Analysis of upstream elements in the HuC promoter leads to the establishment of transgenic zebrafish with fluorescent neurons.
HuC encodes an RNA binding protein homologous to Drosophila elav that serves as an excellent early marker for differentiating neurons. We have characterized the promoter of the zebrafish HuC gene by examining the ability of 5'-upstream fragments to drive expression of green fluorescent protein (GFP) in live embryos. We determined that 2.8 kb of the 5'-flanking sequence is sufficient to restrict GFP gene expression to neurons. The core promoter spans 251 base pairs and contains a CCAAT box and one SP1 sequence but no TATA box is present near the transcription start site. A putative MyT1 binding site and at least 17 E-box sequences are necessary to maintain the neuronal specificity of HuC expression. Interestingly, sequential removal of the putative MyT1 binding site and 14 distal E boxes does not appear to abolish neuronal expression; rather, it leads to a progressive expansion of GFP expression into muscle cells. Further removal of the three proximal E boxes eliminates neuronal and muscle specificity of GFP expression and leads to ubiquitous expression of GFP in the whole body. Identification of key components of the HuC promoter has led to the establishment of a stable zebrafish transgenic line (HuC-GFP) in which GFP is expressed specifically in neurons. We crossed mind bomb (mib) fish with this line to visualize their neurogenic phenotype in live mib(-/-) mutant embryos. This cross illustrates how HuC-GFP fish could be used in the future to identify and analyze zebrafish mutants with an aberrant pattern of early neurons.
Park
H C
HC
Department of Genetic Engineering, Kyungpook National University, Taegu, 702-701, Korea.
Kim
C H
CH
Bae
Y K
YK
Yeo
S Y
SY
Kim
S H
SH
Hong
S K
SK
Shin
J
J
Yoo
K W
KW
Hibi
M
M
Hirano
T
T
Miki
N
N
Chitnis
A B
AB
Huh
T L
TL
D000818:Animals; D030801:Animals, Genetically Modified; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002454:Cell Differentiation; D004247:DNA / Q000235:genetics; D017931:DNA Primers / Q000235:genetics; D051959:ELAV Proteins; D000067778:ELAV-Like Protein 3; D018507:Gene Expression Regulation, Developmental; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000235:genetics; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D009154:Mutation; D009419:Nerve Tissue Proteins / Q000235:genetics*; D009474:Neurons / Q000166:cytology / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000235:genetics*; D012261:Ribonucleoproteins / Q000235:genetics; D015027:Zebrafish / Q000196:embryology* / Q000235:genetics* / Q000378:metabolism; D029961:Zebrafish Proteins
D017931:DNA Primers; D051959:ELAV Proteins; D000067778:ELAV-Like Protein 3; D008164:Luminescent Proteins; D009419:Nerve Tissue Proteins; D016601:RNA-Binding Proteins; D012261:Ribonucleoproteins; D029961:Zebrafish Proteins; C088635:elavl3 protein, zebrafish; D049452:Green Fluorescent Proteins; D004247:DNA; D011505:Protein-Tyrosine Kinases; D017346:Protein Serine-Threonine Kinases