Functional studies of novel genes involved in embryogenesis of the vertebrate Xenopus laevis

In this study, the large-scale whole-mount in situ hybridization was employed to screen an activin-treated ectoderm cDNA library of Xenopus laevis, leading to the isolation of 51 novel cDNA sequences of Xenopus. So this approach turned out to be practicable for identification of new genes from a cDNA library in a large scale. XXBP-1, XMLP and XAT were selected for further functional studies based on their suggestive expression pattern. XXBP-1 is a novel basic leucine zipper transcription factor in Xenopus. It is a maternal factor and expressed on the dorsal blastopore lip and ventral ectoderm with exception of perspective neural plate in gastrula stages. RT-PCR indicated that XXBP-1 is weakly expressed before gastrula stages, afterwards is up-regulated and kept in a persistent level during the embryonic development. Overexpression of XXBP-1 leads to ventralization of the injected embryos as described for BMP-4. Moreover, XXBP-1 and BMP4 interact in a positive feedback loop. Consistent with mediating BMP-4 signaling, the ectopic expression of XXBP-1 partially recovers the expression of epidermal keratin in animal cap assay and converses the dorsolizaiton imposed by truncate BMP receptor I. Thus, we propose that the XXBP-1 is downstream of BMP receptors, and plays roles in the inhibition of neural differentiation. XXBP-1 functions as a transcriptional activator. When overexpressed ventrally, the effect of wild type XXBP-1 can be phencopied by the XXBP1-VP16 containing the activator domain VP16 and the DNA binding domain of XXBP-1. Another fusion construct, XXBP1-Eve containing the DNA binding domain of XXBP-1 and even-skipped repression domain induce neural markers NCAM and nrp1 in animal cap assay and leads to the secondary axis when overexpressed ventrally in a low ratio (10%). This confirms that XXBP-1 acts as an activator, playing negative roles in neural induction. The XMLP may be a new number of the small MLP protein family. Using whole-mount in situ hybridization and RT-PCR, XMLP maternal transcripts were detected during the cleavage stages. After MBT the signals are restricted to the neural plate. Subsequently XMLP is expressed predominantly in the brain, somites, and pronephros. Ectopic expression of XMLP results in eye and axis defects and as well as a change of the expression pattern of Krox 20. Apoptosis was induced by the injection of XMLP. Overexpression of mutant XMLPs indicated that this phenotype is correlated with its putative PSD domain and glycine at position 2. The loss-of-function of XMLP was studied by injection with a morpholino oligo complementary to XMLP mRNA, which revealed the malformations of anterior axis and eye defects. According to extirpation experiments the phenotypes might be correlated with disturbed morphogenetic movements rather than an inhibition of induction process. Overexpression of XCYP26 resulted in a shift of the expression pattern of XMLP, showing the signal stripe of XMLP in injected half of the embryo getting diffuse or even disappeared. This observation suggests that retinoic acid plays an important role in the XMLP regulation. Taken together, XMLP may participate in pattern formation of the embryonic axis and the central nervous system. XAT encodes Xenopus amidinotransferase which shares a highly conserved sequence with the homologues of human, chick and rat. Characterization of embryonic expression indicates that XAT is differentially expressed around the yolk plug including the dorsal blastopore area at early gastrula stages and is extensively expressed in the midline of the neural plate of early neurula stages. Sections reveal that its transcripts are located in the notochord. In the tailbud stage signals are found both in the notochord and the trunk area, whereas faint signals can be found in the cephalic part only.



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