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Dev Biol
2005 Jul 01;2831:253-67. doi: 10.1016/j.ydbio.2005.04.020.
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Identification of target genes for the Xenopus Hes-related protein XHR1, a prepattern factor specifying the midbrain-hindbrain boundary.
Takada H
,
Hattori D
,
Kitayama A
,
Ueno N
,
Taira M
.
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The midbrain-hindbrain boundary (MHB) acts as a local organizer in the development of the CNS in vertebrates. Previously, we identified an MHB-specific bHLH-WRPW transcriptional repressor gene, Xenopus Hes-related 1 (XHR1), which is initially expressed in the presumptive MHB (pre-MHB) region at the early gastrula stage. To better understand the gene cascades involved in MHB formation, we investigated the genes downstream from XHR1 by differential screening using a Xenopus cDNA macroarray and a dexamethasone (DEX)-inducible, dominant-negative transcriptional activator construct of XHR1 (XHR1-VP16-GR). Among the newly identified candidate target genes of XHR1 were Enhancer of split-related genes (ESR1, ESR3/7, and ESR9) and Xenopus laevis cleavage 2 (XLCL2). XHR1-VP16-GR induced the expression of the ESR genes and XLCL2 as well as Xdelta1, Xngnr1, and XHR1 itself in the presence of DEX even after pretreatment with the protein synthesis inhibitor, cycloheximide. This suggests that these genes are direct targets of XHR1. XHR1-knockdown experiments with antisense morpholino oligos and ectopic expression of wild-type XHR1 revealed that XHR1 is necessary and sufficient to repress ESR genes in the pre-MHB region. Misexpression of the ESR genes in the pre-MHB region repressed the MHB marker gene, Pax2, suggesting that the repression of the ESR genes by XHR1 is at least partly required for the early development of the pre-MHB. Our data also show that XHR1 is not activated by Notch signaling, differing from ESR genes. Taken together, we propose a model in which XHR1 defines the pre-MHB region as a prepattern gene by repressing those possible direct target genes.
Fig. 1. Preparations for macroarray screening. (A) Pax2 is downregulated by XHR1-VP16-GR only in the presence of DEX (indicated by arrow). mRNAs for XHR1-VP16-GR (12.5 pg) and nβ-gal were coinjected into the dorsoanimal region of the leftblastomere at the four-cell stage. Half the injected embryos were treated with DEX at the early gastrula stage (stage 10.5–11) (right) and the other half were untreated (left). Following nβ-gal staining (red), WISH was performed with a Pax2 probe at the early neurula stage (stage 13–14; purple). The images are dorsal views with the anterior to the top (the orientation of the following figures is the same as shown here unless otherwise stated). (B) Pax2 expression levels and gastrulation-arrest rates upon DEX treatment at different stages. Embryos were coinjected as described in panel (A) with mRNAs for XHR1-VP16-GR or β-globi n (100 pg) and nβ-gal, treated with DEX from the indicated stages to stage 13–14, and subjected to WISH for Pax2 expression. Colored bars indicate Pax2 expression levels on the injected side. Percentages of embryos with gastrulation arrest are shown on the right side of the graph. N, number of embryos. Each datum is from two or more individual experiments. (C) A schematic representation of the dissection procedure used to isolate the anteriorneuroectoderm containing the pre-MHB region. Embryos were injected with mRNA at the four-cell stage, cut into halves in the middle of the body length at the late gastrula to early neurula stage (stage 12.5–13). The anteriorneuroectoderm (ANE) together with the underlying anteriorendomesoderm (AEM) was dissected and separated by collagenase digestion. (D, E) Dissected fragments containing the pre-MHB region as ascertained by XHR1 expression visualized by WISH (D), or by RT-PCR of extracted total RNA for XHR1 expression (E). Histone H4 was used as the loading control.
Fig. 3. The results of two-color WISH for ESR1 (left column, purple) or XLCL2 (right column, purple) and XHR1 (turquoise). Arrows indicate XHR1 expression in the pre-MHB region.
Fig. 4. The effects of XHR1-VP16-GR on the expression of possible target genes and neuronal markers. (A) ESR1, ESR9, XLCL2, Xdelta1, Xngnr1, and XHR1 are activated by XHR1-VP16-GR in the presence of CHX. Embryos were injected into the dorsal leftblastomere at the four-cell stage with XHR1-VP16-GR mRNA (100 pg), and treated with or without CHX for 30 min before the addition (or not) of DEX at the gastrula stage (stage 11), as indicated. The expression of ESR1 (a), ESR9 (b), XLCL2 (c), Xdelta1 (d), Xngnr1 (e), and XHR1 (f) were analyzed at stage13 by WISH. (B) The effects of XHR1-VP16-GR on the expression of neuronal markers. The expression of Xlim3 (a), Xash3 (b), and β2-tubulin (c) was analyzed at stage 13 by WISH. Fractions indicate the proportion of the presented phenotype per total number: numbers in black, normal expression; numbers in red, upregulation; numbers in green, disrupted expression; numbers in blue, downregulation. Black arrows in Ad,e, pre-MHB region; red arrow in Ba, ectopic expression; black arrow in Bb, reduced expression.
Fig. 5. The effects of gain-of-function or loss-of-function of XHR1 on expression gene. (A) Misexpression of XHR1 represses ESR1 and XLCL2 expression. XHR1 mRNA (12.5–25 pg) was injected into the dorsal leftblastomere at the four-cell stage. ESR1 (a) and XLCL2 (b) expression was examined by WISH at stage 13–14. Each stripe in which primary neurons arise is numbered in the expression of ESR1. 1, Medial stripe; 2, intermediate stripe; 3, lateral stripe. Arrows, reduced expression. (B) Effects of XHR1-MOs on the expression of ESR1 (a–d), ESR9 (e, f), Xdelta1 (g, h), and Pax2 (i–l). XHR1-MOs (a, e, g, i) or XHR1-5mmMO (b, f, h, j) was injected at the four-cell stage, and globin (c, k; negative control) or XHR1 (d, l) mRNA was injected with nβ-gal mRNA at the eight-cell stage. (a′, e′) Magnification of the same embryo as shown in panels a and e. Black arrows, reduced expression; red arrows, ectopic expression. Fractions in black, normal expression; in blue, downregulation; in red, upregulation. Injected doses: MOs, 6 or 3 (i, j) ng; globin or XHR1 mRNA, 3–10 (d), or 0.3–1 (l) pg.
Fig. 6. ESR1 downregulates Pax2 and XHR1 expression. ESR1 mRNA was injected at 100–400 pg per blastomere. Pax2 and XHR1 expression was analyzed at stage 13. Arrows, reduced expression. Fractions in black, normal expression; in blue, downregulation.
Fig. 7. Notch signaling does not activate XHR1 expression. Notch-ICD mRNA was injected at 250–1000 pg per blastomere. The expression of ESR1, XHR1, and Sox2 was analyzed at stage 13. Arrow, reduced expression. Fractions in white, normal expression; in blue, downregulation; in red, upregulation.
Fig. 8. Models of the pre-MHB formation by XHR1. (A) Possible gene interactions. XHR1 demarcates the pre-MHB region by repressing ESR1, ESR3, ESR9, Xngnr1, Xdelta1, XLCL2, and yet identified genes (?). XHR1 is likely to repress own transcription. (B) Prepatterns in the neural plate. Combination of prepatterns by Notch and Xdelta1 (a; expression patterns are simplified) and XHR1 (b) specifies expression patterns of ESR genes (c) and Pax2 (d). See Discussion for more detail.
lbh (limb bud and heart development) gene expression in Xenopus laevis embryo, assayed via double in situ hybridization, (dark blue) , NF stage 15/16, dorsal view, anterior up. ( Arrow indicates the midbrain-hindbrain boundary, marked via hes7.1 expression, in turquoise.)
hes5.1 (hes family bHLH transcription factor 5 ) gene expression in Xenopus laevis embryo, assayed via double in situ hybridization, (dark blue) ,NF stage 15/16, dorsal view, anterior up. (Arrow indicates the midbrain-hindbrain boundary, marked via hes7.1 expression, in turquoise.)
ascl2 (achaete-scute family bHLH transcription factor 2) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 13, dorsal view, anterior up.
