Gain of MEF2D function induces the expression of paraxial mesoderm genes.

<p>(A) ISH of stage 9 vegetally-injected embryos (Mef2D-Flag mRNA) using antisense <i>Brachyury</i> probe (left). Arrows point at punctate staining. MEF2D-Flag mRNA was injected marginally to one cell embryos. RNA was extracted from whole embryos (n = 18) at stages 10.5 and the expression levels of the indicated genes was analyzed by semi quantitative RT-PCR (right). (B) mRNA encoding MEF2-VP16 chimera was injected to one cell embryos. Left panel: Stage 10.5 embryos were analyzed by ISH using antisense probe to <i>brachyury</i>. Right panel: Two blastomere embryos were injected unilaterally with MEF2-VP16 and βGAL mRNA and grown to stage 16. ISH with a probe to <i>myod</i> was performed. The injected side was identified by βGAL staining. (C) MEF2-VP16 mRNA was injected marginally to one cell embryos. RNA was extracted from VMZ explants (n = 18) at stages 10.25 (left) and 14 (right) and analyzed by semi quantitative RT-PCR.</p

MEF2 activity is sufficient to induce the expression of <i>Xnr1</i> that partly mediates the function of MEF2D in mesoderm gene expression.

<p>(A) ISH analysis of hemisected embryos at stage 9 using probes to <i>xnr1</i> (left) and <i>mef2d</i> (right). (B) <i>Mef2-vp16</i> encoding mRNA was injected marginally to one cell embryos. Stage 10 embryos were analyzed by ISH using an <i>Xnr1</i> probe. The animal side is presented. (C) One cell embryos were injected with mRNA encoding <i>Xnr1</i>, MEF2D-AMO or both. Stage 10.5 embryos were analyzed by ISH using antisense <i>Brachyury</i> probe. (D) Injections were performed as in C (each treatment; 18 embryos). RNA was extracted from stage 10.5 embryos and qPCR was performed. Data are presented as means ± SE of three independent experiments with duplicates.</p

A model describing the role of MEF2D in mesoderm gene expression.

<p>Solid arrows indicate activation at the levels of expression and/or activity. Dashed arrow indicates the cooperation between Nodal signaling and MEF2D activity in marginal mesoderm activation. Red arrows indicate conclusions of the present study while blue arrows indicate previous knowledge.</p

Expression pattern of maternal and zygotic MEF2.

<p>(A) Quantitative PCR analysis of MEF2A and MEF2D transcripts from fertilization to gastrula stages. Experiment was performed two independent times. (B) 4 cell embryos were analyzed by ISH using probes <i>to mef2a</i> and <i>mef2d</i>. (C) Left panels: Stage 8 embryos were dissected to animal and vegetal halves. RNA and proteins were extracted. RNA was analyzed by RT-PCR reaction and proteins by Western analysis. Right panel: Immunohistochemistry of stage 8 embryos. Animal-vegetal sections were prepared. Sections were reacted with anti-MEF2 antibodies (orange) and counterstained with hematoxylin. The right panel is an enlargement of a segment of the left panel. (D) Left panel: Hemisected stage 9 embryos were analyzed by ISH using probes <i>to mef2a and mef2d</i>. Right panel: Sections of stage 9 embryos were reacted with anti-MEF2 antibodies (orange) and counterstained with hematoxylin. The right end panel is an enlargement of a segment of the left panel. Arrows point at stained nuclei. (E) Left panel: Stage 10.5 embryos were analyzed by <i>in situ</i> hybridization using a probe <i>to mef2d</i>. Right panel: x3 Mef2-Luc reporter (30 pg) was injected to one cell embryo (20 embryos). At early gastrula stage (10.25), DMZ and VMZ explants were isolated and luciferase was measured. Luciferase activity was normalized to the total protein levels. Data are presented as means ± SE of three independent experiments.</p

FGF signaling and MEF2D cooperate in mesoderm specification.

<p>(A) <i>fgf8</i> mRNA was injected without or with MEF2D AMO to one cell embryos. At stage 8, AC explants were isolated and grown to stage 15. RNA was extracted and gene expression was analyzed by qPCR reaction. Expression levels of each gene induced by FGF8 were arbitrarily set to a value of 1, and values of other treatments were standardized accordingly. Data are presented as means ± SE of two independent experiments with duplicates(B) mRNA encoding dominant negative FGF receptor (FGF-DNR) was marginally injected to one cell embryos and at stage 9 hemisected embryos were analyzed by ISH with a probe to <i>mef2d.</i> (C) mRNA encoding FGF-DNR was marginally injected to one cell embryo at the indicated concentrations and qPCR analysis was performed on isolated RNA from stage 10.5 embryos. Expression levels of each gene in uninjected embryos were arbitrarily set to a value of 1, and values of other treatments were standardized accordingly. Data are presented as means ± SE of two independent experiments with duplicates. (D) <i>fgf8</i> mRNA was injected vegetally to one of four cell embryos. At stage 9 embryos were analyzed by ISH with a probe to <i>mef2d.</i> Upper panel: whole embryos. Lower panel: hemisected embryos. (E) One cell embryos were injected with a plasmid containing x3 Mef-Luc reporter gene without or with <i>fgf8</i> mRNA or FGF-DNR mRNA. At stage 10.5 proteins were extracted and luciferase activity was measured and normalized according to the total amount of proteins. Data are presented as means ± SE of three independent experiments with duplicates. (F) qPCR analysis of control embryos and embryos injected with MEF2D AMO. Five embryos on each group were injected and RNA was extracted at stage 10.5. Expression levels of control embryos were arbitrarily set to a value of 1, and values of injected embryos were standardized accordingly. Data are presented as means ± SE of two independent experiments with duplicates.</p

MEF2D regulates the expression of mesoderm genes.

<p>(A) qPCR analysis of embryos injected with MEF2D AMO or mismatch AMO. 5 embryos on each group were injected and RNA was extracted at stage 9. qPCR was performed as described in “materials and methods”. Expression levels of each gene were arbitrarily set to a value of 1 in the mismatch AMO injected embryos. The values for each gene were standardized accordingly. Data are presented as means ± SE of two independent experiments with duplicates(B) qPCR was performed on stage 10.5 embryos treated as is described in A. (C) Stage 10.5 embryos were analyzed by ISH using antisense probes to <i>brachyury</i>, <i>goosecoid</i> and <i>chordin</i> (left to right). (D) Control and MEF2D AMO-injected embryos were analyzed at stage 14 by RT-PCR (left) and at stage 16 by ISH with antisense probe to <i>myod</i>. (E) Transversal sections of stage 24 control and MEF2D AMO-injected embryos analyzed by immunohistochemistry. The antibody used was anti Myosin heavy chain (MF20) and samples were counterstained with hematoxylin. (F) Co-injection of MEF2D-Flag mRNA with MEF2D-AMO restores the expression of mesoderm and organizer genes. MEF2D AMO was injected alone or together with Mef2D-Flag mRNA into one cell embryos. Embryos were analyzed by RT-PCR (left) and by ISH with antisense probe to <i>goosecoid</i> (right) at stage 10.25.</p

Pairs of primers used in reverse transcriptase qPCR analysis.

<p>Pairs of primers used in reverse transcriptase qPCR analysis.</p

Animal cap- MEF2D-depleted explants do not express mesoderm markers in Nieuwkoop recombinants.

<p>(A) Embryos were injected with <i>vegt</i> 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).</p

Transcriptional Regulation of Mesoderm Genes by MEF2D during Early <i>Xenopus</i> Development

<div><p>In <i>Xenopus</i>, 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 <i>Nodal-related 1</i> gene (<i>Xnr1</i>) that mediates some of MEF2D mesodermal activities. Chromatin immunoprecipitation analysis revealed that MEF2D associates with transcriptional regulatory sequences of the <i>Xnr1</i> gene. Several MEF2 binding sites within the proximal promoter region of <i>Xnr1</i> were identified by their <i>in vitro</i> 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.</p></div

MEF2D associates with <i>Xnr1</i> regulatory elements.

<p>(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: <i>Xnr1</i> 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) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069693#pone.0069693-Hyde1" target="_blank">[47]</a>. 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 <i>mef2d-flag</i> 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.</p