Vonica A et al. (2011), APOBEC2, a selective inhibitor of TGFβ signalin...

XB-ART-42121

Dev Biol 2011 Feb 01;3501:13-23. doi: 10.1016/j.ydbio.2010.09.016.

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APOBEC2, a selective inhibitor of TGFβ signaling, regulates left-right axis specification during early embryogenesis.

Vonica A , Rosa A , Arduini BL , Brivanlou AH .

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The specification of left-right asymmetry is an evolutionarily conserved developmental process in vertebrates. The interplay between two TGFβ ligands, Derrière/GDF1 and Xnr1/Nodal, together with inhibitors such as Lefty and Coco/Cerl2, have been shown to provide the signals that lead to the establishment of laterality. However, molecular events leading to and following these signals remain mostly unknown. We find that APOBEC2, a member of the cytidine deaminase family of DNA/RNA editing enzymes, is induced by TGFβ signaling, and that its activity is necessary to specify the left-right axis in Xenopus and zebrafish embryos. Surprisingly, we find that APOBEC2 selectively inhibits Derrière, but not Xnr1, signaling. The inhibitory effect is conserved, as APOBEC2 blocks TGFβ signaling, and promotes muscle differentiation, in a mammalian myoblastic cell line. This demonstrates for the first time that a putative RNA/DNA editing enzyme regulates TGFβ signaling and plays a major role in development.

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Species referenced: Xenopus laevis
Genes referenced: apobec2 cda dand5 gdf1 gdf3 h2bc21 lefty1 myod1 nodal nodal1 nodal2 nodal5.4 odc1 pnma2 sia1 sia2 tbxt wnt8a
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	Fig. 1. APOBEC2 is a target of TGFÎ² signaling coexpressed with derriÃ¨re in Xenopus embryos. (A) Strategy for identification of genes regulated by derriÃ¨re. Posterior dorsal fragments from wild-type and derriÃ¨re -MO (Der-MO)-injected embryos were isolated at stage 18. (B) TGFÎ² signaling and xA2 expression. Overexpression of a dominant negative type 1 receptor (DN ALK4) reduces expression of marginal xA2 in stage 10 whole embryos (left panel). Overexpressed Xnr1 (30Â pg) and derriÃ¨re (100Â pg) RNA induce xA2 expression in stage 10 (central panel) and 18 (right side panel) animal caps. RT-PCR for xA2, MyoD, and Brachyury as markers of mesoderm induction, and ODC as loading control. (C) Timing of xA2 expression. RT-PCR of embryos collected at the indicated developmental stages. (D) Spatial expression of xA2 in stage 10 embryos. RT-PCR of embryonic explants (VMZ: ventral marginal zone; DMZ: dorsal marginal zone). (Eâ€“M) Comparative expression of xA2 and derriÃ¨re. In situ hybridization for xA2 (Eâ€“I) and derriÃ¨re (Jâ€“M) expression. (E, J) Stage 10 vegetalâ€“dorsal views, arrowhead indicates the forming dorsal lip; (F, K) stage 11 dorso-ventral sections (dorsal to the right). Arrowheads indicate recently involuted mesoderm; (G, L) dorsal views (anterior up). Arrowheads indicate the blastopore; (H, M) stage 16 transversal sections, posterior fragments (dorsal is up). (I) Stage 32 lateral view. Overlap between xA2 and derriÃ¨re occurs at stage 10 (dorsal marginal), stage 11 (involuted mesoderm), and stage 16 (paraxial mesoderm). (Nâ€“R) Expression of zebrafish A2 (zA2). (N, O, P) Seventy-five percent epiboly, in (N) lateral view (dorsal to the right) and (O) dorsal views. The arrowheads in panels N and O indicate the shield. (Qâ€“R) Fourteen somite stage embryo. The inset in panel O (twofold magnification) shows shield cells with nuclear stain. (Q) Lateral view, anterior to the left, (R) dorsal view (anterior up). (P) Embryo stained with the sense probe as negative control. The scale bars in panel E indicates 0.3Â mm, and in panel N, 0.1Â mm.
	Fig. 3. xA2 depletion inhibits Xnr1 and increases derriÃ¨re expression in posterior mesoderm. (Aâ€“H) In situ hybridization for derriÃ¨re (stage 18, A) and Xnr1 (stage 22, B), and double in situ hybridization for derriÃ¨re (C, E, G), or Xnr1 (D, F, H), purple, and LacZ RNA co-injected as tracer (red). Panels Câ€“H are internal views of posterior dorsal fragments, anterior side up, of stage 18 embryos. The left side of each panel is the right side of each embryo. Xnr1 expression was inhibited by xA2 MO (nÂ =Â 36; H). Expression of derriÃ¨re was expanded in anterior direction on the side injected with xA2 MO (nÂ =Â 33, arrowheads in G). The scale bar in panel C represents 0.1Â mm. (I) xA2 depletion synergizes with low levels of overexpressed Xnr1 RNA. RT-PCR of posterior poles injected bilaterally with xA2 MO (10Â ng), Xnr1 RNA (1Â pg), or both. The combination increased derriÃ¨re and MyoD, but not Xbra expression.
	Fig. 2. Effect of APOBEC2 protein depletion in Xenopus and zebrafish. (A, B) Inhibition of in vitro translation by xA2 MO (A) and zA2 MO (B). (Câ€“E) Left-side depletion of xA2 protein randomizes the leftâ€“right axis in Xenopus. Embryos injected on the left side with 10Â ng xA2 MO were stained for light meromyosin at stage 46. (C) Control embryo; (D) right-side MO injection normal embryo; (E) left-side MO injection, inverted heart and abnormally folded intestine. Arrows indicate the direction of the heart outflow tract and intestinal looping. (Fâ€“G) Depletion of zA2 protein prevents heart looping in zebrafish. In situ hybridization with cmlc2 antisense probe for heart muscle on 36â€“40 hpf embryos. Arrows indicate ventricular looping. (Hâ€“Q) xA2 depletion blocks the left-side nodal signal. In situ hybridization for Xnr1 (H, I, L, M, P, Q), and Lefty (J, K, N, O) in purple, and injected LacZ RNA as tracer (Lâ€“Q) in red. Wild-type expression of Xnr1 (H) and Lefty (J) in the left lateral plate mesoderm was inhibited by injection of xA2 MO in the left paraxial mesoderm (L, N). Left-side expression of Xnr1 was rescued by coinjection of GRVP16SMAD2Î´3 RNA (25Â pg RNA, induced at stage 16; P). All views are lateral, except in panel O (dorsal), anterior to the left. Embryos are stage 23 (Xnr1) and stage 24 (Lefty). The scale bar in panel H represents 0.3Â mm.
	Fig. 4. APOBEC2 is a TGFÎ² inhibitor in Xenopus. (Aâ€“F) xA2 prevents cap elongation induced by derriÃ¨re but not Xnr1. RNAs were injected in the animal poles of 2 cell stage embryos (xA2 2 ng, derriÃ¨re 200 pg, Xnr1 30 pg). Caps were explanted at stage 10 and cultured to stage 18. (G, H) Expression of xA2 RNA represses derriÃ¨re-, but not Xnr1-dependent gene induction. RT-PCR of early gastrula (G) and late neurula (H) stage animal caps injected with derriÃ¨re RNA (200 pg), or Xnr1 RNA (20 pg), with or without xA2 RNA (2 ng). (I) xA2 depletion increased mesoderm induction by derriÃ¨re RNA. RT-PCR for mesodermal genes (Xnr2, Xbra and MyoD), with ODC as loading control, in stage 18 animal caps injected with derriÃ¨re RNA (100 pg) and xA2 MO (10 ng). (J) xA2 specifically inhibits DerriÃ¨re-induced transcription. Transcription assays for the Activin/nodal reporter ARE. Transcriptional inhibition by xA2 was statistically significant with derriÃ¨re (200 pg RNA), but not with Xnr1 (20 pg RNA). (K) An intact putative deaminase domain is required for the inhibitory activity of xA2. Wild-type, but not the triple mutant xA2 (Mut xA2, 2 ng RNA), inhibited transcription induced by derriÃ¨re (200 pg RNA). Only the difference in transcriptional activity induced by DerriÃ¨re alone vs. DerriÃ¨re + xA2 wild-type was statistically significant (P < 0.001). (L) xA2 blocks the C-terminal phosphorylation of endogenous Smad2/3 induced by derriÃ¨re. Western blot for total Smad2/3 and C-terminally phosphorylated Smad2/3 in stage 10.5 animal caps. No effect was seen on total Smad2/3 protein levels.
	Fig. 5. mAPOBEC2 inhibits TGFÎ² signaling in C2C12 myoblasts. (A) RT-PCR analysis of mA2 and the muscle differentiation marker Skeletal-Î±-Actin mRNA levels during C2C12 differentiation. (B) Real-time qPCR analysis of the mRNA levels of A2 and the indicated muscle markers in C2C12 cells transfected either with control (siC) or anti-A2 (siA2) siRNAs and differentiated for 2 days in DM. Numbers above the histogram bars indicate the relative decrease in mRNA levels. (C, D) A2, but not AID, inhibits TGFÎ² signaling in C2C12 cells. Cells were transfected with the p3TP-lux reporter in combination with an empty vector (Vector) or an A2 (C) or AID (D) expressing vector. After transfection, cells were cultured with or without 5 ng/ml recombinant TGF-Î²1 for 24 h before harvesting for luciferase assay. Numbers above the histogram bars indicate the fold reduction in luciferase activity.
	Supplementary Figure 1. Sequence and expression of xA2. (A) Sequence of Xenopus, mouse, and zebrafish APOBEC2, and human APOBEC1. The active site ofAPOBEC1 is boxed. Active center residues, where H100, C130 and C134 coordinate Zn, are labeled with asterisks, and residues mutated in MutXA2 are indicated by red arrowheads. (B, C) xA2 expression is decreased in stage 18 posterior poles (B, 2 and 5 ng Der MO) and stage 12 dorsal halves (C, 5 ng Der MO) of Xenopus embryos depleted of Derriere protein. RT-PCR with ODC as loading control.
	Supplementary Figure 2. (Aâ€“E) Blocking reagents for Xenopus, zebrafish, and mouse apobec2. (A) Sequence of the two morpholino oligonucleotides for Xenopus A2 depletion. (B) Sequence of the morpholino oligonucleotide for zebrafish A2 depletion. (C, D) Unilateral PM injection of XA2 MO (10 ng) does not affect antero-posterior development. Dorsal views of control (C) and XA2 MO injection in left PM (D). (E) Effect of different siRNAs (1â€“3) against mouse A2 mRNA on expression of A2 protein levels in C2C12 cells. siA2.2 was chosen for the experiments in Fig. 5. DM: differentiation medium, GM: growth medium. Lanes 1â€“5: endogenous A2. Lane 7: overexpressed A2 (CMV-APOBEC2 transfection). Western blot for mouse A2 and tubulin. (Fâ€“I) Targeted injections for PM and LPM. Embryos injected at the 4 cell stage with fluorescent control MO (lateral subequatorial in the dorsal left blastomere, G) and Cherry-H2B (lateral subequatorial in the ventral left blastomere, H) were fixed at stage 20. Dorsal views, posterior is down. The same embryo in visible (F), green UV channel (G), red UV channel (H), and overlap of green and red (I).
	Supplementary Figure 3. A. xA2 MO coinjected with Xnr1 RNA (1 pg) in left PM. B., C. Xnr1 RNA does not rescue expression of Lefty in the left LPM of xA2-depleted embryos. Double in situ hybridization for Lefty (purple) and injected LacZ RNA (red). Lefty is absent in the left LPM but increased in the midline. B is a left lateral view, C is a dorsal view. Anterior is to the left. D-I. Xnr1 RNA and xA2 MO synergize to increase MyoD expression at stage 18. Double In situ hybridization for MyoD (purple) and LacZ (red) as tracer. D., F., H are dorsal views, anterior to the left. E, G, I are posterior fragments of the same embryos sectioned transversally. xA2 MO alone increased MyoD expression on the injected side stained with Fast Red in addition to the markers stain (n = 25, D, E). Xnr1 RNA alone (1 pg) had no effect (n = 32, F, G), while the combination of RNA and MO significantly increased the lateral expansion of MyoD (n = 29, H, I) and produced a blastopore closure defect (H, right side). Bars in D, F, H indicate the plane of the transversal section. J. Depletion of xA2 protein reduces expression of Xnr1 in posterior mesoderm. RT-PCR for the indicated genes in dorsal posterior fragments of stage 18 embryos. 10 ng xA2MO were injected at the 4 cell stage and targeted at the paraxial mesoderm. K. Expression of the triple mutant xA2 and wild-type tagged xA2 proteins were equal. Embryos (stage 24) were injected with 4 ng RNA and collected at stage 10. L. xA2 does not inhibit the Wnt pathway. Embryos were injected with Siamois reporter gene and Wnt8 RNA (50 pg)+/- xA2 RNA (2ng). Statistical significance was calculated with Studentâ€™s t test. M. xA2 is an inhibitor specific for DerriÃ¨re. The ARE reporter gene (100 pg) was activated by coinjected derriÃ¨re (100 pg), Xnr2 (5 pg), Xnr4 (50 pg), Xnr5 (0.1 pg), or Activin (100pg) RNA, with or without xA2 RNA (2 ng) at the 4 cell stage and measured at stage 10.5. xA2 overexpression had a significant effect only on DerriÃ¨re activity (Studentâ€™s t test).

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