Xtbx6r, a novel T-box gene expressed in the paraxial mesoderm, has anterior neural-inducing activity

Abstract

T-box proteins are important transcriptional regulators in animal development. We searched the Xenopus laevis expressed sequence tag (EST) database using zebrafish tbx24 (Ztbx24) as a query and found a sequence. We then obtained corresponding clones from a neurula cDNA library. This novel gene has a T-box showing 53% homology with Xenopus laevis tbx6 (Xtbx6) and 51% with Ztbx24 at the amino acid level and is relatively close to Xtbx6 indicated by alignment analysis. In situ hybridization showed that it is expressed in the paraxial mesoderm in the caudal region in a very similar manner to Xtbx6, with a slight difference in that the former is emphasized more dorsally and has a more restricted distribution along the antero-posterior axis. From these results we named this gene Xtbx6r (tbx6-related). Xtbx6r or Xtbx6r-En R , when overexpressed in animal caps, induced anterior neural markers but not mesodermal markers. In contrast, Xtbx6r-VP16, Xtbx6 and Ztbx24 induced various mesodermal markers. These results indicate that Xtbx6r is a transcriptional repressor and has activity different from that of Xtbx6 or Ztbx24. Xtbx6r induced Otx2, XAG and Pax6 in animal caps. This activity differed from that of Xbra-En R or Xtbx6-En R , suggesting that some differences in biological activity exist among the tested repressor-type T-box genes. Depletion of Xtbx6r by antisense morpholino oligo produced curved embryos, but did not affect expressions of MyoD, Myf5, XWnt8 or thylacine2, nor inhibited muscle differentiation or segmentation. The results of knockdown and overexpression experiments suggest that Xtbx6r is involved in some morphogenesis in the paraxial mesoderm.

Mechanisms driving neural crest induction and migration in the zebrafish andXenopus laevis

“…3 This VegT/Nodal cascade leads to the zygotic expression of a number of genes, among which are those that encode the T-box transcription factors Eomesodermin, 58 Brachyury (there are in fact three distinct brachyury genes in Xenopus), 59-61 the zygotic splice variant of VegT, known as Antipodean, 62,63 and the T-box transcription factors Tbx6 and Tbx6-related. [64][65][66][67] Recent studies indicate that downregulation of zygotic VegT/Antipodean is We have indicated where a number of the genes that are the primary focus of this review are thought to act. That said, and as noted in the text, it is often the case that genes expression is co-dependent, so that simple linear pathways should be considered skeptically.…”

pMesogenin1 and 2 function directly downstream of Xtbx6 in Xenopus somitogenesis and myogenesis

“…Weak pMesogenin2 expression was detected in the head region by whole-mount in situ hybridization. RT-PCR from cDNAs obtained by dissection of stage 33 embryos into four fragments (head-1, head-2, trunk, and tail; Yabe et al, 2006) revealed that pMesogenin2 was expressed only in the tail region (data not shown) suggesting that the weak signal observed in the head region was non-specific.…”

“…The Hind III-Sma I (526 bp, pMesogenin1) or EcoR I-Xho I (504 bp, Xtbx6) fragment from these plasmids was labeled with 32 P, and subsequent screenings were carried out as described by Yabe et al (2006). GenBank Accession Numbers for Xtbx6 and pMesogenin2genomic D NA sequences are AB462326 and AB462327, respectively.…”

“…In wholemount double in situ hybridization, cRNA probes for pMesognin1 and 2 were labeled with digoxigenin-UTP (Roche, Basel, Switzerland) and that for Xtbx6 was labeled with fluorescein-UTP (Roche). The subsequent protocol was described by Yabe et al (2006).…”

“…RT-PCR was performed as described by Yabe et al (2006), except that PrimeScript Reverse Transcriptase (Takara Bio Inc., Otsu, Japan) was used for reverse transcription. Forward (F) and reverse (R) PCR primers for pMesogenin1, pMesogenin2, Xtbx6,Xtbx6 intron1,Xbra,XmyoD,Xwnt8,chordin,Xvent1,Xvent2,Cact,TMsk, and ODC were as follows: pMesogenin1 (F: 5Ј-GGACTTGTGCAGGAAACTC-CTAG-3Ј, R: 5Ј-AACCTCACGCTCTCT-TGGAGC-3Ј); pMesogenin2 (F: 5Ј-CTT-GGGAAAGCAGCTTCTGG-3Ј, R: 5Ј-AACCTCACGCTCTCTTGGAGC-3Ј);…”