Click here to close
Hello! We notice that you are using Internet Explorer, which is not supported by Xenbase and may cause the site to display incorrectly.
We suggest using a current version of Chrome,
FireFox, or Safari.
Dev Biol
2009 Dec 15;3362:313-26. doi: 10.1016/j.ydbio.2009.09.039.
Show Gene links
Show Anatomy links
Downstream of FGF during mesoderm formation in Xenopus: the roles of Elk-1 and Egr-1.
Nentwich O
,
Dingwell KS
,
Nordheim A
,
Smith JC
.
???displayArticle.abstract???
Signalling by members of the FGF family is required for induction and maintenance of the mesoderm during amphibian development. One of the downstream effectors of FGF is the SRF-interacting Ets family member Elk-1, which, after phosphorylation by MAP kinase, activates the expression of immediate-early genes. Here, we show that Xenopus Elk-1 is phosphorylated in response to FGF signalling in a dynamic pattern throughout the embryo. Loss of XElk-1 function causes reduced expression of Xbra at neurula stages, followed by a failure to form notochord and muscle and then the partial loss of trunk structures. One of the genes regulated by XElk-1 is XEgr-1, which encodes a zinc finger transcription factor: we show that phosphorylated XElk-1 forms a complex with XSRF that binds to the XEgr-1 promoter. Superficially, Xenopus tropicalis embryos with reduced levels of XEgr-1 resemble those lacking XElk-1, but to our surprise, levels of Xbra are elevated at late gastrula stages in such embryos, and over-expression of XEgr-1 causes the down-regulation of Xbra both in whole embryos and in animal pole regions treated with activin or FGF. In contrast, the myogenic regulatory factor XMyoD is activated by XEgr-1 in a direct manner. We discuss these counterintuitive results in terms of the genetic regulatory network to which XEgr-1 contributes.
Fig. 1. Spatiotemporal distribution of XElk-1 mRNA and protein. (A) Schematic representation of XElk-1 functional domains. See text for details. (B) Temporal expression of XElk-1; transcripts are maternally expressed and are present throughout early development. These data are representative of two independent experiments. Error bars show standard deviations of technical replicates of a single experiment. (C) Spatial distribution of XElk-1 mRNA in transverse sections of an albino embryo at the neurula stage reveal XElk-1 expression in notochord and somitic mesoderm. (D) Comparison of the amino acid sequences of human (h), Xenopus laevis (Xl) and Xenopus tropicalis (Xt) Elk-1 in the vicinity of human serine 383, which is phosphorylated by MAP kinase. (E) Western blot analysis of XElk-1 phosphorylation in response to FGF. Animal caps derived from embryos that had been injected with 400 pg myc-tagged XElk-1 mRNA were treated with 200 ng/ml bFGF for the indicated times and then subjected to western blotting using an antibody specific for phosphorylated XElk-1 (î±-pElk-1) or a pan-Elk-1 antibody (î±-Elk-1). Levels of phosphorylated XElk-1 are high at time zero, perhaps as a response to dissection ([Christen and Slack, 1999] and [Krain and Nordheim, 1999]) but decline by 45 min unless bFGF is present. (Fâ J) Immunocytochemical analysis of phosphorylated XElk-1 protein. (F, G) Sagittal sections of Xenopus embryos at the early gastrula stage. Control embryos incubated in the absence of primary antibody show only non-nuclear staining in cells of the yolky vegetal hemisphere. Use of an antibody directed against phosphorylated XElk-1 protein reveals staining in nuclei of the animal hemisphere and of the ventral and dorsal mesoderm, especially in the vicinity of the dorsal blastopore lip (arrow, G). (Hâ J) Whole mount immunocytochemistry showing the distribution of phosphorylated XElk-1 at gastrula (H), neurula (I) and tailbud (J) stages. pXElk-1 is enriched in the dorsal midline and extends towards the presumptive forebrain as gastrulation proceeds (arrows, H, I). At the early tailbud stage high levels of pXElk-1 are present in the presomitic mesoderm (arrow, J).
Fig. 2. Impairment of embryonic development by interference with Elk-1 activity. (A) The XElk-1 constructs used in these experiments. Top: myc-tagged XElk-1; middle: myc-tagged XElk-1-EnR; bottom: î EtsXElk-1-EnR. (B) Injection of XElk-1-myc mRNA (0.5 ng into each cell of the two-cell stage; total 1 ng) has no effect on early Xenopus development. (C, D) A non-tagged version of XElk-1 also has no effect on development. Compare C (uninjected) with D (injected). (Eâ G) Interference with XElk-1 activity by means of a dominant-interfering construct disrupts Xenopus development. (E) Uninjected embryos. (F) Embryos injected with 750 pg RNA encoding XElk-1-EnR; such embryos form a dorsally curved trunk and open neural folds. (G) Embryos injected with 750 pg RNA encoding î EtsXElk-1-EnR appear normal. (Hâ J) Trunk and anterior defects caused by XElk-1-EnR (I) are rescued, at least partially, by co-expression of X-Elk1 mRNA (J). (Kâ N) Notochord (marked by MZ15) and muscle (marked by 12/101) differentiation is diminished in embryos in which XElk-1 activity is perturbed. Antibody staining was carried out on 50-î¼m sections of stage 35 embryos previously injected with 750 pg RNA encoding XElk-1-EnR. Little notochord can be detected in embryos expressing XElk-1-EnR (L; compare with control embryo in K). Muscle differentiation is also greatly inhibited (N; compare with control embryo in M). (O, P) Xbra expression is also greatly inhibited in embryos injected with 750 pg RNA encoding XElk-1-EnR (O; compare with control embryos in P). All results were obtained in three independent experiments with over 200 embryos examined per treatment, except for data in panels C and D, which were derived from two experiments with 60 embryos in each case.
Fig. 3. An antisense morpholino oligonucleotide targeted against X. tropicalis Elk-1 has less effect on gene expression than does XElk-1-EnR but does interfere with muscle and notochord differentiation. (A, C) Embryos injected with a control morpholino oligonucleotide showing expression of Xbra (A) and XtEgr-1 (C). (B, D) Embryos injected with an antisense morpholino oligonucleotide directed against XtElk-1. Note delay in blastopore closure caused by the MO (compare A and B). Expression of Xbra is only slightly affected by XtElk-1 MO (B) but expression of XtEgr-1 is more significantly reduced. (E, F) Expression levels of XtMyoD are substantially decreased by XtElk-1 MO. (E) An embryo injected with a control MO; (F) an embryo injected with an antisense morpholino oligonucleotide directed against XtElk-1. (G, J) Control embryos at tailbud stages stained with 12/101 (G) or MZ15 (J). (H, K) Embryos injected with an antisense morpholino oligonucleotide targeted against XtElk-1. Note the reduction in muscle (H) and notochord (K) differentiation. (I, L) Rescue of the effects of the MO targeted against XtElk-1. Muscle (I) and notochord (L) formation are both restored in embryos injected with XElk-1 mRNA as well as XtElk-1 MO. Results in G-L were obtained in three independent experiments. Eighty-five embryos were injected with our control MO, of which 90% appeared normal; 109 were injected with XtElk-1 MO, of which 13% appeared normal; and 118 were injected with XtElk-1 MO together with XElk-1-myc mRNA, of which 79% appeared normal.
Fig. 5. Effects of gain and loss of XEgr-1 function on early Xenopus development. (A–D) XEgr-1 over-expression perturbs transcription of Xbra in the marginal zone. Embryos were injected at the one-cell stage with the indicated doses of XEgr-1 mRNA. They were cultured to stage 11 and then analyzed by in situ hybridization for expression of Xbra. As levels of XEgr-1 increase, transcription of Xbra decreases. (E–G) Similar experiments reveal that XMyoD is up-regulated in such embryos. (E) An embryo showing the combined endogenous expression of both Xbra and XMyoD. (F) An embryo previously injected with XEgr-1 mRNA into one blastomere at the four-cell stage showing down-regulation of Xbra. (G) An embryo previously injected with XEgr-1 mRNA into one blastomere at the four-cell stage showing the combined expression of Xbra and XMyoD. Note the down-regulation of Xbra and XMyoD in the marginal zone and the ectopic expression of XMyoD in the animal hemisphere. (H–K) Embryos injected with 1 ng XEgr-1 mRNA were allowed to develop to stage 26, when formation of muscle and notochord was analyzed using antibodies 12/101 and MZ15, respectively. Over-expression of XEgr-1 causes reduction in both tissues. (L–S) Loss of Xenopus tropicalis Egr-1 activity causes elevated levels of Xtbra. X. tropicalis embryos were injected with 30 ng XtEgr-1 MO2 and analyzed for expression of Xtbra throughout gastrula and neurula stages. Marginal zone expression of Xtbra is unaffected during gastrula stages (L–N, P–R) but by stage 15 Xtbra is higher in embryos lacking XtEgr-1 than in control embryos (O, S). Loss of X. tropicalis Egr-1 activity causes down-regulation of XtMyoD at the late gastrula stage. Expression of XtMyoD is normal in uninjected embryos (T) and embryos injected with a control morpholino oligonucleotide (U) but is down-regulated in embryos injected with antisense morpholino oligonucleotides directed against X. tropicalis Egr-1 (V, W). (X) Inhibition of X. tropicalis Egr-1 function causes down-regulation of XtMyoD and up-regulation of Xtbra. Embryos were left uninjected (Uninj), were injected with a control morpholino oligonucleotide (Con) or were injected with antisense morpholino oligonucleotides directed against X. tropicalis Egr-1 (MO1, MO2). They were assayed for expression of XtMyoD at the neurula stage. Note the down-regulation of XtMyoD in embryos in which XtEgr-1 function is compromised. Expression of Xtbra is up-regulated in embryos injected with XtEgr-1 MO2 (which yields a stronger phenotype than MO1). (Y–EE) Loss of XEgr-1 function causes trunk defects and perturbs notochord and muscle formation. X. tropicalis embryos were injected with 30 ng control MO (Y, BB, DD), 30 ng XtEgr-1 MO1 (Z) or 20 ng XtEgr-1 MO2 (AA, CC, EE) and cultured to stage 26. Embryos lacking XtEgr-1 display defects in trunk development, which are particularly marked in embryos injected with XtEgr-1 MO2. Muscle (BB, CC) and notochord (DD, EE) differentiation are perturbed in such embryos. The figure shows representative results from three independent experiments: n > 70.
