September 1, 2009;
Vegetally localized Xenopus trim36 regulates cortical rotation and dorsal axis formation.
Specification of the dorsoventral axis in Xenopus depends on rearrangements of the egg
vegetal cortex following fertilization, concomitant with activation of Wnt/beta-catenin signaling. How these processes are tied together is not clear, but RNAs localized to the vegetal cortex during oogenesis are known to be essential. Despite their importance, few vegetally localized RNAs have been examined in detail. In this study, we describe the identification of a novel localized mRNA, trim36
, and characterize its function through maternal loss-of-function experiments. We find that trim36
is expressed in the germ plasm and encodes a ubiquitin ligase of the Tripartite motif-containing (Trim) family. Depletion of maternal trim36
using antisense oligonucleotides results in ventralized embryos and reduced organizer
gene expression. We show that injection of wnt11
mRNA rescues this effect, suggesting that Trim36
functions upstream of Wnt/beta-catenin activation. We further find that vegetal microtubule
polymerization and cortical rotation are disrupted in trim36
-depleted embryos, in a manner dependent on Trim36
ubiquitin ligase activity. Additionally, these embryos can be rescued by tipping the eggs 90 degrees relative to the animal-vegetal axis. Taken together, our results suggest a role for Trim36
in controlling the stability of proteins regulating microtubule
polymerization during cortical rotation, and subsequently axis formation.
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Fig. 1. Expression of trim36 in Xenopus. (A) RT-PCR for trim36 at different Nieuwkoop and Faber (NF) stages; `-RT' was processed in the absence of reverse transcriptase. ornithine decarboxylase (odc) was included as a loading control. (B-E) Whole-mount in situs using antisense trim36 probe. (B) Stage I oocytes, (C) stage VI (left) and stage IV (right) oocytes, (D) 4-cell embryos (vegetal view) and (E) neurula embryos (dorsal/anterior view). (F-H) In situs for trim36 on sections; insets in F and G are low-power views, inset in H is trim36 sense probe. (F) Stage 7 sagittal section, (G) stage 11 sagittal section and (H) adult testis.
Fig. 3. Dorsal marker expression in trim36-depleted embryos. Dorsal genes and β-catenin protein in control (A,C,E,G) and trim36-depleted (B,D,F,H) embryos. (A,B) In situs for eomes in stage 12 embryos. Dorsal view, anterior is to the top. Arrow in A indicates eomes expression in the anterior notochord. (C,D) In situs for myod in stage 12 embryos. Dorsal view, anterior is to the top. (E,F) In situs for nr3 in stage 9.5 embryos. Vegetal view, dorsal is to the top. (G,H) Immunostaining for β-catenin in stage 8 embryos. Animal pole view of cleared embryos, dorsal is to the right. Arrow in G indicates nuclearβ -catenin. (I) Quantitative real-time PCR of dorsal (sia and nr3) and ventral (szl) markers in control (Un) and trim36-depleted (trim36-) embryos at stage 10.5.
Fig. 5. trim36-depleted embryos are rescued by wnt11 mRNA.trim36 was depleted by the injection of antisense oligos into oocytes and 50 pg wnt11 mRNA was injected prior to fertilization by the host-transfer method. gsc (A,C,E) and nr3 (B,D,F) expression in stage 10.5 embryos; (A,B) uninjected; (C,D) trim36-depleted (trim36-); and (E,F) trim36-depleted + 50 pg wnt11 mRNA (trim36-; + wnt11). (G) Histogram showing the distribution of phenotypes (see key) in uninjected embryos, trim-36-depleted embryos and trim36-depleted embryos rescued by wnt11 injection (from two experiments). (H,I) Quantitative real-time PCR analysis of nr3 expression in control, depleted and rescued embyros injected with 50 pg wnt11 (H) or 1.0 ng dngsk3b (I) (green bars).
pygopus Encodes a nuclear protein essential for wingless/Wnt signaling.