December 1, 2001;
Early patterning of the prospective midbrain-hindbrain boundary by the HES-related gene XHR1 in Xenopus embryos.
The molecular mechanisms that govern early patterning of anterior neuroectoderm
(ANE) for the prospective brain
region in vertebrates are largely unknown. Screening a cDNA library of Xenopus ANE led to the isolation of a Hairy and Enhancer of split- (HES)-related transcriptional repressor gene, Xenopus HES-related 1 (XHR1
is specifically expressed in the midbrain
) region at the tailbud
stage. The localized expression of XHR1
was detected as early as the early gastrula
stage in the presumptive MHB
region, an area just anterior
to the involuting dorsal mesoderm
that is demarcated by the expression of the gene Xbra
. Expression of XHR1
was detected much earlier than that of other known MHB
genes, XPax-2 and En-2, and also before the formation of the expression boundary between Xotx2
and Xgbx-2, suggesting that the early patterning of the presumptive MHB
is independent of Xotx2
and Xgbx-2. Instead, the location of XHR1
expression appears to be determined in relation to the Xbra
expression domain, since reduced or ectopic expression of Xbra
altered the XHR1
expression domain according to the location of Xbra
expression. In functional assays using mRNA injection, overexpression of dominant-negative forms of XHR1
in the MHB
region led to marked reduction of XPax-2 and En-2 expression, and this phenotype was rescued by coexpression of wild-type XHR1
. Furthermore, ectopically expressed wild-type XHR1
near the MHB
region enhanced En-2 expression only in the MHB
region but not in the region outside the MHB
. These data suggest that XHR1
is required, but not sufficient by itself, to initiate MHB
marker gene expression. Based on these data, we propose that XHR1
demarcates the prospective MHB
region in the neuroectoderm
in Xenopus early gastrulae.
[+] show captions
Fig. 3. Whole-mount in situ hybridization analysis of XHR1 expression during early development. (A) Dorsal view of a stage 10.5 embryo. The animal side is to the top. XHR1 expression is detected in a narrow area of the dorsal side. (B) Comparison of the expression domains of Sox-2 and XHR1. A stage 10.5 embryo was bisected sagittally into two hemispheres and was then hybridized with Sox-2 (left panel) or XHR1 (right panel). The animal side is to the top. The inset shows the higher magnification view of XHR1 expression in the sensorial layer. (C) Dorsal view of a stage 12 embryo. Anterior is to the top. The expression domain is sharpened and reduced in the midline, giving a V-shaped form. (D) Dorsal view of a stage 15 embryo. Anterior is to the top. XHR1 expression is confined to a limited area in the anterior neuroectoderm. (E) Dorsal view of a stage 17 embryo stained with XHR1 (brown) and En-2 (purple). Anterior is to the top. The expression of both XHR1 and En-2 overlaps. (F) Dorsoanterior view of a cleared stage 18 embryo. XHR1 is expressed in the prospective eye region (white arrowhead) and possible pineal gland region (white arrow) too. (G) Lateral view of a cleared stage 28 embryo. Anterior is to the left. XHR1 expression is restricted in the MHB region with slight expression in forebrain.
Fig. 4. Comparison of expression of XHR1 with that of XFGF-8 and En-2 at tailbud stages. (A–C) Lateral view of stages 36–37 embryos hybridized for XHR1, XFGF-8, and En-2. (A′–C′) Horizontal sections of the embryos in (A–C). Anterior is to the left. Arrowheads, MHB; mb, midbrain; hb, hindbrain; ey, eye; ot, otic vesicle; st., developmental stage.
Fig. 5. XHR1 expression in the prospective MHB region at gastrula stages. Expression of XHR1 was compared with that of Xbra during gastrula stages by whole-mount in situ hybridization and sections. (A–D) Gastrula embryos hybridized for XHR1 (left panel), for Xbra (right panel), and for both (middle panels). All embryos are dorsally viewed, and animal or anterior is to the top. (E–G) Sagittal sections of embryos hybridized for both XHR1 and Xbra. Only dorsal halves are shown, and animal is to the top. The boundary between the ectoderm and mesoderm is indicated by dotted lines, and was determined based on the differences in nuclear density of germ layers visualized by DAPI staining (lower panels). Arrowheads, delimitation of XHR1 expression; white arrowheads, delimitation of Xbra expression; st., developmental stage.
Fig. 6. (A–C) Comparison of expression of XHR1 with that of Xotx2 and Xgbx-2 at gastrula stages. (A) A stage 11 embryo was bisected sagittally into two hemispheres and was then hybridized for XHR1 (left panel) or Xotx2 (right panel). Animal is to the top. (A′) Higher magnification of (A). The expression domain of XHR1 does not correspond to that of Xotx2 in the ectoderm. (B, C) Embryos at stages 11 or 11.5 hybridized for XHR1 (left panel), for Xgbx-2 (right panel), and for both (middle panel). All embryos are dorsally viewed, and animal is to the top. (D–F) Possible regulation of XHR1 expression by the Xbra-expressing region. Embryos were injected with mRNA or DNA as indicated together with nβ-gal mRNA (30 pg/embryo) for a lineage tracer. Double in situ hybridization with XHR1 and Xbra probes was performed after nβ-gal staining. (D) Injection of globin mRNA as negative control. (E) Injection of XFD mRNA (60 pg/embryo). Xbra expression was reduced in a nβ-gal-stained region as indicated by an arrowhead. (F) Injection of CSKA-eFGF (10 pg/embryo). In situ staining in nβ-gal-positive region means Xbra expression as indicated by asterisks. Two typical phenotypes are shown. Dotted lines, the boundary between ectoderm and mesoderm that was determined based on the differences in nuclear density of germ layers visualized by DAPI staining (data not shown; see Fig. 5E–G); st., developmental stage; thick arrows, shifted XHR1 expression; thin arrows, XHR1 expression; white asterisks, Xbra expression.
Fig. 8. nrp-1 expression in XHR1 or XHR1-VP16 RNA-injected embryos. Injected embryos at the tailbud stage (stages 24/25) were stained with nβ-gal (red) and then hybridized for nrp-1 (purple). RNA, 100 pg, for wild-type XHR1 (A, B) or 50 pg of RNA for XHR1-VP16 (C, D) were injected. (B, D) Sections of stained embryos. Right panel, staining with DAPI. Sections are at the levels indicated by solid bars in (A, C). Solid bar (B, D), midline; broken line, the boundary between neural and mesodermal tissues; bracket, an area lacking nrp-1 expression in the neural tissue; nt, notochord; ey, eye; cg, cement gland.