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Fig. 1. Whole-mount in situ hybridization for ets-1 and ets-2 transcripts; RT/PCR quantification in the different regions of the oocyte. (AI ers-7 rranscnpr derection in oocytes of different developmental stages. usmg a digoxlgenm labeled antisense riboprobe. Note the apparent uniform distributIOn of the stalnmg in small oocytes (white arrowheads) and the confining of the transcripts to rhe anrmal pole of larger oocytes (red arrowheads). (B) ets- 2 transcflpts detection in shed eggs, cut into halves prior to hybridization with a digoxlgenin- labeled antisense ets-2 probe. Note the confmement of the signal to the anrmal pole. IC) RT/ PCR analysIs of ets-l and ers-2 transcripts in rhe animal pole, Intermediare zone and vegetal pole of the oocyte. (1.21ets-l detection; (3,41ets.2 detection: (5,6) control on 185 rRNA: (7,8)
control on XI-Twi mRNA Odd numbers correspond to ethldium bromide staimng: even
numbers correspond ro southern hybfldizations with f32Pl labeled specific probes. AC, RT/PCR camed out on total RNA extracted from animal caps, (thickness,., 113of the oocyte diameter). In, RT/PCR on rotal RNA extracted from the mtermediate zone (thickness,=, 113of the oocyte diameter); VC, RT/PCR on total RNA extracted from the vegetal pole (thickness = 1/3 of the oocyte diameter);(+), positive control,(-), negarive control (PCR without template DNA).
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Fig. 2. Whole-mount in situ hybridization for ets-1 and ets-2 in early embryogenesis. (A) Localization of ets-1 transcript in the animal pole of a 4-cell embryo. The cleavage furrows are clearly indicated by residual dark-brown pigmentation. (B). Same experiment conducted for ets-2 transcripts. (C) Control experiment in which a 2-cell stage embryo was cut into halves prior to hybridization to exclude possible artifacts resulting from a poor penetration of the anti-digoxigenin antibody (ets-1 antisense probe). An, animal pole; Veg, vegetal pole. (D) ets-1 mRNA localization in a 32-cell stage blastula, showing the presence of transcripts In the animal blastomeres and in the upper part of vegetal blastomeres (blue arrowheads). (E) Same experiment carried out on a 32- cell stage embryo cut into halves prior to hybridization. Note the presence of the transcripts (dark-blue staining) in the roof of the blastocoel and in the marginal zone. An, animal; Veg, vegetal; BI, blastocoel. (F,G,H) Whole-mount inSitu localization of ets-1 (F) and ets-2 (G,H) in stage 8 (F and H) and stage 6 (G) embryos. In addition to the animal pole cytoplasmic localization of the transcripts, notice the nuclear signal which can be observed in stage 8 embryos, at the level of the vegetal part, in the almost colorless cytoplasmic background. The specificity of this signal is assessed by the fact that no nuclear staining is observed in embryos prior to MBT (compare F and H with G) and by the fact that a sense probe does not give any signal (not shown). The proliferative activity of these cells is demonstrated by the presence of duplicated nuclei (white arrowheads in F) and a mitotlc figure (white arrowhead in H), in cells which have not yet undergone cytokinesis. (I,J) 10um thick sections of whole-mount hybridizations on a 32-cell stage (ets-1) and 16-cell stage (ets-2) embryos respectively. The transcripts are essentially located below the cortex of the animal blastomeres, but a significant level of both ets-1 and ets-2 mRNAs is also observed at the level of the germ plasms (red arrows). AB, animal blastomere; BI, blastocoel; VB, vegetal blastomere. Bar, 100 um.
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ets1 ( v-ets avian erythroblastosis virus E26 oncogene homolog 1) gene expression in Xenopus laevis embryos, NF stage 6, assayed by in situ hybridization, transverse section, animal pole up.
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ets2 ( v-ets avian erythroblastosis virus E26 oncogene homolog 2) gene expression in Xenopus laevis embryos, NF stage 5, assayed by in situ hybridization, section view, animal up.
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Fig. 3. Ets-1 transcript localization in Xenopus laevis gastrula (stage 101121. (A) Whole-mount In situ hybridizarson with antisense and sense probes (left and right embryo respectively). Ers-l transcripts dre observed as a Clfcumblastoporal collar (blue staining in the left embryo), whereas no signa/is observed with the sense probe. (BI Sect/on of astage 101/1 embryo showing the dorso-blastoporaf lip (red arrowhead) and rhe blastocoel (BL). The ets-l stainmg is mamly observed in the upper eplthebal layer of the marginal zone, and to a lesser extent In the deep marginal zone. However, cells rolling around the dorsoblastoporal lip do not seem to be labeled. In agreement with the absence of staining in a narrow cordon immediately adjacent fa the blastoporal lip in the left embryo(A) Bar, 100um.
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Fig. 4. Wholemount in situ detection of ets-1 transcripts in Xenopus laevis neurula ( st 15/18). (A) Stage 15 ( right embryo) and stage 18 ( left embryo). Note the presence of segregated cell masses on the external side of the neural plate, corresponding (to) the mandibular (m) hyoid (h) and branchial (b) segments of neural crest cells. Also note the thin stained line on the internal side of the elevating neural fold ( white arrowheads in stage 15 neuraula) and the corresponding labelling of the midline in the closing neural tube (white arrowheads in stage 18) embryo. (B) Horizontal section through a stage 17/78 Xenopus laevis embryo. Due to the curvature of the AlP axis, the section is almost transverse at the level of the neural plate. Segregated neural crest cell masses are conspicuously stamed on both sides of the neural plate. A clear staining is also observed in the lateral plate mesoderm. Ch, chord: LPM, lateral plate mesoderm: NC neural crest: NP, neural plate. Bar, 100 um.
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Fig. 5. Whole-mount in situ detection of ets-1 transcripts in pre-migratory and migrating neural crest cells. (A) Stage 19/20 embryo. antero-dorsal view: the mandibular (m), hyoid (h) and branchial (b) neural crest cell segments are strongly labeled; also note the punctuated label at the level of the border between prosencephalon and mesencephalon (red arrowheads). The black star shows a diffuse faint labeling in front of the most anterior part of the neural rube, normally deprived of neural crest cells and which could correspond to the hypophyseal anlage. (B) Stage 21/22 embryo. antero-dorsal view: the neural crest cells have now started their migration towards the ventral side of the embryo. The mandibular segment starts to surround the forming eye vesicle (E). Note the increase in the staining at the border between pro-and mesencephalon (red arrowheads), with respect to (A). Also notice the strong labeling at the midline of the neural tube. (C) Stage 19/20 embryo, antero-ventral view (right side embryo) and antero-dorsal view (Ieft side embryo). in addition to the labeling at the periphery of the neural tube already depicted (left side embryo), a strong staining is observed in the ventro-anterior region (white star on the right side embryo), in the putative area for heart and blood island. The faint and diffuse labeling in front of the neural tube is still clearly observed (black star). (D.E) Stage 22/23 embryos, lateral and dorsal views respectively: the neural crest cells are now populating the mandibular hyoid and branchial arches (D). The dorsal view clearly shows a labeling at the level of the vagal and truncal neural crest cells (E). b,h,m, branchial, hyoid and mandibular neural crest cell segments or arches: P,M,R, pro-, meso and rhombencephalon, respectively. E. eye vesicle.
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ets1 ( v-ets avian erythroblastosis virus E26 oncogene homolog 1) gene expression in Xenopus laevis embryos, NF stage 19/20, assayed by in situ hybridization, antero-lateral view,, dorsal up.
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ets1 (v-ets avian erythroblastosis virus E26 oncogene homolog 1) gene expression in Xenopus laevis embryos, NF stage 21/22, as assayed by in situ hybridization, lateral view, anterior left, dorsal up.
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ets1 ( v-ets avian erythroblastosis virus E26 oncogene homolog 1) gene expression in Xenopus laevis embryos, NF stage 22/23, as assayed by in situ hybridization, lateral view, anterior left, dorsal up.
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ets1 ( v-ets avian erythroblastosis virus E26 oncogene homolog 1) gene expression in Xenopus laevis embryos, NF stage 22/23, as assayed by in situ hybridization, lateral view, anterior left, dorsal up.
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Fig. 6. Ets-1 and ets-2 are expressed in the cranial, vagal, and dorsal root ganglia. (A) Whole-mount hybridization with an ets-1 probe on stage 29/30 embryos showing a conspicuous expression of ets-1 in the cranial ganglia and in the vagal and truncal neural crest cells (respectively VC and TC). Also note the staining at the level of the condensing pronephros, in the hemangioblast precurors, in the ventral region (red arrowhead), and in the forming hyaloid artery (white arrow). (B) Whole-mount hybridization with an ets-2 probe on stage 29/30 embryos showing the same expression in cranial ganglia, vagal, and truncal neural crest cells. The punctuated signal in the trunk neural crests is strongly evocative of the future dorsal-root ganglia (DRG). Also note the presence of ets-2 transcripts at the level of the forming pronephros rudiment and in the forming lens. At that stage, ets-1 is not expressed in the forming lens(A). (C) Whole-mount hybridization with an ets-2 probe on a stage 36/37 embryo, showing that in addition to gV and gVII-VIII, the vagal complex (GIX and X) is now labeled. (D,E) Whole-mount hybridization on stage 29/30 embryos (dorsal view) with ets-1 and ets-2 probes respectively, showing the conspicuous expression at the level of the future DRGs. (F,G) Horizontal sections of whole-mount hybridizations with an ets-2 probe, on stage 29/30 and 36/37 embryos respectively, showing the intense labeling of the cranial ganglia and vagal complex. Note that the lens, which is labeled at stage 29/30, is no longer stained at stage 36/37. In (A to E) embryos were cleared in Murray's reagent. AV, auditory vesicle; CG. cement gland: E, eye: gV, fifth cranial ganglion (trigeminal or gasserian); gVII-VIII,seventh and eighth cranial ganglia (geniculate and auditory); glX, ninth cranial ganglion (glossopharyngeal): gX, tenth cranial ganglion (vagus or pneumogastric); L. lens: P,pronephros. Bars, 100.um.
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ets2 (v-ets avian erythroblastosis virus E26 oncogene homolog 2) gene expression in Xenopus laevis embryos, NF stage 29/30, as assayed by in situ hybridization, lateral view, anterior left, dorsal up.
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ets2 ( v-ets avian erythroblastosis virus E26 oncogene homolog 2) gene expression in Xenopus laevis embryos, NF stage 36/37, as assayed by in situ hybridization, lateral view, anterior left, dorsal up.
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Fig. 7, Ets-1 expression in forming blood vessels; ets-1 and ets-2 expressions in the dorsal fin and posterior end of the embryo. (A,B) Horizontal sections of stage 39/40 embryos showing the expression of ers-l in forming blood vessels: aortic arches (largearrows In A) and intersomiric arteries (small red arrowheads in BJ. (C,D) Presence of ets-2 transcripts in the dorsal fin (large red arrow in the coronal section of C) and in the tip of the tail of a whole-mount stained embryo (stage 35/36) !D). CG, cement gland: Ch, chord; NT. neural tube: Ph, pharynx. Bars, 100 }Jm
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Fig. 8. Ets-2 expression in the pronephric tubules and growing pronephric duct. (A) Whole-mount 10 situ hybridIzation with an ets-2 specific probe, on stage 37/38 embryo. The pronephric tubules (PT) and the Wolffian duct (WD) are strongly labeled. (B) Transverse section of a stage 37/38 embryo after whole-mount hybridization, showing the strong labeling of the epithelial structure of the Wolff/an duct. Dorsal is to the left. (C) Horizontal section of a stage 37/38 embryo after whole-mount hybridization, showing the labeling of the pronephric tubules and Wolffian duct. Anterior is to the left. Bars, 100 p.m. Ch, notochord; NT. neural tube; PT. pronephric tubule: S, somite; WD, Wolffian duct.
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Fig. 9. In situ hybridization on sagittal sections of Xenopus gut from late developmental stages by using 35S-Iabeled ets-2 probes. (A,C,D,E,G) Antisense probe. (B,F,H) Control experiments using a sense probe. (A,B,C) Anterior part of small intestine from stage 60 animal. (D) Stomach of stage 62 Xenopus. (E,F) Pyloric portion of stomach from stage 62 tadpole. (G,H) Rectum of stage 60 animal. Ets-2 transcripts are located in both epithelial and muscular layers e. epithelium: mI. muscular layer: ct. connective tissue. Bar, 40.um.
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Fig. 10. In situ hybridization on sagittal sections of immature ovary and in Xenopus adult organs by using 35S-labeled ets-2probes. (A,B,C,E) Antisense probe; (D,F) control experiments with sense probe. (A) Ets-2 transcripts are detected In oogonia Included in ovary of stage 62 female. (B) Stage 60 tadpole showing the presence of ets-2 transcripts In the kidney tubules. (C,D) Ets-2 transcripts are detected In adult kidney. (E,F) Ets-2 is expressed In muscles and in spinal ganglia at the level of developing forelimb bud in stage 56/57 tadpoles, o, oogonium; kt. kidney tubules. sg, spinal ganglion; m, muscle; n, nerve. Bar, 40 um
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