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Figure 2. Spatial and temporal analysis of ashwin expression. Dorsal is toward the top, unless otherwise indicated. A: Stage (st.) 10; ashwin is expressed throughout the dorsal circumblastoporal zone. An arrow indicates the dorsal lip. B: A st. 12, dorsal view; expression is detected throughout the dorsal ectoderm and marginal zone. C: St. 12, ventral view; expression is excluded from the ventral hemisphere except for the circumblastoporal ring. D: Embryo hybridized with a sense probe. E: A st. 18, dorsal view; expression is visible in the neural ectoderm. F: A st. 18, frontal view reveals expression in anterior neural folds. G: A st. 18, midline sagittal dissection; expression is strongest in anterior and posterior neural domains but also is detected in the notochord and prechordal plate. H-K: At st. 22. H: Expression is detected throughout the developing nervous system and optic vesicles. I: Sense probe hybridization. J: Frontal view. K: Forebrain dissection; expression is detected in the ventral aspect of the brain and throughout the eye anlagen. L: At st. 26; ashwin is expressed throughout the central nervous system and in the tail bud. M: At st. 36; ashwin is expressed strongly in the brain and moderately in the posterior axial region. D, dorsal; V, ventral; A, anterior; P, posterior. N: Northern analysis of ashwin expression in Xenopus embryos at various embryonic stages. Each lane shows RNA isolated from two embryos at each of the stages indicated. The ashwin transcripts were visualized using a radiolabeled PCR probe. Two transcripts at 1.5 and 2.1 kb are expressed both maternally and zygotically. Levels of 18S and 28S rRNA are included to indicate the total amount of RNA loaded in each lane.
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Figure 3. Phenotypes resulting from overexpression of ashwin. A–C: Embryos were injected in the animal pole with 0.2 ng of mRNA encoding ashwin. A: At stage (st.) 37, a set of three headless embryos at the bottom; the top embryo is a sibling control embryo injected with 0.2 ng of β-galactosidase mRNA (LacZ). B: Representative embryos displaying ashwin-induced split axes at st. 20 and st. 26. The embryo on the left also has open neural folds. C: Stage 22, dorsal view; these embryos display defects in neural tube closure (arrow) and epidermal integrity (arrowhead). In one embryo, the neural folds have not formed and the primary axis is split (asterisk). D,E: Embryos injected in the animal pole with 0.2 ng of myc-ashwin. Distribution of myc-ashwin was visualized using immunohistochemistry, and embryos were cleared in benzyl benzoate: benzyl alcohol (BB:BA) to reveal interior structures. Arrows indicate cement glands. The embryo with little myc-ashwin in the anterior region has a relatively normal head (D), whereas the embryo in E has anterior defects and stronger anterior myc-ashwin expression. F: Stage 22; cross-sections showing thickening of abnormal epithelium; a section of a control embryo (LacZ) is included for comparison. G,H: Embryos injected equatorially in two dorsal blastomeres at the four-cell stage with 0.2 ng of myc-ashwin and stained to reveal myc-ashwin distribution. G: Strong expression of myc-ashwin in neural ectoderm leads to distended heads and defects in neural tube closure. H: Strong expression of myc-ashwin in dorsal mesoderm leads to anterior truncations. I: Embryo injected with 0.2 ng of ashwin mRNA in two ventral blastomeres at the four-cell stage; this embryo shows the maximal extent of abnormalities obtained with ventral injections.
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Figure 4. Anterior defects in embryos overexpressing ashwin. Embryos were injected in the animal pole at the two-cell stage with 0.2 ng of mRNA encoding either ashwin or MT-ashwin; results for both constructs were indistinguishable. Control embryos were injected with β-galactosidase mRNA (LacZ). Embryos were cultured until st. 30, when they were fixed and processed for immunohistochemistry with the Tor-70 antibody to visualize the notochord (No). A,B: The anterior limit of the notochord (arrow) is located at the level of the midbrain in LacZ embryos (A) and at the far anterior end of an Ashwin embryo (B). C: A sagittal section of a LacZ embryo showing the forebrain (FB), pigmented retina (RPE), and otic vesicle (OV). D: A similar section through an Ashwin embryo shows that all tissue anterior to the notochord is a solid mass. E,F: Tadpoles expressing β-galactosidase (LacZ) or myc-ashwin reveal characteristic defects appearing at later stages. E: A cross-section through the eye reveals that the right optic cup (OC; arrow) is small and underdeveloped, whereas the left optic cup has failed to extend away from the midline. The right lens (L) is rudimentary. Overall, anterior patterning is displaced so that the heart (H) appears at the same level as the eye; normally, the heart appears at the level of the otic vesicle, as seen in the LacZ embryo at left in F. F: Sections through more posterior regions of control (LacZ) and myc-ashwin–injected embryos show a less severe phenotype in the region of the hindbrain compared with more anterior regions. The dorsal neural tube is thin, and the notochord has split (central arrows). The otic vesicle (arrow at right) is frequently the most anterior structure visible in ashwin-injected embryos with anterior truncation.
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Figure 5. Region-specific neural gene expression in embryos overexpressing ashwin. A–D:Otx2 expression in control (A,B) and embryos injected with 200 pg ashwin mRNA (C,D). Expression is normal at stage (st.) 18 in 100% of embryos; by st. 28, embryos overexpressing ashwin have lost much of their otx2 expression. Virtually all embryos examined show either a reduction (51%) or loss (49%) of otx2 expression at this stage. E: A neurula-stage embryo injected unilaterally with 200 pg of ashwin mRNA and hybridized in situ for Krox20 and HoxB9. HoxB9 expression is unchanged (arrowheads); the R5 stripe of Krox20 expression is lost on the injected side. F,G: Embryos injected with 200 pg of β-galactosidase mRNA (F) or ashwin mRNA (G) and hybridized in situ for en-2 and Krox-20 at st. 28. Both R3 and R5 stripes of Krox-20 are visible (lines), and expression is also detected in the third branchial arch (arrow). Asterisk indicates en2 expression.
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Figure 6. Secondary axis formation in embryos overexpressing ashwin. A,B: Two-cell stage embryos were microinjected in the animal pole with either 0.25 ng of ashwin mRNA (A) or 1 ng of ashwin DNA (B). The split axis phenotype is generated at a similar frequency by either RNA or DNA injections. C–F: Embryos were injected as described with 0.25 ng of ashwin RNA. C,D: Expression of Xnot1 in control early gastrulae (LacZ; C) and gastrulae injected with 0.25 ng ashwin mRNA (D). There is no difference in Xnot1 expression between ashwin- and control-injected embryos. E,F: Expression of gsc in control early gastrulae (E) and gastrulae after injection of 0.25 ng of ashwin mRNA (F). Expression of gsc is reduced in embryos overexpressing ashwin. G,H: Sections through the trunk of stage 20 embryos expressing either LacZ (G) or MT-Ashwin (H). MT-ashwin was injected at a concentration of 0.2 ng into two blastomeres at the two-cell stage. Ectopic MT-ashwin is detected with anti-myc antibody (red–orange staining). The notochord (No) is split into two in the Ashwin embryo (arrows). No, notochord; NT, neural tube. I,J: Embryos injected with 0.2 ng of mRNA encoding myc-ashwin, stained to visualize myc-ashwin, and cleared to reveal interior structures. I: Dorsal view revealing outline of archenteron in early midgastrula embryo; archenteron shape is characteristic of over 75% of injected embryos. J: Dorsal view of midgastrula embryo shows bifurcation at leading edge of archenteron. Arrows indicate tips of archenteron.
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Figure 7. Cell lineage analysis of embryos injected with ashwin mRNA. Embryos were injected with 200 pg of myc-ashwin mRNA in the animal pole of one blastomere at the two-cell stage, fixed at early gastrula stages, hybridized in situ using the gsc probe, and then processed for immunohistochemistry using the anti-myc antibody. A,B: Single embryo photographed before (A) and after (B) immunohistochemistry; myc-ashwin is distributed throughout the dorsal lip. C,D: Second embryo photographed before (C) and after (D) immunohistochemistry; myc-ashwin is present only on one side of the dorsal lip. Dashed lines indicate the edge of the region expressing myc-ashwin; arrows indicate position of the dorsal lip.
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Figure 8. Disruption of ashwin expression by means of morpholino oligonucleotide (MO) -mediated knockdown leads to lethality, gastrulation defects, and apoptosis. A: Frequencies of lethality at gastrulation and gastrulation defects in embryos injected with MO directed against ashwin. Embryos were injected with 10 ng of MOs against the ashwin exon I splice junction (Ash Ex1 MO), the ashwin translation start site (Ash 5′ MO), or the five-base mispair of the exon I MO (Ex 1 mispr MO). Additional embryos were injected with 10 ng of Ash Ex1 MO and 0.1 ng of Ashwin mRNA, to determine whether ashwin mRNA could rescue the effects of the Ex1 MO. Typical gastrulation defect elicited by Ash Ex1 MO is shown in B. B–E: Terminal uridine nucleotide end labeling (TUNEL) assays of gastrula embryos injected with either 10 ng of Ash Ex1 MO (B), 200 pg of ashwin mRNA (C), 10 ng of control MO (D), or 200 pg of β-galactosidase mRNA (E). Asterisks indicate the position of the dorsal blastopore lip.
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Figure 9. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of gene expression in animal cap ectoderm overexpressing ashwin. A: Animal caps were isolated from late blastula embryos injected with 200 pg of mRNA encoding either β-galactosidase (lac, lane 2), ashwin (A, lane 3), or noggin (N, lane 4). Animal caps were also collected from embryos co-injected with 0.2 ng of ashwin mRNA and 0.25 ng of noggin mRNA (A + N, lane 5). Animal caps were collected for RT-PCR when control embryos reached stage (st.) 18. WE, whole embryo (lane 1). –RT, control sample prepared without reverse transcriptase (lane 6). EF-1α primers flank an intron, so any contaminating genomic DNA can be detected in each sample (n = 4 independent experiments). B: Expression of BMP4-inducible genes in animal caps overexpressing ashwin. Animal caps were isolated from embryos injected with 200 pg of either LacZ mRNA (Lac, lane 2) or Ashwin mRNA (Ash, lane 3) and collected for RT-PCR when controls reached st. 11. BMP4-inducible gene expression in animal caps overexpressing LacZ (Lac, lane 4) or noggin (nog, lane 5). Msx1 was also down-regulated in animal caps overexpressing noggin (data not shown) (n = 3 independent experiments).
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c2orf49 (chromosome 2 open reading frame 49) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 10, blastoporal view, dorsal up.
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c2orf49 (chromosome 2 open reading frame 49) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 18, dorsal view, anterior right.
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c2orf49 (chromosome 2 open reading frame 49) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 18, midline sagittal dissection, lateral view, dorsal up, anterior right.
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c2orf49 (chromosome 2 open reading frame 49) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 18, dorsal view, anterior right.
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c2orf49 (chromosome 2 open reading frame 49) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 26, , lateral view, dorsal up, anterior right.
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