January 1, 2006;
XBP1 forms a regulatory loop with BMP-4 and suppresses mesodermal and neural differentiation in Xenopus embryos.
The active form of the Xenopus X-box binding protein 1 (xXBP1
) partially synergizes and partially antagonizes with BMP-4 signaling. xXBP1
overexpression inhibits mesoderm
differentiation and formation of neural tissues. A functional knockdown promotes differentiation of lateral
and dorsal mesoderm
but not of ventral mesoderm
and of neuroectoderm
. We show that the active form of xXBP1
and early neurula
stage embryos is generated by removal of exon 4 and not by an endoribonuclease activity in the endoplasmic reticulum. The N-terminal region of xXBP1
which contains the basic leucine-zipper also contains a nuclear localization signal and both, the N-terminal as well as the C-terminal regions are required for xXBP1
function. The effects of xXBP1
are in part correlated to a regulatory loop between xXBP1
and BMP-4. xXBP1
and BMP-4 stimulate mutually the transcription of each other, but xXBP1
inhibits the BMP-4 target gene, Xvent-2. Both, in vitro and in vivo assays demonstrate that xXBP1
interacts with BMP-4 and Xvent-2B promoters. GST-pulldown assays reveal that xXBP1
can interact with c-Jun
, the transcriptional co-activator p300
and with the BMP-4 responsive Smad1
. On the other hand
also binds to the inhibitory Smads, Smad6
, that can act as transcriptional co-repressors. Based on these data, we conclude that xXBP1
might function as an inhibitor of mesodermal and neural tissue
formation by acting either as transcriptional activator or as repressor. This dual activity depends upon binding of co-factors being involved in the formation of distinct transcription complexes.
[+] show captions
Fig. 4. Regulatory circuit between xXBP1, BMP-4, Xvent-2 and ADMP as revealed by whole mount in situ hybridization (Aã¢â â K) and RT-PCR (L and M). (A) Expression of BMP-4 in a control embryo. (B) Ectopic expression of BMP-4 induced by dorsal injection of xXBP1. (C) Expression of xXBP1 in a control embryo. (D) Transcription of xXBP1 is enhanced in response to dorsal injection of BMP-4 mRNA. (E) Expression of Xvent-2 in a control embryo. Transcription of Xvent-2 is inhibited by either dorsal injection (F) or ventral injection (G) of xXBP1 mRNA. (H) Expression of ADMP in a control embryo. (I) Dorsal overexpression of xXBP1 depletes ADMP transcripts. (K) Dorsal overexpression of ADMP upregulates xXBP1 as compared to control (J). Dorsal side of each embryo is up. (L) RT-PCR analysis on the transcription of BMP-4 and Xvent-2 at stage 11 in response to overexpression of xXBP1 at different doses. (M) Transcription of xXBP1 at stage 11 in response to BMP-4 injection into two dorsal blastomeres at 4-cell stage.
Fig. 2. Functional knockdown of xXBP1 with antisense morpholino oligo caused increase of early mesoderm and promoted dorsal and paraxial mesoderm differentiation. (A) Antisense morpholino oligo against xXBP1 (MOxXBP) specifically inhibits xXBP1 translation in an in vitro assay. Lane 1, no plasmid added; 2, pBK-CMV-xXBP1 plasmid only; 3, pBK-CMV-xXBP1 plasmid and MOxXBP; 4, pBK-CMV-xXBP1 plasmid and control morpholino oligo (ctrlMO). (B) Embryos injected with 50 ng of MOxXBP into four blastomeres at 4-cell stage. (C) Embryos injected with 50 ng of ctrlMO. (D) Embryos injected with 50 ng of MOxXBP and 200 pg of xXBP1 mRNA. (E) Embryos injected with 50 pg of BMP-4 mRNA. (F) Embryos injected with 50 pg of BMP-4 mRNA and 50 ng of MOxXBP. (G) Embryos injected with 40 pg of ADMP mRNA. (H) Embryos injected 40 pg of ADMP mRNA and 50 ng of MOxXBP. (I) Embryos injected with 50 ng of ctrlMO or MOxXBP were collected at stage 11, 18 and 30, respectively, and examined for expression of marker genes with RT-PCR. RT-: control reaction without reverse transcriptase as a control for the absence of endogenous DNA contamination; ODC: loading control. These are the same as in the following figures.
Fig. 6. Overexpression of xXBP1 deletion mutants caused different phenotypic changes. (A) Diagram of deletion mutants used for overexpression. (B) Embryos injected with 50 pg of MT-xXBPDC mRNA into four blastomeres at 4-cell stage and (C) a control embryo at stage 32. (D) Embryos injected with 30 pg of MT-xXBPDC and (E) a control embryo at stage 46. (F) Embryos at stage 32 injected with 2.5 ng of MT-xXBPDN mRNA. (G) Embryos of stage 32 injected with 2.5 ng of NLS-MT-xXBPDN mRNA into four blastomeres at 4-cell stage.