Gammill LS and Sive H (2001), otx2 expression in the ectoderm activates anter...

XB-ART-7824

Dev Biol 2001 Dec 01;2401:223-36. doi: 10.1006/dbio.2001.0470.

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otx2 expression in the ectoderm activates anterior neural determination and is required for Xenopus cement gland formation.

Gammill LS , Sive H .

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We previously showed that otx2 regulates Xenopus cement gland formation in the ectoderm. Here, we show that otx2 is sufficient to direct anterior neural gene expression, and that its activity is required for cement gland and anterior neural determination. otx2 activity at midgastrula activates anterior and prevents expression of posterior and ventral gene expression in whole embryos and ectodermal explants. These data suggest that part of the mechanism by which otx2 promotes anterior determination involves repression of posterior and ventral fates. A dominant negative otx2-engrailed repressor fusion protein (otx2-En) ablates endogenous cement gland formation, and inhibits expression of the mid/hindbrain boundary marker engrailed-2. Ectoderm expressing otx2-En is not able to respond to signals from the mesoderm to form cement gland, and is impaired in its ability to form anterior neural tissue. These results compliment analyses in otx2 mutant mice, indicating a role for otx2 in the ectoderm during anterior neural patterning.

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Species referenced: Xenopus
Genes referenced: ag1 bmp4 cer1 chrd egr2 en2 gsc hesx1 muc2 myc ncam1 neurog1 neurog2 nog nrp1 otx2 ptgds sia1 tbx2 tbxt ventx1.2 ventx2 zic1

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	FIG. 1. otx2 activates anterior and represses posterior and ventral gene expression in whole embryos. (A) Albino embryos were injected in one cell at the two-cell stage with 100 pg otx2-GR RNA mixed with 60 pg lacZ RNA. Injected embryos were incubated without or with dexamethasone (dex) beginning at midgastrula (stage 11.5) and until early neurula (stage 13), midneurula (stage 15), or hatching (stage 30). (B) Gene expression in embryos injected with otx2-GR and incubated without (#1113230; or with (#1113090; dex was assayed by whole-mount in situ hybridization. Embryos are oriented with anterior at the top. Dorsal to the right: ad, g, h, m. Dorsal view: e, f, i, q. Arrowheads point to altered patterns of gene expression. otx2 activity at gastrula stages induced ectopic NCAM expression ventrally (Gammill and Sive, 2000) and expanded NCAM expression dorsally, often to the extent that the eye was no longer apparent on the injected side (data not shown). General neural markers N-tubulin (Richter et al., 1988) and nrp-1 (Knecht et al., 1995) behaved similarly (data not shown). Anterior neural: (a) lipocalin, st. 30, -dex; (b) lipocalin, st. 30, +dex; (c) XBF-1, st. 32, -dex; (d) XBF-1, st. 32, +dex. Mid/hindbrain boundary: (e) gbx-2, st. 13, -dex; (f) gbx-2, st. 13, +dex; (g) engrailed-2, st. 32, -dex; (h) engrailed-2, st. 32, +dex. Posterior neural: (i), Krox20, st. 16, -dex; (j) Krox20, st. 16, +dex; (k) N-tubulin, st. 15, -dex; (l) N-tubulin, st. 15, +dex. Ventral ectoderm: (m) GATA-2, st. 13, -dex; (n) GATA-2, st. 13, +dex; (o) Vox, st. 13, -dex; (p) Vox, st. 13, +dex. Mesoderm: (q) goosecoid, st. 13, -dex; (r) goosecoid, st. 13, +dex; (s) brachyury, st. 13, -dex; (t) brachyury, st. 13, +dex.
	FIG. 5. otx2-En interferes with cement gland formation. (A) Embryos were injected at the two-cell stage with otx2-En RNA without or with otx2 RNA, targeting the future anterior/ventral ectoderm by injecting superficially in the animal pole. lacZ RNA was included as a lineage tracer. Embryos were allowed to develop until hatching (stage 30). (B) Embryos were injected with 100 pg otx2-En (a), 100 pg otx2-En plus 100 pg otx2 (b), 100 pg otx2-En plus 200 pg otx2 (c), or 100 pg globin (d) RNA mixed with 120 pg lacZ RNA. Injected embryos were stained for ﰇ-galactosidase activity (blue) and scored for cement gland formation by morphology when lineage tracer was present anteriorly (white arrowheads). (C) Embryos from (B) were scored for absent or minimal cement gland formation (defined as no or very few pigmented cells; G absent,gray bars), reduced cement gland formation (a small, round patch of pigmented cells; G reduc.,hatched bars), or normal cement glands (oblong shape, scored relative to size of average control cement gland; ormal,black bars). The data represent four separate rescue experiments. Rescued cement glands often included some ectopic cement gland formation due to the presence of otx2 RNA.
	FIG. 2. otx2 alters ectodermal patterning in isolated ventral ectoderm. (A) Embryos were injected in the ventral portion of both blastomeres at the 2-cell stage with 100 pg otx2-GR RNA mixed with 500 pg GFP RNA. At midgastrula (stage 11.5), ventral ectoderm exhibiting GFP fluorescence was dissected and cultured without or with dexamethasone (dex) for 3.5 h until early neurula (stage 13.5). (B) Explants of ventral ectoderm (ventral) from embryos injected with 100 pg otx2-GR were incubated without (2) or with (1) dex and assayed by RT-PCR. Expression of general neural [nrp-1, neurogenin (ngnr-1)], cement gland (XCG-1), anterior neural (opl, Xanf-1), mid/hindbrain boundary [engrailed-2 (en-2)], ventral ectodermal (XVent-1, BMP4), and dorsal mesendodermal [brachyury, (Xbra), goosecoid (gsc), and cerberus (cer)] markers were examined by RT-PCR using EF-1a expression as a loading control. Lane 1, ventral ectoderm, 2dex; lane 2, ventral ectoderm, 1dex; lane 3, stage 13.5 whole embryo.
	FIG. 3. The transcriptional activation domain of otx2 resides in the carboxyl terminus. (A) Deletions were created in MT-otx2 at the restriction endonuclease sites indicated in otx2. otx2 is 864 nucleotides and 288 amino acids in length. Deletions were created from the 39 end since the homeobox is located close to the 59 end (Pannese et al., 1995). The number of amino acids deleted in each construct is indicated. (B) Embryos were injected at the two-cell stage with 100 pg of each deleted MT-otx2 RNA. Animal caps were dissected at late blastula (stage 9) and cultured until midneurula (stage 15). (C) Animal caps from deleted MT-otx2-injected embryos were assayed by RT-PCR for the cement gland markers XCG-1 and XAG-1 and the neural marker NCAM using EF-1a as a loading control. Lane 1, uninjected; lane 2, full-length MT-otx2; lane 3, NgoMI deleted MT-otx2; lane 4, BsmBI deleted MT-otx2; lane 5, NsiI deleted MT-otx2; lane 6, StuI deleted MT-otx2; lane 7, NgoMI to BsmBI internally deleted MT-otx2. (D) Embryos injected at the two-cell stage with 100 pg of each MT-otx2 deletion RNA were harvested at midgastrula (stage 11.5) and assayed by immunocytochemistry with an anti-myc antibody. Each panel shows a close-up of myc-positive cells, demonstrating expression and nuclear localization of the truncated proteins.
	FIG. 4. otx2-En interferes with otx2 activity. (A) 162 amino acids, including the activation domain (“A,” vertical hatching), were deleted from the carboxyl terminus of otx2 (top) and fused to the engrailed minimal repressor domain (“EnR”, gray shading) to create otx2-En (bottom). (B) Embryos were injected with otx2-En with or without otx2 or siamois RNA at the two-cell stage. Animal caps were dissected at late blastula (stage 9) and harvested at early gastrula (stage 10.5; siamois, D) or midneurula (stage 15; otx2, C). (C) Animal caps from embryos injected with 500 pg otx2-En, 50 pg otx2, or both were analyzed by RT-PCR for expression of the cement gland markers XCG-1 and XAG-1, using EF-1a as a loading control. Differences in the amount of injected RNA were normalized using CAT RNA. Lane 1, 500 pg CAT; lane 2, 500 pg otx2-En; lane 3, 50 pg otx2 plus 500 pg CAT; lane 4, 50 pg otx2 plus 500 pg otx2-En; lane 5, whole embryo. (D) Animal caps from embryos injected with 500 pg otx2-En, 50 pg siamois, or both were assayed by RT-PCR for expression of the organizer specific markers chordin, noggin, and goosecoid using EF-1a as a loading control. Differences in the amount of injected RNA were normalized using CAT RNA. Lane 1, 500 pg CAT; lane 2, 500 pg otx2-En; lane 3, 50 pg siamois plus 500 pg CAT; lane 4, 50 pg siamois plus 500 pg otx2-En; lane 5, stage 10.5 whole embryo.
	FIG. 6. otx2-En disrupts cement gland and anterior neural gene expression. (A) Albino embryos were injected in one blastomere at the two-cell stage with 100 pg otx2-En or globin control RNA plus 60 pg lacZ RNA. Embryos were allowed to develop until early neurula (stage 13), midneurula (stage 15), or late neurula (stage 18), as appropriate for the marker assayed. (B) Gene expression in otx2-En injected embryos was analyzed by whole-mount in situ hybridization. (a) globin injected, XCG stage 13; (b) otx2-En injected, XCG stage 13; (c) otx2-En injected, XCG stage 15; (d) close-up of embryo in c. Small arrows mark XCG-1 expressing cells surrounded by bgal positive cells; (e) globin injected, En-2 stage 15; (f) otx2-En injected, En-2 stage 15; (g) globin injected, Krox20 stage 18; (h) otx2-En injected, Krox20 stage 18.
	FIG. 7. otx2 activity in the ectoderm is required for cement gland formation and is important for neural induction. (A) Embryos at the two-cell stage were injected with 200 pg otx2-En or 200 pg globin RNA mixed with 250 pg GFP RNA. At initial gastrula (stage 101), animal caps were dissected from embryos with evenly distributed GFP fluorescence and conjugated to anterior dorsal mesoderm from uninjected midgastrula (stage 11.5) embryos. The conjugates were harvested when the animal cap tissue reached tailbud (stage 20) equivalent. (B) Stage 101 animal caps (“ecto”) from embryos injected with 200 pg otx2-En (“OEn”) or 200 pg globin (“G”) were conjugated to mesoderm (“meso”) from uninjected embryos or cultured in isolation. When animal cap tissue reached tailbud stage equivalent, the conjugates were harvest for RT-PCR analysis for XCG-1, XAG-1, NCAM, Xanf-1, and EF-1a as a loading control. Lane 1, otx2-En ectoderm 1 mesoderm; lane 2, globin ectoderm 1 mesoderm; lane 3, otx2-En ectoderm; lane 4, globin ectoderm; lane 5, mesoderm; lane 6, st. 20 whole embryo.