APOBEC2, a selective inhibitor of TGFβ signaling, regulates left-right axis specification during early embryogenesis.
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.
PubMed ID: 20880495
PMC ID: PMC3038383
Article link: Dev Biol.
Grant support: R01 HD032105 NICHD NIH HHS , R03 HD057334 NICHD NIH HHS , R03 HD057334-01A2 NICHD NIH HHS
Genes referenced: apobec2 cda dand5 gdf1 gdf3 lefty myod1 nodal nodal1 odc1 t
Morpholinos referenced: apobec2 MO1 apobec2 MO2
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|Fig. 1. APOBEC2 is a target of TGFβ signaling coexpressed with derrière in Xenopus embryos. (A) Strategy for identification of genes regulated by derrière. Posterior dorsal fragments from wild-type and derrière -MO (Der-MO)-injected embryos were isolated at stage 18. (B) TGFβ 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 (30 pg) and derrière (100 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). (E–M) Comparative expression of xA2 and derrière. In situ hybridization for xA2 (E–I) and derrière (J–M) expression. (E, J) Stage 10 vegetal–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 derrière occurs at stage 10 (dorsal marginal), stage 11 (involuted mesoderm), and stage 16 (paraxial mesoderm). (N–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. (Q–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 0.3 mm, and in panel N, 0.1 mm.|
|Fig. 3. xA2 depletion inhibits Xnr1 and increases derrière expression in posterior mesoderm. (A–H) In situ hybridization for derrière (stage 18, A) and Xnr1 (stage 22, B), and double in situ hybridization for derrière (C, E, G), or Xnr1 (D, F, H), purple, and LacZ RNA coinjected as tracer (red). Panels C–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 (n = 36; H). Expression of derrière was expanded in anterior direction on the side injected with xA2 MO (n = 33, arrowheads in G). The scale bar in panel C represents 0.1 mm. (I) xA2 depletion synergizes with low levels of overexpressed Xnr1 RNA. RT-PCR of posterior poles injected bilaterally with xA2 MO (10 ng), Xnr1 RNA (1 pg), or both. The combination increased derrière 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). (C–E) Left-side depletion of xA2 protein randomizes the left–right axis in Xenopus. Embryos injected on the left side with 10 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. (F–G) Depletion of zA2 protein prevents heart looping in zebrafish. In situ hybridization with cmlc2 antisense probe for heart muscle on 36–40 hpf embryos. Arrows indicate ventricular looping. (H–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 (L–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 GRVP16SMAD2δ3 RNA (25 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 0.3 mm.|