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Figure 1. eg5 expression pattern in developing Xenopus embryos. Whole-mount in situ hybridization analysis of spatio-temporal eg5 expression. A–D: Dorsal view, anterior on the left. E: Dorso-lateral view. H, J: Lateral view, anterior on the left, dorsal at the top. F, G, I: Transversal sections. Dashed lines in C, E, and H indicate the sites of transverse section shown in F, G, and I, respectively. A: eg5 transcripts are first detected since the late gastrula stage (stage 12.5) in the dorsal region of embryos at the neural plate and neural plate borders (arrowheads). B–D: During neurulation (stages 14–18), eg5 is expressed in the neural plate borders and in prospective neural crest (arrowheads). No expression is detected in the neural plate midline (arrow). The transversal section of a stage-16 midneurula embryo (F) shows eg5 expression in neural plate (black bracket) and in the neural crest region (red brackets). The transversal section of stage-21 embryo (G) shows eg5 transcripts in the dorsal region of dermatomes (arrowheads). No specific labeling is observed in the sense probe control (C, inset). E: Stage-21 embryos show eg5 expression in the head region, Rohon-Beard sensory neurons (arrows), and the epidermis in a spotted pattern. H: Stage-24 embryos show eg5 expression at the somites, optic vesicle, profundal ganglia primordium (encircled), and epidermis external layer (I). J: The tailbud-stage embryos show the expression at the branchial arches, somites, brain, and epidermis. n, notochord; nt, neural tube; s, somites; b, brain; ba, branchial arches; op, optic vesicle. K–M: Double in situ hybridizations for eg5 (purple) and foxd3 (turquoise), sox2 (turquoise), and xk81a (turquoise). Dorsal view, anterior on the left. K'–M': Transverse sections of St. 15 neurula embryos displayed in K–M showing that eg5 expression overlaps with foxd3 in the neural crest territory (K', black brackets: foxd3 neural crest expression), and is complementary with sox2 and xk81a expressions (L', M'). L': Red brackets, neural crest region; black bracket, neural plate. M': Black brackets, neural crest region. N–P: RT-PCR analysis of eg5 expression in developing embryos. O: Quantification of the gel shown in N, the results are expressed as Relative intensity (sample/ef1α x10). P: RT-PCR analysis of eg5, foxd3, sox2, myod and xk81a expression in neural crest, intermediate mesoderm, neural plate and non-neural ectoderm explants. Explants were dissected out from stage-14 embryos (see Experimental Procedures section). ef1α, loading control. np, neural plate; n, notochord; nc, neural crest; m, intermediate mesoderm; nne, nonneural ectoderm; ac, animal cap (Stage 9).Download figure to PowerPoint
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Figure 2. eg5 is required for early neural crest specification in Xenopus embryos. A–E: In vitro and in vivo efficiency of eg5 antisense morpholino oligonucleotide (eg5MO). A: Efficiency of eg5 antisense morpholino oligonucleotide. eg5MO injected and control embryos were cultured until stage 14 and homogenized for total protein extraction. Eg5-GFP protein was detected by Western blotting using an anti-green fluorescent protein (GFP) antibody. Each lane was loaded with 100 μg of total protein. eg5MO inhibits translation of eg5 in a dose-dependent manner. eg5MO concentration is expressed in ng/embryo. B–E: Dorsal views of Xenopus laevis embryos under the fluorescence stereo microscope, anterior side is on the left. White arrowheads indicate the injected side. B', D', E': Fluorescence and clear field images of each embryo are shown in merged images. B, B': Embryo injected with mRNA encoding δC-eg5GFP (1 ng/embryo) showing GFP fluorescence on the treated side. C, C': Embryo injected with δC-eg5GFP mRNA (1 ng/embryo) and control antisense morpholino oligonucleotide (CoMO, 20 ng/embryo). D, D', E, E': Embryos injected with δC-eg5GFP mRNA (1 ng/embryo) and eg5MO (C and C', low dose (l), 10 ng/embryo; D and D', high dose (h), 22 ng/embryo. No embryo shows GFP fluorescence at a high dose of eg5MO. F–O: Analysis of eg5MO effects on neural crest early specification. Dorsal views of Xenopus laevis embryos, anterior side is on the left. Black arrowheads indicate the injected side. F, G: eg5MO-injected embryos show inhibition of foxd3 and snail2 neural crest markers, respectively. H, I: The expression of the neural plate marker sox2 is reduced and the epidermal marker xk81a is expanded on the eg5MO-treated side. Brackets indicate the width of the neural plate (H) and the width of the neural plate plus the neural crest domain (I). J: Embryo labeled by double in situ hybridization for sox2 and xk81a genes showing the reduction of prospective neural crest (black brackets) and neural plate (red brackets) domains in the eg5MO-injected side. Green brackets indicate neural crest and neural plate territories together. This result is identical to H and I. K: The neural plate border specification marker pax3 show reduced expression in the eg5MO-injected side. L, M: Coinjection of eg5MO and Xenopus tropicalis eg5 mRNA rescues foxd3 and sox2 expression, respectively. N: Embryo injected with eg5MO and xr11, the Xenopus bcl2 orthologue. The antiapoptotic factor xr11 did not rescue the expression of foxd3 in the neural crest. O: CoMO-injected embryos show normal expression of foxd3.Download figure to PowerPoint
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Figure 3. Overexpression of eg5 mRNA leads to an increase in neural crest markers. A–F: Dorsal views of Xenopus laevis embryos, anterior side is on the left. The injected side is indicated by an arrowhead. Embryos were injected into one blastomere at the 8–16-cell stage with 4 ng of eg5 mRNA and the expression of several markers was analyzed by in situ hybridization. A–C: eg5-injected embryos show increased expression of foxd3 and snail2 and an expanded expression of neural plate marker sox2 (C). D: eg5 overexpression produced a slight reduction in the expression of xk81a epidermal marker. E: Embryo labeled by double in situ hybridization for sox2 and xk81a genes showing the expansion of prospective neural crest (black brackets) and neural plate (red brackets) domains in the eg5 mRNA-injected side. Green brackets indicate neural crest and neural plate territories together. This result is identical to C and D. Dashed line, embryo midline. F: The neural plate border specification marker pax3 show increased expression in the eg5 mRNA-injected side.Download figure to PowerPoint
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Figure 4. Analysis of eg5 temporal requirements in neural crest development. A: Evaluation of early effect of monastrol on Xenopus embryos development. Sixteen-cell-stage embryos were incubated in increasing concentration of monastrol. Embryos show normal segmentation up to 500 μM monastrol, while 750 μM arrested development of almost 50% of embryos. Number of embryos for each treatment are in parenthesis. B, C, E, F, H, I: Dorsal view of embryos. Anterior side is on the right. D, G; J–L: Lateral views of embryos, anterior side is on the right. B, D: Embryos incubated in NAM solution containing monastrol show an almost complete depletion of foxd3 (arrows in B) and sox10 (arrow in D) expression compared to DMSO-incubated embryos (B, F). C: Embryos incubated in monastrol show normal paraxis mesodermal marker expression, compared with sibling control (F). H, I: Embryos were grafted at stage 11.5 on the right neural fold with a monastrol-soaked bead (H) or DMSO-soaked bead (I) and fixed at stage 15. Monastrol-soaked beads grafted in stage 11.5 produced a decrease in the expression of neural crest markers foxd3 (H) while no effect on foxd3 was observed when control DMSO containing beads were grafted on embryos (H). Arrowhead, treated side. J: Stage-17 embryos were grafted on the right neural crest region with a 100-μM monastrol-soaked bead. Embryos were cultured until stages 21–23 and the expression pattern of sox10 marker shows arrested neural crest cell migration and the accumulation of these cells laterally to the hindbrain. The leading edge of migration is indicated by dotted lines. K, L: Non-grafted embryos and embryos grafted with control DMSO-soaked beads show normal neural crest migration. M–R: Neural crest explants were dissected including the underlying mesoderm from stage-11.5 and stage-16 embryos. M, P: Groups of explants were fixed immediately (t0) after excision at stages 11.5 or 16. Groups of explants were cultured until stage 15 (N, O) or stage 22 (Q, R) in the presence of monastrol or DMSO vehicle. O, R: The neural crest explants isolated at stage 11.5 and cultured until stage 15 or dissected at stage 16 and incubated until stage 22 in the presence of 100 μM monastrol lose foxd3 and sox10 expression, respectively.Download figure to PowerPoint
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Figure 5. eg5 does not control cell proliferation or apoptosis during neural crest specification. A–H: Dorsal views of Xenopus laevis embryos, anterior side is on the left. The injected side is indicated by an arrowhead (A–F). Embryos were injected into one blastomere at the 8–16-cell stage with 4 ng of eg5 mRNA (A, C, E) or 22 ng of eg5MO (B, D, F), incubated until stage 14 and fixed. A, B: eg5 mRNA- or eg5MO-injected embryos were incubated in the presence of HUA (hydroxyurea and aphidicolin) inhibitors mix until stage 14 and the expression of foxd3 was analyzed by in situ hybridization. eg5 produced an expanded foxd3 labeling (A) and eg5MO blocked the expression of foxd3 marker in the neural crest (B). The presence of HUA produced no changes in the foxd3 phenotype after eg5 gain- or loss-of-function. Wholemount anti- phospho H3 immunohistochemistry (C, D) and TUNEL (E–H) labeling were performed as indicated in the Experimental Procedures section. C', D': The injected side is recognized by the fluorescence of the lineage tracer fluorescein dextran. G: Embryos were incubated in 100 μM monastrol from stage 11.5 to stage 14, fixed and processed for TUNEL. Sibling embryos were incubated in parallel in a solution containing DMSO (H). H': Positive control performed by DNAase treatment and subsequent TUNEL labeling. I, J: Quantification of phospho-H3 and TUNEL labeling for eg5-, eg5MO-microinjected embryos and monastrol treatment of embryos (see Experimental Procedures section). J, green bars: Quantification of the effector caspases 3/7 activity in neural crest explants lysates dissected out at stage 14. Caspase activity was expressed as sample:control ratio. No significant changes were observed in cell proliferation or apoptosis caused by the microinjection of eg5 mRNA and egMO or by incubation in the specific inhibitor monastrol.Download figure to PowerPoint
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Figure 6. eg5 is required for the formation of neural crest derivatives formation. A–C: Craniofacial cartilages preparations of stage-46 tadpoles, Alcian blue staining. Ventral view, anterior side is at the top. Injected side is indicated by an arrowhead. A: Control morpholino CoMO-injected embryos show no effects on the treated side. B: eg5MO injected side of embryos (arrowhead) present a marked reduction in Meckel's, ceratohyal and ceratobranchial cartilages. C: The coinjection of eg5MO and Xenopus tropicalis eg5 mRNA rescues normal cartilages morphology. D: Schematic representation of eg5MO effects on Xenopus head cartilages. E, E', E'': eg5MO-injected embryos show altered Rohon-Beard sensory neurons and cranial ganglia precursors derivatives. Notice that tbx2 expression pattern was altered in the injected side of embryos. M, Meckel's cartilage; CH, ceratohyal cartilage; CB, ceratobranchial cartilage; Ir, infrarostral cartilage; BH, basihyal cartilage; Pr, profoundal placode; L, lens placode; Ll, lateral line placode; V, trigeminal placode; Cg, cement gland; RB, Rohon-Beard sensory neurons. A-D, black arrowhead, injected side. E, E', white arrowhead, injected side; black arrow, cranial ganglia precursors; black arrowhead, lens placode; Otp, otic placode.Download figure to PowerPoint
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Figure 7. Hierarchical relationships between eg5 and ihh cell signaling pathway genes. A–H: Dorsal view of embryos. Injected side is indicated by an arrowhead. Anterior side is on the left. eg5 mRNA-injected embryos show increased expression of neural crest marker foxd3 (A) and eg5MO-injected embryos show inhibition of foxd3 (B). C, D: ihhMO-injected embryos show inhibition of foxd3 expression (C). The coinjection of ihhMO and eg5 mRNA rescues foxd3 expression on neural crest (D). E, F: gli3MO strongly reduces foxd3 marker (E), and this effect is not rescued by the coinjection of eg5 mRNA (F). G, H: The coinjection of ihh mRNA cannot rescue foxd3 inhibition caused by eg5MO (G). The effect of eg5MOwas rescued by the coinjection of gli3 mRNA (H). The results suggest that eg5 is downstream of ihh and upstream of gli3 in the genetic cascade that controls neural crest specification.Download figure to PowerPoint
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