Yoon J et al. (2011), xCITED2 Induces Neural Genes in Animal Cap Expl...

XB-ART-44512

Exp Neurobiol 2011 Sep 01;203:123-9. doi: 10.5607/en.2011.20.3.123.

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xCITED2 Induces Neural Genes in Animal Cap Explants of Xenopus Embryos.

Yoon J , Kim JH , Lee OJ , Yu SB , Kim JI , Kim SC , Park JB , Lee JY , Kim J .

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Neural tissue is arisen from presumptive ectoderm via inhibition of bone morphogenetic protein (BMP) signaling during Xenopus early development. Previous studies demonstrate that ectopic expression of dominant negative BMP4 receptor (DNBR) produces neural tissue in animal cap explants (AC) and also increases the expression level of various genes involved in neurogenesis. To investigate detail mechanism of neurogenesis in transcriptional level, we analyzed RNAs increased by DNBR using total RNA sequencing analysis and identified several candidate genes. Among them, xCITED2 (Xenopus CBP/p300-interacting transcription activator) was induced 4.6 fold by DNBR and preferentially expressed in neural tissues at tadpole stage. Ectopic expression of xCITED2 induced anterior neural genes without mesoderm induction and reduced BMP downstream genes, an eye specific marker and posterior neural marker. Taken together, these results suggest that xCITED2 may have a role in the differentiation of anterior neural tissue during Xenopus early development.

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Species referenced: Xenopus
Genes referenced: a2m actl6a bmp4 chrd cited2 crebbp egr2 ep300 gata2 hoxb9 krt12.4 mixer ncam1 neurod1 odc1 otx2 rax tbxt ventx1 zic3

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	Fig. 1. Blocking of BMP4 signaling induced the expression of xCITED2 in animal cap explants of Xenopus embryos. (A) Animal caps were dissected from stage 8~9 embryos that had been injected at the one cell stage or two-cell stage with 2 ng of DNBR RNAs or animal cap explants dissected from un-injected embryos were treated with activin (50 ng/ml). Animal caps were harvested at stage 24 and RT-PCR analysis was performed. Pan-neural marker: NCAM, mesoderm marker: Actin, epidermis marker: XK81. ODC serves as mRNA loading control. (B) Alignment of xenopus xCITED2 with human and mouse homologues of CITED2. Yellow indicated that Amino-acids which are conserved between xenopus CITED2 (gene bank number: NP_001088289.1) and other members (human: NP_006070.2, mouse: NP_034958.2). All family members contain transcription activating domain (CR-2 domain) in C-terminal region. This diagram was generated by the VectorNTI 8.0.
	Fig. 2. Temporal and spatial expression pattern of xCITED2 in Xenopus development. (A)Temporal Expression pattern of xCITED2 was analyzed using RT-PCR at various stages as indicated. ODC serves as loading control. xCITED2 was expressed from Oocyte and maintained until tail-bud stages. (B) Spatial expression pattern of xCITE2 was also analyzed by RT-PCR with dissected parts as indicated from stage 28 embryos.
	Fig. 3. xCITED2 induced neurogenesis in Xenopus Animal cap. (A) Expression level of HA-xCITED2 was confirmed by western-blot analysis. 1 ng of HA-xCITED2 RNAs were injected at 1 cell stage embryo and harvested at stage 12. Immuno-bloting was performed using anti-HA probe. (B) 1 ng of xCITED2 RNAs were injected at 1 cell stage and dissected animal cap at stage 8 and incubated in animal cap media until stage 12 or stage 24 (C). RT-PCR was performed to analyze various genes expression. ODC was used as loading control, ventral marker: Xvent1/2, GATA2, PV.1, endoderm marker: mixer and Edd, mesoderm marker: Xbra, Actin, organizer marker: chordin and neural marker: Zic3, NCAM, Krox20.
	Fig. 4. xCITED2 induced anterior neural gene. (A) 1 ng of xCITED2 RNAs were injected alone or co-injected with 1 ng of DNBR RNAs at 1 cell stage and dissected animal cap at stage 8 and incubated in animal cap media containing activin (50 ng/ml) or none as indicated until stage 24. RT-PCR was performed to analyze various genes expression. Pan-Neural marker: NCAM, NeuroD and Xneurogenine-1, anterior neural marker: OTX2, posterior neural marker: HoxB9, eye specific marker: Rx1. ODC was used as loading control. (B) The lines represent the angle of sectioning for a and b. Transverse section through xCITED2 injected embryo or control embryo.

References [+] :

Andrews, Isolation and expression of a novel member of the CITED family. 2000, Pubmed