the experiment. Animal cap explants from control or AMO-injected embryos were dissected at stage 9 and combined with stage 9 vegetal explants for 3 hours (n = 20). Following a co-culture period, explants were separated and AC explants grown to stage 12. RNA was extracted and analyzed by semi quantitative RT-PCR. Right panel: Expression of mesoderm genes was induced in control AC explants previously combined with vegetal explants (left lane) but was barely induced in AC explants from AMO-injected embryos (middle lane).

Mentions: Next, we asked whether MEF2D functions as a mediator of vegetal signals in the initiation of mesoderm gene expression. Injection of vegt mRNA induced the expression of pro-mesodermal genes in control AC explants [28], yet expression of these genes by vegt mRNA was prevented in MEF2D-knockdown embryos (Figure 6A). Injected MEF2D AMO prevented the expression of Xnr1 and Xbra at a late blastula stage and myf5 and muscle actin at a late gastrula stage. We therefore conclude that MEF2D is necessary for VegT-induced expression of mesoderm genes.


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

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

Animal cap- MEF2D-depleted explants do not express mesoderm markers in Nieuwkoop recombinants.(A) Embryos were injected with vegt mRNA without or with MEF2D AMO. AC explants (10 explants per treatment) were dissected at stage 8 and were allowed to grow to stage 9 (left) or to stage 14 (right). RNA was extracted and semi quantitative RT-PCR was performed. (B) Left panel: Scheme of the experiment. Animal cap explants from control or AMO-injected embryos were dissected at stage 9 and combined with stage 9 vegetal explants for 3 hours (n = 20). Following a co-culture period, explants were separated and AC explants grown to stage 12. RNA was extracted and analyzed by semi quantitative RT-PCR. Right panel: Expression of mesoderm genes was induced in control AC explants previously combined with vegetal explants (left lane) but was barely induced in AC explants from AMO-injected embryos (middle lane).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3716644&req=5

pone-0069693-g006: Animal cap- MEF2D-depleted explants do not express mesoderm markers in Nieuwkoop recombinants.(A) Embryos were injected with vegt mRNA without or with MEF2D AMO. AC explants (10 explants per treatment) were dissected at stage 8 and were allowed to grow to stage 9 (left) or to stage 14 (right). RNA was extracted and semi quantitative RT-PCR was performed. (B) Left panel: Scheme of the experiment. Animal cap explants from control or AMO-injected embryos were dissected at stage 9 and combined with stage 9 vegetal explants for 3 hours (n = 20). Following a co-culture period, explants were separated and AC explants grown to stage 12. RNA was extracted and analyzed by semi quantitative RT-PCR. Right panel: Expression of mesoderm genes was induced in control AC explants previously combined with vegetal explants (left lane) but was barely induced in AC explants from AMO-injected embryos (middle lane).
Mentions: Next, we asked whether MEF2D functions as a mediator of vegetal signals in the initiation of mesoderm gene expression. Injection of vegt mRNA induced the expression of pro-mesodermal genes in control AC explants [28], yet expression of these genes by vegt mRNA was prevented in MEF2D-knockdown embryos (Figure 6A). Injected MEF2D AMO prevented the expression of Xnr1 and Xbra at a late blastula stage and myf5 and muscle actin at a late gastrula stage. We therefore conclude that MEF2D is necessary for VegT-induced expression of mesoderm genes.

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