Isolation of Early Neurogenesis Genes with Xenopus cDNA Microarray

Saet-Byeol Yu; Ok-Joo Lee; Young-Ju Park; Sung-Young Lee; Seung-Hwan Lee; Jaeho Yoon; Yoo-Seok Hwang; Jong-Il Kim; Jae-Yong Lee; Jae-Bong Park; Sung Chan Kim; Jaebong Kim

doi:10.5625/lar.2010.26.1.109

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Yu, Lee, Park, Lee, Lee, Yoon, Hwang, Kim, Lee, Park, Kim, and Kim: Isolation of Early Neurogenesis Genes with Xenopus cDNA Microarray

Original Article

Lab. Anim. Res. 2010;26(1):109-115.

Published online: 17 March 2010

DOI: https://doi.org/10.5625/lar.2010.26.1.109

Isolation of Early Neurogenesis Genes with Xenopus cDNA Microarray

Saet-Byeol Yu¹, Ok-Joo Lee¹, Young-Ju Park¹, Sung-Young Lee¹, Seung-Hwan Lee¹, Jaeho Yoon¹, Yoo-Seok Hwang², Jong-Il Kim³, Jae-Yong Lee¹, Jae-Bong Park¹, Sung Chan Kim¹, Jaebong Kim^1,^∗

¹Department of Biochemistry, College of Medicine, Hallym University, Chuncheon, Korea

²Department of Anatomy, School of Medicine, Kyungpook National University, Daegu, Korea

³Department of Biochemistry and Molecular Biology, Seoul National University College of Medicine, Seoul, Korea

^∗Corresponding author: Jaebong Kim, Department of Biochemistry, College of Medicine, Hallym University, 39 Hallymdaehakgil, Chuncheon, Kangwono 200-702, Korea Tel: +82-33-248-2544 Fax: +82-33-244-8425 E-mail: jbkim@hallym.ac.kr

Received 9 March 2010 Revised 15 March 2010 Accepted 18 March 2010

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

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.

Keywords: Xenopus, BMP inhibition, neurogenesis, Affymetrix gene chips

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Figure 1.

Analysis of neuro-specific genes involved in early stage of neurogenesis by Affiamtrix chip. Schematic diagram of experimental procedures (A). RT-PCR confirmation to examine the quality of samples with well-known genes (B and C). EF1α serves as RNA loading controls.

Figure 2.

Identification of neuro-specific transcripts and confirmation of EST gene expression by RT-PCR. Increased expression of ESTs during early neurogenesis (A). Temporal expression of EST genes. EF1α and Histone4 serve as RNA loading control (B). Identification of neuro-specific EST genes by Affimatrix chip (C).

Figure 3.

Amino acid sequences of subcloned two EST genes. Amino acid sequences of ORF (open reading frame) for subcloned two EST genes, 1946 (A) and 15853 (B).

Figure 4.

Characterization of a EST gene 1946 (CYP26c). The exrpession pattern of 1946 (CYP26c) induced by DNBR and RA (A), AP-1(JunD/c-Fos) (B). Spatial expression pattern of CYP26c examined by in situ hybridization at late stages (st.27 and st.30) (C). A model for role of CYP26c (D).

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