Vegetally localized Xenopus trim36 regulates cortical rotation and dorsal axis formation

Abstract

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.

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. 2.

Depletion of maternal trim36. (A) Quantitative real-time PCR of trim36 expression in oligo-injected oocytes. Samples were normalized to odc levels and displayed as a percentage relative to the uninjected oocyte (Un oo) sample. Oocytes were injected with 3.0 or 4.0 ng of trim36-3 oligo. (B) Phenotype of an uninjected stage 24 embryo. (C) Phenotypes of sibling trim36-depleted embryos. (D) In situ hybridization of sizzled (szl) in an uninjected control embryo. (E) szl expression in a trim36-depleted embyro. (F) H&E-stained section of a control embryo, with the neural tube (n.t.) and notochord (n.o.) indicated. (G) H&E-stained section of a trim36-depleted embryo; notochord and neural tube are absent, and a somite muscle mass persists in the midline (arrow). (H,I) Notochord marker Tor70 (green) and somite marker 12/101 (red) in control (H) and trim36-depleted (I) embryos. (J) Histogram showing the distribution of phenotypes (see key) in trim36-depleted embryos (from two experiments).

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 nuclear β-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. 4.

Specificity of trim36 depletion. (A-C) Phenotypes of representative control uninjected (Un, A), trim36-depleted (trim36-, B) and rescued (trim36-; + trim36 RNA, C) embryos. (D) Quantitative real-time PCR of dorsal (sia and nr3) and ventral (szl) markers in rescue experiments (green bars). N.D., not determined.

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).

Fig. 6.

Trim36 regulates vegetal microtuble formation and cortical rotation. (A-C) Immunostaining of microtubules at 80 minutes post fertilization in control (A, uninjected), trim36-depleted (B, trim36-) and β-catenin-depleted (C, βcat-) eggs. (D-F) Immunostaining of cytokeratin at 80 minutes post fertilization in control (D, uninjected), trim36-depleted (E, trim36-) and β-catenin-depleted (F, βcat-) eggs. (G-I) Immunostaining of microtubules at 80 minutes post fertilization in control (G, uninjected), trim36-depleted (H, trim36-) and rescued (I, trim36-; + trim36 RNA) eggs. Vegetal views are shown in A-I, viewed with a 63× objective. (J-O) Representative phenotypes of uninjected (J,K), trim36-depleted (trim36-; L,M) and β-catenin-depleted (βcat-; N,O) embryos. (J,L,N) Normal orientation, (K,M,O) tilted 90°. (P-S) Frames from time-lapse movies of cortical rotation in DiOC6(3)-stained eggs. Controls (P,Q) and trim36-depleted (R,S) embryos at time 0 (t0) and +10 minutes (t+10′). Arrows indicate the starting points of indicated germ-plasm islands, arrowheads indicate the final position of the same islands. Images were taken with a 10× objective.

Fig. 7.

Trim36 ubiquitin ligase activity is required to rescue the dorsal axis in trim36-depleted embryos. (A) Auto-ubiquitylation of Trim36. Immunoblotting of Trim36 immune complexes from lysates of control uninjected embryos (Un), trim36-injected embryos (1 ng trim36), HA-ubiquitin-injected embryos (1.0 ng HA-ub) or embryos injected with both trim36- and HA-ub. Top panel shows blotting using anti-HA, lower panel shows blotting using Trim36 antisera. (B) Trim36 CA/HA is deficient in auto-ubiquitylation. Immunoprecipitation and blotting as in A. (C-F) Immunostaining of microtubules at 80 minutes post fertilization. (C) Uninjected eggs (Un), (D) trim36-depleted eggs (trim36-) and (E,F) trim36-depleted embryos injected with either 200 pg wild-type trim36 RNA (E) or 200 pg trim36 CA/HA (F). (G) Quantitative real-time PCR analysis of nr3 expression in control embryos, trim-36-depleted embryos and trim36-depleted embyros injected with wild-type trim36 RNA or with trim36 CA/HA RNA. Embryos in G are siblings of the embryos in C-F.