Limits...
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.

Show MeSH
MEF2D associates with Xnr1 regulatory elements.(A) Chromatin immunoprecipitation (ChIP): Embryos were injected with mRNA encoding MEF2D-Flag and at stage 10, crosslinked sheared chromatin was prepared. Chromatin was immunoprecipitated with anti-Flag (polyclonal, Sigma) or with pre immune serum (control) and was subjected to a qPCR reaction with several pairs of primers (left). Expression of the injected MEF2D-Flag protein was analyzed by Western blot (right). (B) Upper panel: Xnr1 promoter sequence (proximal region) with highlighted putative binding sites of MEF2. PE-proximal element; IE1, 2-Intermediate element 1, 2; DE-distal element; TBX1, 2- T box binding sites (VegT) [47]. Arrows show the two transcription start site and “M” the translation initiation codon. Lower panel: EMSA of each of the MEF2 binding elements coupled with protein extracts of stage 9 control embryos as well as embryos injected with mef2d-flag mRNA. Anti-flag antibody (1 µl, 0.1 µg/µl) was included in some reaction mixtures while unlabeled homologous double stranded oligonucleotides in 100 fold excess over the probe was included in others, as indicated. Unbound probes are not shown. Arrow indicates the MEF2D-DNA complex. Arrowhead indicates the Anti MEF2-MEF2D-DNA complex. (C) 293T HEK cells were transfected as indicated. Thirty six hours later, proteins were extracted and luciferase activity was measured and was normalized to total protein levels. Activity of the reported gene with an empty vector was set to a value of 1 and values of other treatments were standardized accordingly. Means of two independent experiments are presented.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3716644&req=5

pone-0069693-g008: MEF2D associates with Xnr1 regulatory elements.(A) Chromatin immunoprecipitation (ChIP): Embryos were injected with mRNA encoding MEF2D-Flag and at stage 10, crosslinked sheared chromatin was prepared. Chromatin was immunoprecipitated with anti-Flag (polyclonal, Sigma) or with pre immune serum (control) and was subjected to a qPCR reaction with several pairs of primers (left). Expression of the injected MEF2D-Flag protein was analyzed by Western blot (right). (B) Upper panel: Xnr1 promoter sequence (proximal region) with highlighted putative binding sites of MEF2. PE-proximal element; IE1, 2-Intermediate element 1, 2; DE-distal element; TBX1, 2- T box binding sites (VegT) [47]. Arrows show the two transcription start site and “M” the translation initiation codon. Lower panel: EMSA of each of the MEF2 binding elements coupled with protein extracts of stage 9 control embryos as well as embryos injected with mef2d-flag mRNA. Anti-flag antibody (1 µl, 0.1 µg/µl) was included in some reaction mixtures while unlabeled homologous double stranded oligonucleotides in 100 fold excess over the probe was included in others, as indicated. Unbound probes are not shown. Arrow indicates the MEF2D-DNA complex. Arrowhead indicates the Anti MEF2-MEF2D-DNA complex. (C) 293T HEK cells were transfected as indicated. Thirty six hours later, proteins were extracted and luciferase activity was measured and was normalized to total protein levels. Activity of the reported gene with an empty vector was set to a value of 1 and values of other treatments were standardized accordingly. Means of two independent experiments are presented.

Mentions: We next characterized the effects of gain of MEF2 activity. To induce ectopic MEF2 activity, a transcript encoding MEF2D-Flag protein was marginally-injected to vegetal blastomeres of 4 cell embryos. Injection of this transcript induced the expression of a MEF2-dependent reporter gene (x3 MEF2-Luc) (see figure 8C). Injected mef2d-flag mRNA caused ectopic punctate staining of Xbra in the vegetal region as observed in in situ hybridization analysis (Figure 4A, left panel). MEF2D-Flag slightly increased the expression of endogenous mesoderm and organizer genes as shown by RT-PCR analysis (Figure 4A, right panel). In a second approach to induce ectopic MEF2 activity, a transcript encoding the potent HSV VP16 activation domain and the DNA binding domain of MEF2C (mef2-vp16) was marginally injected to embryos (Figure 4B). The constitutively active MEF2-VP16 chimera protein induced prominent ectopic vegetal expression of Xbra in gastrula stage embryos. Injection of mef2-vp16 mRNA into one of two blastomeres expanded the expression domain of myod in the injected half of the embryo at the neurula stage. Marginal zone injection of mef2-vp16 mRNA induced the expression of Xnr1 in gastrula VMZ explants and of myod and muscle actin in neurula VMZ explants. Overall, these results indicated that exogenously-expressed MEF2 protein induced ectopic expression of paraxial 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)

MEF2D associates with Xnr1 regulatory elements.(A) Chromatin immunoprecipitation (ChIP): Embryos were injected with mRNA encoding MEF2D-Flag and at stage 10, crosslinked sheared chromatin was prepared. Chromatin was immunoprecipitated with anti-Flag (polyclonal, Sigma) or with pre immune serum (control) and was subjected to a qPCR reaction with several pairs of primers (left). Expression of the injected MEF2D-Flag protein was analyzed by Western blot (right). (B) Upper panel: Xnr1 promoter sequence (proximal region) with highlighted putative binding sites of MEF2. PE-proximal element; IE1, 2-Intermediate element 1, 2; DE-distal element; TBX1, 2- T box binding sites (VegT) [47]. Arrows show the two transcription start site and “M” the translation initiation codon. Lower panel: EMSA of each of the MEF2 binding elements coupled with protein extracts of stage 9 control embryos as well as embryos injected with mef2d-flag mRNA. Anti-flag antibody (1 µl, 0.1 µg/µl) was included in some reaction mixtures while unlabeled homologous double stranded oligonucleotides in 100 fold excess over the probe was included in others, as indicated. Unbound probes are not shown. Arrow indicates the MEF2D-DNA complex. Arrowhead indicates the Anti MEF2-MEF2D-DNA complex. (C) 293T HEK cells were transfected as indicated. Thirty six hours later, proteins were extracted and luciferase activity was measured and was normalized to total protein levels. Activity of the reported gene with an empty vector was set to a value of 1 and values of other treatments were standardized accordingly. Means of two independent experiments are presented.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0069693-g008: MEF2D associates with Xnr1 regulatory elements.(A) Chromatin immunoprecipitation (ChIP): Embryos were injected with mRNA encoding MEF2D-Flag and at stage 10, crosslinked sheared chromatin was prepared. Chromatin was immunoprecipitated with anti-Flag (polyclonal, Sigma) or with pre immune serum (control) and was subjected to a qPCR reaction with several pairs of primers (left). Expression of the injected MEF2D-Flag protein was analyzed by Western blot (right). (B) Upper panel: Xnr1 promoter sequence (proximal region) with highlighted putative binding sites of MEF2. PE-proximal element; IE1, 2-Intermediate element 1, 2; DE-distal element; TBX1, 2- T box binding sites (VegT) [47]. Arrows show the two transcription start site and “M” the translation initiation codon. Lower panel: EMSA of each of the MEF2 binding elements coupled with protein extracts of stage 9 control embryos as well as embryos injected with mef2d-flag mRNA. Anti-flag antibody (1 µl, 0.1 µg/µl) was included in some reaction mixtures while unlabeled homologous double stranded oligonucleotides in 100 fold excess over the probe was included in others, as indicated. Unbound probes are not shown. Arrow indicates the MEF2D-DNA complex. Arrowhead indicates the Anti MEF2-MEF2D-DNA complex. (C) 293T HEK cells were transfected as indicated. Thirty six hours later, proteins were extracted and luciferase activity was measured and was normalized to total protein levels. Activity of the reported gene with an empty vector was set to a value of 1 and values of other treatments were standardized accordingly. Means of two independent experiments are presented.
Mentions: We next characterized the effects of gain of MEF2 activity. To induce ectopic MEF2 activity, a transcript encoding MEF2D-Flag protein was marginally-injected to vegetal blastomeres of 4 cell embryos. Injection of this transcript induced the expression of a MEF2-dependent reporter gene (x3 MEF2-Luc) (see figure 8C). Injected mef2d-flag mRNA caused ectopic punctate staining of Xbra in the vegetal region as observed in in situ hybridization analysis (Figure 4A, left panel). MEF2D-Flag slightly increased the expression of endogenous mesoderm and organizer genes as shown by RT-PCR analysis (Figure 4A, right panel). In a second approach to induce ectopic MEF2 activity, a transcript encoding the potent HSV VP16 activation domain and the DNA binding domain of MEF2C (mef2-vp16) was marginally injected to embryos (Figure 4B). The constitutively active MEF2-VP16 chimera protein induced prominent ectopic vegetal expression of Xbra in gastrula stage embryos. Injection of mef2-vp16 mRNA into one of two blastomeres expanded the expression domain of myod in the injected half of the embryo at the neurula stage. Marginal zone injection of mef2-vp16 mRNA induced the expression of Xnr1 in gastrula VMZ explants and of myod and muscle actin in neurula VMZ explants. Overall, these results indicated that exogenously-expressed MEF2 protein induced ectopic expression of paraxial 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