Isolation of Early Neurogenesis Genes with Xenopus cDNA Microarray / 한국실험동물학회지

Neurogenesis is the process that develops neuroectoderm from ectoderm. Bone morphogenetic protein (BMP) inhibition in ectodermal cells is necessary and sufficient for neurogenesis in Xenopus embryos. To isolate genes involved in early neurogenesis, Xenous Affymetrix gene chips representing 14,400 genes were analyzed in early stage of neuroectodermal cells that were produced by inhibition of BMP signaling with overexpression of a dominant-negative receptor. We identified 265 candidate genes including 107 ESTs which were newly expressed during the early neurogenesis by blocking BMP signaling. The candidates of 10 ESTs were selected and examined for upregulation in neuroectoderm. Five EST genes were confirmed to be upregulated in neuroectoderm and examined for time-dependent expression patterns in intact embryos. Two EST genes were cloned and identified as a homology of CYP26c (Xl.1946.1.A1_at) and Kielin containing VWC domain (Xl.15853.1.A1_at). One of them, CYP26c, was further characterized for its transcriptional regulation and role of anterior-posterior patterning during neurogenesis. Taken together, we analyzed and characterized genes expressed in early neurogenesis. The results suggest that neurogenesis by inhibition of BMP provides useful system to isolate genes involved in early events of neurogenesis during early vertebrate embryogenesis.

Spatiotemporal regulation of fibroblast growth factor signal blocking for endoderm formation in Xenopus laevis

We have previously shown that the inhibition of fibroblast growth factor (FGF) signaling induced endodermal gene expression in the animal cap and caused the expansion of the endodermal mass in Xenopus embryos. However, we still do not know whether or not the alteration of FGF signaling controls embryonic cell fate, or when FGF signal blocking is required for endoderm formation in Xenopus. Here, we show that FGF signal blocking in embryonic cells causes their descendants to move into the endodermal region and to express endodermal genes. It is also interesting that blocking FGF signaling between fertilization and embryonic stage 10.5 promotes endoderm formation, but persistent FGF signaling blocking after stage 10.5 restricts endoderm formation and differentiation.

Screening of Interacting Proteins with PV.1 as Downstream Factors of BMP Signal / 대한해부학회지

Homeodomain transcription factors functioning downstream of BMP ventral pathway have been reported to share similar domain of roles in mesoderm patterning along the dorsal-ventral axis. To elucidate the differential role of PV.1 in the aspect of relationship between dorsal and ventral region, we tried to screen PV.1- interacting proteins. Twenty-four PV.1-interacting proteins were identified by yeast two-hybrid screening. Xvent-2 and Xclaudin-6 among these, went under domain study. The C-terminus of PV.1, more specifically 197-241 region was found to interact with Xclaudin-6. Meanwhile Xvent-2 has mild affinity to overall C-terminal region of PV.1. At the same time it was found that Xvent-2 homodimerizes and also binds to Xclaudin-6.

Transcriptional Regulation of the Xbr-1a/Xvent-2 Gene by BMP-4 Signaling during Xenopus Embryonic Development / 대한해부학회지

BMP-4 signaling is mediated through Smad proteins which may translocate to the nucleus to activate transcription. Little is known about how BMP-4 signaling regulates the transcription of its target genes, e.g., Xvent genes. Therefore, we isolated the genomic clone of a BMP-4 responsive homeobox gene, Xbr-1a/Xvent-2. This clone contains a promoter and three exons for the entire coding region. Using the primer extension, we identified the transcription initiation site corresponding to position -64 bp upstream to the ATG codon of the Xvent-2 gene. The promoter was linked to the luciferase reporter gene, and promoter activity determined by luciferase assay. The temporal promoter activity peaked between embryonic stages 13~17, in agreement with its temporal mRNA expression in the whole embryo. Through the serial deletion mutation, the upstream -235 bp of the promoter retains the full transcriptional activity, and is regulated by BMP-4 signaling. The present results suggest that the BMP-4 responsive element is located on the upstream 235 bp of the promoter.

CGRP Immunoreactivities Following Artificail Ureteral Calsulosis and Ureteral Chemical Irritation in the Rat / 대한해부학회지

The aim of the present study was to investigate the CGRP-IR pattern in the ureter in stone-implanted rats and ureteral formalin irritated rats. Artificial ureteral stone rats were made according to Giamberardino's guide lines. For urethral formalin irritated rats, a small amount of diluted formalin was instilled into the ureter. The behavioral characteristics were observed and recorded with CCTV and analyzed, statistically. The rats showing characteristic visceral episode were sacrificed three days after stone implantation operation and after overnight chemical irritation followed by immunostaining of the ureter with anti-CGRP on the ureter In the artificial ureteral stone rats, CGRP IR fiber pattern showed variable changes on the upper portion of the stone implanted ureter. i.e., complete depletion, reticular pattern, and reticular pattern with more increased CGRP immunoreactivity were observed. In contrast, more-distinct CGRP IR fibers formed a reticular pattern but were not increased in density in the formalin irritated ureter. The results show the variable changing pattern of CGRP immunoreactivity in the ureter after artificial urethral calculosis, and the constant CGRP immunoreactivity in the ureter after chemical ureteral irritation.