Exp Neurobiol 2011; 20(3): 123-129
Published online September 30, 2011
© The Korean Society for Brain and Neural Sciences
Jaeho Yoon1,#, Jung-Ho Kim1,#, Ok-Joo Lee2, Saet-Byeol Yu3, Jong-Il Kim3, Sung Chan Kim1, Jae-Bong Park1, Jae-Yong Lee1 and Jaebong Kim1*
1Department of Biochemistry, College of Medicine, 2Facial Skeleton Bio Institute, Hallym University, Chuncheon 200-702, 3Genomic Medicine Institute (GMI), Medical Research Center, Seoul National University, Seoul 110-799, Korea
Correspondence to: #These authors equally contributed to this work.
*To whom correspondence should be addressed.
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Neural tissue is arisen from presumptive ectoderm via inhibition of bone morphogenetic protein (BMP) signaling during
Keywords: xCITED2, DNBR, neurogenesis, Xenopus
Neural induction is a process of neural cell formation in ectoderm during embryogenesis (Kessler and Melton, 1994; Gould and Grainger, 1997; Hemmati-Brivanlou and Melton, 1997; Sasai and De Robertis, 1997; Munoz-Sanjuan and Brivanlou, 2002; De Robertis and Kuroda, 2004). In
The CITED protein (CBP/p300-interacting transcription activator) family has 4 subtypes, CITED1 called as MSG1, CITED2 (MGR1), CITED3 and CITED4 (MRG2) (Andrews et al., 2000). All family proteins have CR2 domain which is a highly conserved transcription activating domain (Shioda et al., 1997). Since CITED protein does not have DNA-binding motif, it has been studied as a transcriptional co-activator of CBP (Yahata et al., 2000). CITED1 increases transcriptional activity through interacting with CBP and SMAD4 but CITED2 does not have SMAD4 binding motif. CITED2 enhances transcription with other proteins such as Lhx2 which contains LIM domain (Glenn and Maurer, 1999). Previous studies addressed that CITED family proteins play a role in heart, liver development and anterior-posterior patterning (Goodman and Smolik, 2000). Although CITED2 protein has been studied minutely in mammalian cell, the role of CITED2 is not fully understood during
In this study, we found that
The xCITED2 ORF sequence is appeared in NCBI GenBank under the accession number NM_001094820. Open reading frame (ORF) was amplified by PCR using cDNA library of stage 12
All synthetic mRNAs used for microinjection were produced by
Total RNA was extracted from whole embryo or cultured animal explants with TRIzol reagent (Life Technologies, Inc.) following the manufacturer's instruction. RT-PCR was performed with a Superscript pre-amplification system (Invitrogen). PCR was performed as follows: first, a denaturation step of 94℃ for 5 minutes; second, 94℃ for 1 minute; third, each annealing temperature, for 1 minute ; fourth, 72℃ for 1 minute; fifth, repeat second, third and fourth steps 19-30 cycles of amplification was performed as described at the
Embryos were injected at the one cell stage with RNA constructs as described, and frozen at stage 11. They were then homogenized in lysis buffer (50 mM Tris [pH 7.4]), 150 mM NaCl, 1% NP-40, 0.25% sodium deoxycholate, 0.1% SDS, 50 mM NaF and 1 mM Na3VO4) containing of 1 mM PSMF, 15 mM glycerophosphate, 1 X proteinase inhibitor cocktail (Calbiochem). Cell lysates were cleared by centrifugation. Proper amount of lysate was boiled in sample buffer, and resolved by electrophoresis in 10% SDS-polyacrylamide gels. HA-tagged xCITED2 proteins were visualized after western blotting using rabbit polyclonal anti HA (Santa Cruz, sc-Y11) using ECL Western blotting detection reagents (Amersham Pharmacia Biotech).
We screened for the expressed RNA in DNBR-injected AC using total RNA sequencing (Jones and Woodland, 1987; Karsten et al., 2008). We analyzed about 22,000 contigs of the expressed sequences (bigger than 300 nucleotides) and found several uncharacterized genes induced by DNBR during early
In order to confirm the induction of xCITED2 in DNBR-treated sample, RT-PCR analysis was performed with AC obtained from embryos in various conditions. As shown Fig. 1A, the expression level of xCITED2 was increased in the samples treated with either DNBR or Activin at stage 24, but not in the untreated control AC. The control AC differentiated to epidermis (as shown by enhanced expression of XK81 in Fig. 1A). As we have known, either activin- or DNBR-treated samples differentiated to neural tissues (as shown by NCAM expression in Fig. 1A). We cloned and sequenced the full-length cDNA of xCITED2 into pCS2 vector using PCR amplification. xCITED2 encodes 225 amino acids and contains highly conserved CR-1 and CR-2 domain as indicated in Fig. 1B. CR-2 is a Glu/Asp-rich carboxy-terminal domain consisted of 55 amino acids and it has been known to acts as a transcription activating domain.
To investigate temporal expression of xCITED2 during
Together, DNBR induced expression of xCITED2 in AC and its spatial expression pattern in whole embryos suggest that xCITED2 has a role in neural development of
To perform gain of functional studies, we constructed HA-tagged xCITED2 because specific antibody for xCITED2 was commercially not available. Expression level of HA-xCITED2 was checked by western blotting using anti-HA probe (Fig. 3A). Over-expression of HA-xCITED2 induced a neural specific gene, Zic3 without organizer (chordin), mesoderm (Xbra) and endoderm (Mixer) gene induction in AC, but expression levels of BMP downstream genes (PV.1, Xvent1 and Xvent2) were reduced at stage 12. Consistently, neural specific genes (NCAM and Krox20) were expressed without either dorsal (Actin) or ventral (Globin) mesoderm, and endoderm (Edd) formation by xCITED2 in AC at stage 24. The results suggest that xCITED2 play a role in neurogenesis without inducing dorsal mesoderm.
We found that xCITED2 was induced by DNBR and over-expression of xCITED2 induced neural genes in AC. Because DNBR-induced neural tissue is anterior, we examined whether xCITED2-induced neural tissue was also anterior. As shown in Fig. 4, xCITED2 induced an anterior specific neural marker; OTX2. OTX2 was similarly expressed in either DNBR or activin-treated AC. Overexpression xCITED2 further increased OTX2 transcripts in either DNBR or activin-treated AC. At the same conditions, xCITED2 reduced a posterior specific neural marker; HoxB9 which was induced by activin treatment in AC. In addition, other neural markers NCAM, NeuroD and Xneurogenin-1 were also induced, but an eye specific marker; Rx1 was dramatically reduced by co-injection of xCITED2.
The results suggest that xCITED2 induces anterior neural tissue in AC of
Previous studies have demonstrated that inhibition of BMP4 signaling leads to induce neurogenesis (Hawley et al., 1995). To understand the mechanism of neurogenesis, we investigated DNBR-induced genes using total RNA sequencing analysis. We analyzed total 21,891 expressed sequences and found 506 transcripts were up-regulated more than 3-fold. Up-regulated transcripts included a number of well-known genes involved in neurogenesis such as Zic, OTX and Sox family genes. In addition, we found several uncharacterized genes in neurogenesis processes (data not shown).
In this works, we examined the xCITED2 which was one of uncharacterized genes. xCITED2 has been well known to interact with CBP/p300 and act as a co-transcriptional activator. Although physiological function of xCITED2 has been fully understood in mammalian cell, the role of xCITED2 in early development has been elusive. During chick development, CITED2 is expressed in an anterior region of primitive streak, presomitic and lateral plate mesoderm, in the head-fold (future forebrain) and head mesoderm (Schlange et al., 2000). This spatial expression pattern suggests that CITED2 may functions in neurogenesis. In this work, we showed that xCITED2 was expressed in head and dorsal region and overexpression of xCITED2 resulted in anterior neural induction in AC. In addition, we constructed xCITED2-
Interestingly, CBP/p300 which is major binding partner of xCITED2 is expressed in anterior neural region in dorsal side during