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We have identified the Xenopus homologue of Drosophila Enhancer of Zeste using a differential display strategy designed to identify genes involved in early anterior neural differentiation. XEZ codes for a protein of 748 amino acids that is very highly conserved in evolution and is 96% identical to both human and mouse EZ(H)2. In common with most other Xenopus Pc-G genes and unlike mammalian Pc-G genes, XEZ is anteriorly restricted. Zygotic expression of XEZ commences during gastrulation, much earlier than other anteriorly localized Pc-G genes; expression is restricted to the anterior neural plate and is confined later to the forebrain, eyes and branchial arches. XEZ is induced in animal caps overexpressing noggin; up-regulation of XEZ therefore represents a response to inhibition of BMP signalling in ectodermal cells. We show that the midbrain/hindbrain junction marker En-2,and hindbrain marker Krox-20, are target genes of XEZ and that XEZ functions to repress these anteroposterior marker genes. Conversely, XEZ does not repress the forebrain marker Otx-2. XEZ overexpression results in a greatly thickened floor of the forebrain. These results implicate an important role for XEZ in the patterning of the nervous system.
Fig. 3. Wholemount in situ hybridisation of XEZ. Wholemount in situ hybridisation of XEZ DIG labelled antisense RNA probe shows that expression of XEZ is restricted to the anterior nervous system. (A) Stage 14 embryo expression is restricted to the anterior nervous system (indicated by arrow) (B) Stage 20 embryo and (C) Stage 27 embryo, the XEZ transcripts are detected in the anterior nervous system. In (B) and (C) the expression pattern appears to be graded with the strongest expression in the most anterior regions. (D) Stage 35 embryo shows that expression of XEZ later becomes restricted to the forebrain, eye and branchial arches. The XEZ expression is highest in the first branchial arch, less in the second and is greatly reduced in the third and fourth arches. The sense probe control showed no staining pattern (data not shown).
Fig. 2. Temporal expression profile of XEZ. RT-PCR showing expression pattern of XEZ transcripts in Xenopus laevis unfertilised egg and embryo stages. Significant maternal expression is not detected. Zygotic expression of XEZ commences at low levels in the late blastula stages, increasing at mid to late gastrula stages, peaking in the early neurula stages and is still detectable at stage 41. Stage 41 cDNA is used for the linearity control and ODC is used as a loading control.
Fig. 4. XEZ expression is anteriorly restricted. Dissection series of stage 25 Xenopus laevis embryos analysed by RT-PCR. Stage 25 embryos were dissected as shown in the insert picture into three equivalent axial pieces, A, anterior; M, middle; P, posterior; the endoderm was removed. Duplicate samples 1 and 2 both consisted of 5 embryos. Analysis shows the expression of XEZ is highest in the anterior nervous system, is reduced in the middle section and is barely detectable in the posterior in both dissected embryo pools. Whole embryo control cDNA at stage 25 was used for the linearity and EF1α was used as a loading control. Since the sections of neural tissue were equal in terms of regional identity, but only approximately equivalent in terms of amount of biological material, it is not reasonable to equalize loading of cDNA between samples (as shown by the EF1α signal).
Fig. 5. XEZ is induced by noggin. RT-PCR showing induction of XEZ in noggin-injected animal caps. Animal caps were taken from stage 9 embryos previously injected at the 1 cell stage with noggin mRNA. The animal caps were cultured to stage 13, 20, 28 and 32. RT-PCR analysis shows that at stage 13 XEZ expression is highly induced in noggin animal caps compared to a much lower level of expression in control animal caps. However, this high level of expression is not maintained and XEZ expression is only slightly higher in animal caps overexpressing noggin compared to control caps at stages 20, 28 and 32. Stage 32 whole embryo cDNA was used as input for the linearity and EF1α was used as a loading control.
Fig. 6. Overexpression of XEZ results in thickening of the floor plate region in the forebrain of in Xenopus laevis embryos. Embryos were injected with XEZ mRNA and cultured to stage 37. The embryos were then wax embedded, sectioned at 6 μm and stained with eosin and hematoxylin. (A) Section shows thickening of the floor of the midbrain (arrow) compared to (B) a normal control embryo. v, ventricle; nt, neural tube; fp, floor plate; ph, pharynx; ep, epidermis; e, eye.
Fig. 7. Repression of En-2 by XEZ. Embryos were uni-laterally co-injected at the two cell stage with XEZ mRNA and FLDX. The FLDX was used as a marker for the injected side. The embryos were cultured to stage 19 and subjected to wholemount in situ hybridisation using an En-2 antisense RNA DIG-labelled probe. The injected side is indicated by an arrow. (A) Complete ablation of En-2 expression on XEZ injected side. (B) Anterior shift of En-2 expression on XEZ injected side. (C) Repression of En-2 expression on XEZ injected side. (D) Normal control. The En-2 sense control probe showed no staining pattern (data not shown).
Fig. 8. XEZrepresses En-2. (A) Xenopus laevis embryos were uni-laterally co-injected at the 2-cell stage with XEZmRNA and FLDX. The embryos were cultured to stage 19 and subjected to wholemount in situ hybridisation using the midbrain-hindbrain junction marker En-2 antisense RNA probe. The embryos were scored as having reduced expression of En-2when the level of En-2 staining was lower (or absent) than control embryo normal variation. The embryos showing reduced-expression were also scored for any shift in the anterior or posterior register of the En-2 marker staining relative to the uninjected side. Numbers in brackets are actual numerical scores. (B) XEZ represses En-2, Krox-20 but not other neural markers. Embryos were injected at the 1 cell stage with XEZmRNA, cultured to stage 19 and subjected to RT-PCR. The figure shows that the expression of En-2 and Krox-20 are repressed compared to uninjected control, but other neural markers are not affected. EF1α was used as a loading control and the uninjected sample used for linearity input cDNA.