Kuo JS et al. (1998), Opl: a zinc finger protein that regulates neura...

XB-ART-14645

Development 1998 Aug 01;12515:2867-82. doi: 10.1242/dev.125.15.2867.

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Opl: a zinc finger protein that regulates neural determination and patterning in Xenopus.

Kuo JS , Patel M , Gamse J , Merzdorf C , Liu X , Apekin V , Sive H .

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In order to study the mechanism of neural patterning in Xenopus, we used subtractive cloning to isolate genes activated early during this process. One gene isolated was opl, (odd-paired-like) that resembles the Drosophila pair-rule gene odd-paired and encodes a zinc finger protein that is a member of the Zic gene family. At the onset of gastrulation, opl is expressed throughout the presumptive neural plate, indicating that neural determination has begun at this stage while, by neurula, opl expression is restricted to the dorsal neural tube and neural crest. opl encodes a transcriptional activator, with a carboxy terminal regulatory domain, which when removed increases opl activity. opl both sensitizes animal cap ectoderm to the neural inducer noggin and alters the spectrum of genes induced by noggin, allowing activation of the midbrain marker engrailed. Consistent with the later dorsal neural expression of opl, the activated form of opl is able to induce neural crest and dorsal neural tube markers both in animal caps and whole embryos. In ventral ectoderm, opl induces formation of loose cell aggregates that may indicate neural crest precursor cells. Aggregates do not express an epidermal marker, indicating that opl suppresses ventral fates. Together, these data suggest that opl may mediate neural competence and may be involved in activation of midbrain, dorsal neural and neural crest fates.

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Species referenced: Xenopus laevis
Genes referenced: actl6a ascl2 cat.2 eef1a2 egr2 elavl1 en2 gal.2 gata2 gli3 hoxb9 hoxd1 krt12.4 muc2 muc2l myc ncam1 neurog1 nog otx2 pax3 shh snai2 tac1 tbx2 zic1

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	Fig. 2. (A) Northern analysis of Xenopus embryos. One embryo equivalent per lane was analyzed for opl RNA (top row) at various embryonic stages shown. Ethidium-stained 28S rRNA is a loading control (bottom row).
	Fig. 3. Whole-mount in situ analysis of opl expression. Embryos were analyzed by in situ hybridization as described in Methods. Embryo orientations are indicated by A, anterior; P, posterior; D, dorsal and V, ventral; arrowhead, dorsal lip of blastopore
	Fig. 4. Sections of embryos analyzed by whole-mount in situ hybridization for opl expression. Embryos were fixed, embedded and sectioned as described in Methods. Sectioning planes are shown in schematic diagrams above panels. Black arrowheads indicate position of dorsal blastopore lip. Black arrows indicate opl expression in roofplate. White arrows indicate spinal crest expression. Brackets indicate alar plate expression in forebrain/midbrain regions. D, dorsal; V, ventral. Scale bar, 25 mm.
	Fig. 5. Double whole-mount in situ analysis of opl. In situ analysis was as described in Methods with opl stained light blue and all other markers (otx-2, XCG, slug, en-2, krox20) are stained purple. (A) opl plus otx-2, a marker of forebrain and cement gland (Blitz and Cho, 1995; Pannese et al., 1995), bracket shows otx-2 expression in cement gland primordium; (B) opl plus XCG, a cement gland marker (Sive et al., 1989), bracket shows XCG expression in cement gland; (C) opl plus slug, a neural crest marker (Mayor et al., 1995), bracket shows slug domain contained within opl domain; (D) opl plus en-2, a marker of the midbrain/hindbrain regions (Hemmati-Brivanlou and Harland, 1989) and krox20, a marker of presumptive rhombomeres 3 and 5 in the hindbrain (Bradley et al., 1993). White arrowhead indicates en-2 expression, black arrowheads indicate krox20 expression, white bar is posterior limit of early opl expression.
	Fig. 7. opl is a nuclear protein with a regulatory domain in the COOH-terminal. (A) opl protein transactivates reporter constructs in tissue culture. Transient transfection reporter assays were performed as described in Methods, using the human pancreatic tumor BXPC cell line. The reporter construct is shown schematically: three Gli3 DNA-binding sites are located 5to a basal promoter (TATA box) and luciferase reporter gene (Vortkamp et al., 1995) Data shown are the averages of three independent experiments with corresponding error bars. In the absence of the Gli3-binding sites, neither opl nor oplDC activated a reporter construct (not shown). DNA mixes for transient transfections are as follows, Left panel, reporter plus CS2+ vector; middle panel, reporter plus CS2+opl ORF right panel, reporter plus pCS2-oplDC. (B) Immunocytochemistry of injected opl and oplDC proteins. Albino embryos were injected with 500 pg of synthetic MTopl or MToplDC RNA at 2- to 4-cell stages and analyzed at stage 11 by immunostaining with anti-myc 9E10 antibody as described in Methods. Views of the animal hemispheres are shown, left panel, MTopl-injected embryo, right panel, MToplDC-injected embryo.
	Fig. 8. oplDC can activate neural crest and dorsal neural tube marker genes in animal caps. (A) Experimental scheme. Wild-type embryos were injected in both blastomeres with 200 pg of indicated RNAs at the 2-cell stage. Injection of CAT RNA served as negative control by equalizing the injected RNA dose. Pools of 15-20 animal caps were isolated from injected embryos at late blastula (stage 9) and incubated in saline until harvest at tailbud (stage 22) for RT-PCR analysis. See Methods for details. (B) Expression of marker genes in animal caps. Uninjected embryos serve as control for baseline expression level. Xash3 (Zimmerman et al., 1993), neurogenin (Ma et al., 1996)and NCAM (Kintner and Melton, 1987) are neuralspecific markers; slug (Mayor et al., 1995) is a neural crest marker; pax3 (Espeseth et al., 1995) marks the dorsal neural tube, shh marks the floorplate (Ekker et al., 1995); XK81 (Jonas et al., 1985) and GATA2 (Walmsley et al., 1994) are markers of the ventral ectoderm; EF1a (Krieg et al., 1989) served as loading control. Samples processed without RT did not show EF1a signal after PCR (not shown). Data from one representative experiment is shown; similar results were obtained from three experiments. Lane 1, stage 22 embryo; lane 2, CAT-injected animal caps; lane 3, oplDC-injected caps; lane 4, opl-injected caps.
	Fig. 9. oplDC sensitizes the ectoderm to induction by noggin. Wildtype embryos were injected in both blastomeres with indicated RNAs at the two-cell stage, using 200 pg globin or oplDC, and 0.1 pg. 1 pg or 10 pg noggin (Smith and Harland, 1992) RNAs, as diagrammed in Fig. 8A, except injection of globin RNA served as negative control by equalizing the injected RNA dose in all lanes. Pools of 15-20 animal caps were isolated from injected embryos at late blastula (stage 9) and incubated in saline until harvest at tailbud (stage 22) for RT-PCR analysis. See Methods for details. Expression of neural markers NCAM (Kintner and Melton, 1987) and Xash3 (Zimmerman et al., 1993) was analysed. EF1a served as loading control (Krieg et al., 1989). Samples processed without RT did not show EF1a signal after PCR (not shown). Data from one representative experiment is shown, similar results were obtained from three experiments. Lane 1, globin-injected caps; lane 2, caps injected with 200 pg oplDC RNA; lane 3, caps injected with 0.1 pg noggin RNA; lane 4, caps injected with 1 pg noggin RNA; lane 5, caps injected with 10 pg noggin RNA; lane 6, caps injected with 0.1 pg noggin RNA plus 200 pg oplDC RNA; lane 7, caps injected with 1 pg noggin RNA plus 200 pg oplDC RNA; lane 8, caps injected with 10 pg noggin RNA plus 200 pg oplDC RNA.
	Fig. 10. opl synergizes with noggin to activate more posterior neural markers in animal caps. Wild-type embryos were injected in both blastomeres with indicated RNAs at the 2-cell stage, using 200 pg CAT, opl or oplDC, and 10 pg noggin (Smith and Harland, 1992) RNAs, as diagrammed in Fig. 8A. Injection of CAT RNA served as negative control by equalizing the injected RNA dose. Pools of 15-20 animal caps were isolated from injected embryos at late blastula (stage 9) and incubated in saline until harvest at tailbud (stage 22) for RT-PCR analysis. See Methods for details. Expression of a set of neural markers expressed along the anteroposterior (A/P) or dorsoventral (D/V) neural axes was analysed. Uninjected embryos serve as control for baseline expression level. NCAM is a general neural marker (Kintner and Melton, 1987). Expressed in the indicated anterior (A)-to-posterior (P) series: XCG is a cement gland marker, the most anterior ectodermal tissue (Sive et al., 1989); otx-2 is a forebrain and cement gland marker (Blitz and Cho, 1995; Pannese et al., 1995); en2 is a marker of mid/hindbrain boundary (Hemmati-Brivanlou and Harland, 1989); krox20 marks rhombomeres 3 and 5 in the presumptive hindbrain (Bradley et al., 1993); HoxD1 is a hindbrain (posterior to rhombomere 4) and spinal cord marker (Kolm and Sive, 1995a), and HoxB9 is a spinal cord marker (Wright et al., 1990). Expressed in the indicated dorsal (D) to ventral (V) series of markers, slug (marking the neural crest, (Mayor et al., 1995)), pax3 (marking the dorsal neural tube, (Espeseth et al., 1995)), NK2 (marking the ventrolateral neural tube, (Saha et al., 1993)) and shh (marking the floorplate, (Ekker et al., 1995)). M-actin (Mohun et al., 1984) is a mesodermal marker; EF1a (Krieg et al., 1989) served as loading control. Samples processed without RT did not show EF1a signal after PCR (not shown). Data from one experiment are shown, comparable results were obtained in four independent experiments. Lane 1, stage 22 embryo; lane 2, uninjected animal caps; lane 3, noggin plus CAT-injected; lane 4, oplDC-injected; lane 5, noggin plus oplDC-injected; lane 6, noggin plus opl-injected.
	Fig. 11. opl induces cellular aggregates without cell division. (A) Experimental scheme. Wild-type embryos were injected in one cell at the 2-cell stage with 500 pg of oplGR or oplDC RNA plus 100 pg lacZ RNA as lineage tracer. Injected embryos were incubated in saline alone or with dexamethasone (dex) and/or hydroxyurea plus aphidicolin (HUA) starting at early gastrula (stage 10) until late neurula (stage 19). See Methods for details. (B-H) Morphology and histology of opl-induced cellular aggregates. b-gal staining is obscured by pigment. (B-E) Anterior (A) is to the left, dorsal is to the top. (B) Embryo injected with oplGR RNA without dex addition; (C,D) embryos injected with oplGR RNA and treated with dex from stage 10 until harvest. Arrows point to cellular aggregates in ventrolateral ectoderm. (E) Embryo injected with oplGR RNA and treated with HUA (see Methods) plus dex from stage 10 until harvest. (F-H) Embryos sectioned and stained after oplDC injection. (F) +dex, transverse section of stage 19 embryo through trunk region. Regions magnified in G and H are boxed and indicate uninjected and injected sides of the embryo respectively, as judged by b-gal staining. (G) Uninjected side; (H) injected side. The width of the ectoderm is bracketed in G and H. D, dorsal; V, ventral.