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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. |
