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In amphibians and teleosts, early embryonic axial development is driven by maternally deposited mRNAs and proteins, called dorsal determinants, which migrate to the presumptive dorsal side of the embryo in a microtubule-dependent manner after fertilization. Syntabulin is an adapter protein that binds to kinesin KIF5B and to the transmembrane protein Syntaxin1. In zebrafish, a mutation in Syntabulin causes complete embryo ventralization. It is unknown whether Syntabulin plays an analogous role during early development of other species, a question addressed here in Xenopus laevis. in situ hybridization of syntabulin mRNA was carried out at different stages of Xenopus development. In oocytes, syntabulin transcripts were localized to the vegetal cortex of large oocytes and the mitochondrial cloud of very young oocytes. We extended the zebrafish data by finding that during cleavage Xenopus syntabulin mRNA localized to the germ plasm and was later expressed in primordial germ cells (PGCs). This new finding suggested a role for Syntabulin during germ cell differentiation. The functional role of maternal syntabulin mRNA was investigated by knock-down with phosphorothioate DNA antisense oligos followed by oocyte transfer. The results showed that syntabulin mRNA depletion caused the complete loss of dorso-anterior axis formation in frog embryos. Consistent with the ventralized phenotype, syntabulin-depleted embryos displayed severe reduction of dorsal markers and ubiquitous transcription of the ventral marker sizzled. Syntabulin was required for the maternal Wnt/β-Catenin signal, since ventralization could be completely rescued by injection of β-catenin (or syntabulin) mRNA. The data suggest an evolutionarily conserved role for Syntabulin, a protein that bridges microtubule motors and membrane vesicles, during dorso-ventral axis formation in the vertebrates.
Fig. 2. Xenopus syntabulin is a vegetally localized maternal mRNA. (A) Whole-mount in situ hybridization on stage VI oocytes using an antisense probe showing that maternal syntabulin (sybu) transcripts are localized at the center of the vegetal pole. The inset shows the sense probe negative control. (B) In situ hybridization on albino stage VI oocytes showing that syntabulin transcripts form a granular pattern in the vegetal cortex; note arrow in the hemisection shown in the inset. The germinal vesicle is indicated by a dotted line in inset. (C) In situ hybridization of sybu at different stages of oogenesis (I–V) showing that maternal transcripts localize early (stage I, arrow) in the mitochondrial cloud, and in the cortex of the vegetal pole at later stages. Dotted line in stage I oocyte marks the nucleus. An=animal pole; Veg=vegetal pole; GV=germinal vesicle.
Fig. 3. Maternal syntabulin mRNA is present during early stages of development, and associates with germ plasm and germ cells. (A) Semiquantitative RT-PCR showing syntabulin (sybu) mRNA levels at different developmental stages. Histone 4 (H4) mRNA was used as loading control. (B, C) In the cleaving embryo, syntabulin mRNA associates to germ plasm islands, that occupy a characteristic position close to the cleavage furrow (arrows). (D) During gastrulation, syntabulin transcripts are detected in a small subset of endodermal cells (arrow), corresponding to the known location of germ cell precursors. (E) At stage 33, syntabulin is detected in different tissues such as the olfactory placode (op), the neural tube (nt) and the germ cells. (F) Expression pattern of an established germ cell marker, Xpat, at stage 33. Note the similar localization between syntabulin and Xpat in the germ cell territory.
sybu (syntabulin) gene expression in Xenopus laevis embryo, NF stage2 (2-cell) , as assayed by in situ hybridization, vegetal view.
sybu (syntabulin) gene expression in Xenopus laevis embryo, NF stage 4 (8-cell), as assayed by in situ hybridization, vegetal view.
sybu (syntabulin) gene expression in Xenopus laevis embryo, NF stage 4 (8-cell), as assayed by in situ hybridization, blastoporal view, dorsal up.
Fig. 4. Depletion of maternal syntabulin causes embryo ventralization and decreases the expression of dorsal genes. (A) Wild type oocyte showing normal syntabulin expression. (B) 91% (n=12) of oocytes injected with 10 ng of phosphorothioate-modified DNA antisense oligo targeting syntabulin (sybu AS) showed no expression of the maternal mRNA 48 h after microinjection. Hybrids between mRNA and DNA are efficiently degraded by RNase H. (C) Depletion of syntabulin in injected oocytes assessed by quantitative RT-PCR (qPCR). Groups of five oocytes were used. Injection of different amount of sybu AS (5 or 10 ng) caused a strong decrease of mRNA levels in a dose-dependent fashion. WT indicates uninjected oocytes. (D, E) Representative wild type and sybu AS-injected embryos obtained from host transfer experiments. Note the complete absence of axial structures in sybu-depleted embryos. (F) In wild type embryos, the ventral marker sizzled (szl) is expressed in a ventral domain at stage 12. (G) Age-matched sybu-depleted embryos (sybu-AS) display radial ectopic szl expression, indicating strong expansion of ventral tissues. (H) In wild type embryos at stage 22, szl has an anterior and a posterior domain of expression. (I) sybu-depleted embryos have a radially expanded szl ventro-posterior expression domain. Strong expansion of ventraltissue was observed in 70% of the embryos (n=36). Arrowheads point to the blastopore. (J) Quantification of the ventralization phenotype after syntabulin depletion. Note the high penetrance of complete ventralization (dorso-anterior index 0) in antisense injected embryos (84%); data from 4 independent host transfer experiments (n=61). (K–N) qRT-PCR analyses showing significant decreases in the dorsal markers siamois, Xnr3 and chordin, and a strong increase of the ventral marker sizzled after maternal knockdown of syntabulin. Error bars indicate the standard error from three independent host transfer experiments
Fig. 5. The Syntabulin maternal depletion phenotype is rescued by sybu mRNA or by β-catenin mRNA. (A) Expression of the neural plate marker Sox2 in wild type early neurula embryos, n=16/16. (B) Syntabulin maternal depletion eliminated almost all Sox2 expression, with the exception of a weak residual ring of expression around the blastopore (inset, arrowhead), n=15/15. (C) Injection of β-catenin mRNA at 4-cell stage efficiently rescued expression of Sox2 in sybu depleted host transfer embyos, n=12/12. (D–G) The sybu AS ventralized phenotype was rescued both by syntabulin-UTR+ mRNA (n=7) and by β-catenin mRNA (n=12) at the tailbud stage.
Fig. 6. Rescue of D-V patterning genes in Syntabulin-depleted embryos by syntabulin or β-catenin mRNA injection. (A) Expression of the Spemann organizer marker chordin (n=13/13) in wild type embryos at stage 10. (B) Chordin is eliminated by maternal depletion of syntabulin (n=9/9). (C) Injection of sybu mRNA partially rescues its own depletion (n=6/10), demonstrating specificity of the knock-down. (D) chordin expression is rescued, and somewhat expanded with respect to wild type, by β-catenin mRNA (n=14/14). (E–H) Maternal depletion of syntabulin increases expression of the ventral marker sizzled and this is counteracted by the injection of syntabulin-UTR+ or β-catenin mRNAs.
