Lab Anim Res.  2010 Mar;26(1):109-115. 10.5625/lar.2010.26.1.109.

Isolation of Early Neurogenesis Genes with Xenopus cDNA Microarray

• Yu SB
• Lee OJ
• Park YJ
• Lee SY
• Lee SH
• Yoon J
• Hwang YS
• Kim JI
• Lee JY
• Park JB
• Kim SC
• Kim J

• KMID: 1458948
• DOI: http://doi.org/10.5625/lar.2010.26.1.109

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.

Keyword

Xenopus; BMP inhibition; neurogenesis; Affymetrix gene chips

MeSH Terms

Bone Morphogenetic Proteins
Clone Cells
DNA, Complementary
Ectoderm
Embryonic Development
Embryonic Structures
Expressed Sequence Tags
Female
Neural Plate
Neurogenesis
Oligonucleotide Array Sequence Analysis
Pregnancy
Up-Regulation
Vertebrates
Xenopus
Bone Morphogenetic Proteins
DNA, Complementary

Figure

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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.

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  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).

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  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).

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  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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