A novel POU domain gene, zebrafish pou2: expression and roles of two alternatively spliced twin products in early development.
POU domain proteins are a large family of transcriptional regulatory proteins, many of which are implicated in the control of gene expression during early development. We describe here the cloning and expression of zebrafish pou2, a novel POU domain gene related to the mouse germ-line-specific transcription factor oct-3. Zebrafish pou2 is maternally expressed, and the transcripts are present from the one-cell stage to the gastrula stage. In situ hybridization analyses revealed that the transcripts were present in all blastomeres until the midblastula stage and that the expression was restricted to the epiblast during gastrulation. We found that alternatively spliced transcripts, t-pou2 RNAs, were also expressed in the embryos. In contrast to the Pou2 product, the t-Pou2 product lacks DNA-binding activity because of its incomplete POU domain structure. To examine the roles of the Pou2 and t-Pou2 products, we increased their expression in the embryo by microinjection of synthetic pou2 and t-pou2 RNAs into the fertilized eggs at the one-cell stage. Most embryos that developed from the eggs injected with pou2 RNA did not show any obvious developmental defects. In contrast, overexpression of the t-Pou2 product greatly affected the embryonic development: There was strong developmental retardation or arrest due to the incomplete gastrulation. In the affected embryos, expression of zebrafish T gene was reduced and the hypoblast formation was disturbed. Temporal and spatial expression patterns and the effects of overexpression of these products on development are consistent with the idea that the Pou2 and t-Pou2 proteins are involved in early development of zebrafish embryos. They may be involved in the proliferation of blastomeres in undetermined state at the blastula stage and/or the early cell commitment events at the gastrula stage. Also, our results indicate that different products generated as a result of alternative splicing from the same gene possess distinct functional capacities.
Takeda
H
H
Laboratory of Gene Technology and Safety, Institute of Physical and Chemical Research (RIKEN), Tsukuba, Japan.
Matsuzaki
T
T
Oki
T
T
Miyagawa
T
T
Amanuma
H
H
D017398:Alternative Splicing; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D003001:Cloning, Molecular; D046508:Culture Techniques; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins; D008969:Molecular Sequence Data; D050814:Octamer Transcription Factor-3; D050809:POU Domain Factors; D011485:Protein Binding; D012333:RNA, Messenger / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism; D015027:Zebrafish / Q000196:embryology / Q000235:genetics*; D029961:Zebrafish Proteins
D004268:DNA-Binding Proteins; D050814:Octamer Transcription Factor-3; D050809:POU Domain Factors; C085437:Pou5f3 protein, zebrafish; D012333:RNA, Messenger; D014157:Transcription Factors; D029961:Zebrafish Proteins; D004247:DNA