Liu X et al. (2017), Dual roles of Akirin2 protein during Xenopus ne...

XB-ART-53120

J Biol Chem 2017 Apr 07;29214:5676-5684. doi: 10.1074/jbc.M117.777110.

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Dual roles of Akirin2 protein during Xenopus neural development.

Liu X , Xia Y , Tang J , Ma L , Li C , Ma P , Mao B .

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To ensure correct spatial and temporal patterning, embryos must maintain pluripotent cell populations and control when cells undergo commitment. The newly identified nucleoprotein Akirin has been shown to modulate the innate immune response through epigenetic regulation and to play important roles in other physiological processes, but its role in neural development remains unknown. Here we show that Akirin2 is required for neural development in Xenopus and that knockdown of Akirin2 expands the expression of the neural progenitor marker Sox2 and inhibits expression of the differentiated neuronal marker N-tubulin. Akirin2 acts antagonistically to Geminin, thus regulating Sox2 expression, and maintains the neural precursor state by participating in the Brg1/Brm-associated factor (BAF) complex mediated by BAF53a. Additionally, Akirin2 also modulates N-tubulin expression by acting upstream of neuronal differentiation 1 (NeuroD) and in parallel with neurogenin-related 1 (Ngnr1) during terminal neuronal differentiation. Thus, our results reveal a novel model in which Akirin2 precisely coordinates and temporally controls Xenopus neural development.

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Species referenced: Xenopus laevis
Genes referenced: actl6a akirin1 akirin2 banf1 gmnn neurod1 neurog1 neurog2 nkx6-2 sox2 tubb2b
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	FIGURE 1. Expression pattern of Akirin2 during early Xenopus development. (A) RT-PCR analysis of Akirin2 expression at different stages (St.0 to St.30). H4 is an internal reference. NC, negative control without reverse transcriptase in the RT reaction. (B-I) Whole-mount in situ hybridization of Akirin2. The Akirin2 transcript is detected in the animal pole at St.3 (B) and St.6.5 (C) (B and C, lateral view, animal pole to the top). At St.15, Akirin2 is expressed at the neural plate (np) (D, dorsal view). At late neurulation, Akirin2 is most abundant in branchial arches (br) and the optic vesicle (opt) (E, dorsal view; F, lateral view; G, frontal view). At the tailbud stage, Akirin2 is mainly expressed in the branchial arches (br), optic vesicle (opt), otic vesicle (ot), pronephric tubule (pt) and neural tube (nt) (H and I, lateral view). (J, K) Transverse sections of stage 30 embryo.
	FIGURE 2. Akirin2 knockdown disrupts neural development in Xenopus. (A-C) Morphology of tadpoles (stage 37/38) injected with standard MO (25 ng), Akirin2 MO (25 ng), Akirin2 MO (25 ng) and Akirin2 mRNA (0.6 ng). Embryos were injected in both blastomeres at the two-cell stage and raised to tadpole stage. (D-I) Akirin2 MO (25 ng) with or without Akirin2 mRNA (0.6 ng) was injected into one cell of four-cell-stage embryos, and whole-mount in situ hybridization with probes of Sox2, Nkx6.2 and N-tubulin was processed at St.14-16. LacZ mRNA was co-injected to trace the injected sides (stained red on the left sides). (J) Quantification of the effects of the injection of Akirin2 MO or co-injection of Akirin2 MO and Akirin2 mRNA on the expression of Sox2, Nkx6.2 and N-tubulin as shown in (D-I)
	FIGURE 3. Akirin2 interacts with BAF53a. (A, B) Co-IP assays of exogenous Akirin2 and BAF53a proteins in HEK293 cells. The cells were transfected with the indicated plasmids, and the cell extracts were immunoprecipitated and immunoblotted with the indicated antibodies. (C) A schematic map of the Akirin1-Akirin2 fusion truncations with amino acid numbers indicated. (D) Interactions of various Akirin1-Akirin2 fusion constructs with BAF53a in co-IP experiments. WCL, whole cell lysate; IB, immunoblot; IP, immunoprecipitation; FL, full-length.
	FIGURE 4. BAF53a and Akirin2 involve in Xenopus neural development. (A-D) Co-injection with BAF53a mRNA (1.0 ng) rescues the expansion of Sox2 and Nkx6.2 in the Akirin2 morphants. LacZ mRNA was co-injected to trace the injected sides (stained red on the left sides). (E) Quantification of the effects of the injection of Akirin2 MO or co-injection of Akirin2 MO and BAF53a mRNA on the expression of Sox2, Nkx6.2, as shown in (A-D). (F-K) Co-injection with BAF53a mRNA (1.0 ng) or Akirin2 mRNA (1.0 ng) rescues the expansion of Sox2 and Nkx6.2 in the BAF53a morphants. LacZ mRNA was co-injected to trace the injected sides (stained red on the left sides). (L) Quantification of the effects of the injection of BAF53a MO or co-injection of BAF53a MO and BAF53a mRNA or Akirin2 mRNA on the expression of Sox2, Nkx6.2 as shown in (F-K).
	FIGURE 5. Akirin2 and Geminin antagonistically regulate Sox2 expression. (A, B) Co-IP assays of exogenous Akirin2 and Geminin proteins in HEK293 cells. The cells were transfected with the indicated plasmids, and the cell extracts were immunoprecipitated and detected with the indicated antibodies. (C) Interactions of various Akirin1-Akirin2 fusion constructs with Geminin in co-IP experiments. (D) A schematic map of the Geminin truncations with amino acid numbers indicated. (E) Interactions of various Geminin truncation constructs with Akirin2 in co-IP experiments. (F) BAF53a enhances the interaction of Akirin2 and Geminin. (G-K) Co-injection with either Akirin2 or BAF53a mRNA rescues the expansion of Sox2 in the Geminin misexpression embryos, whereas this effect was reversed by co-injection of BAF53a MO or Akirin2 MO. LacZ mRNA was co-injected to trace the injected sides (stained red on the left sides). (L) Quantification of the effect of the injection of mRNA or co-injection of MO as shown in (G-J). WCL, whole cell lysate; IB, immunoblot; IP, immunoprecipitation; FL, full-length.
	FIGURE 6. Akirin2 functions in parallel with Ngnr1 during early neurongenesis in Xenopus. (A, B) Co-injection with Akirin2 mRNA (0.6 ng) rescues the reduction of NeuroD in the Akirin2 morphants. LacZ mRNA was co-injected to trace the injected sides (stained red on the left sides). (C-F) Co-injection with Akirin2 MO (0.6 ng) inhibits the activation of NeuroD (C, D) and N-tubulin (E, F) in the Ngnr1 mixexpression embryos. LacZ mRNA was co-injected to trace the injected sides (stained red on the left sides). (G, H) Co-injection with Akirin2 MO (0.6 ng) cannot inhibit the activation of N-tubulin in the NeuroD mixexpression embryos. LacZ mRNA was co-injected to trace the injected sides (stained red on the left sides). (I) Quantification of the effects of the injection of A2 MO, Ngnr1 mRNA, NeuroD mRNA or co-injection of A2 MO mRNA and Akirin2/Ngnr1/NeuroD mRNA on the expression of NeuroD or N-tubulin as shown in (A-H)
	FIGURE 7. Proposed model for the coordination and temporal control of Xenopus neural development by Akirin2. In neural progenitor cells, Akirin2 acts antagonistically to Geminin in regulating Sox2 expression, and it maintain the neural precursor state after being recruited into BAF by BAF53a. Akirin2 also modulates N-tubulin expression by acting upstream of NeuroD, functioning in parallel with Ngnr1 during terminal neuronal differentiation when BAF53a and Geminin disappear.
	akirin2 (akirin 2) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 3, animal pole view.
	akirin2 (akirin 2) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 7, animal pole up.
	akirin2 (akirin 2) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 15, dorsal view, anterior left.
	akirin2 (akirin 2) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 21, dorsal view, anterior left.
	akirin2 (akirin 2) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 21, F: lateral view, anterior left, dorsal up; G: anterior view, dorsal up.

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