Promoter analysis in living zebrafish embryos identifies a cis-acting motif required for neuronal expression of GATA-2.
We have used zebrafish embryos to dissect the promoter activity of a gene with a complex expression pattern during embryogenesis. GATA-2 is a transcription factor required for hematopoiesis and is dynamically expressed in hematopoietic tissues and in the central nervous system. Using constructs containing zebrafish GATA-2 genomic flanking sequences and the green fluorescent protein (GFP) reporter gene, we demonstrate that distinct regulatory domains are required for hematopoietic, enveloping layer (EVL), and neuronal expression of GATA-2. During gastrulation, GFP expression is confined to the ventral ectoderm and lateral mesoderm and is lacking in the dorsal shield. Cells derived from the regions expressing GFP give rise to hematopoietic progenitors, EVL cells, and neurons. Deletion analysis of the 7.3-kb GATA-2 promoter region revealed that a 1.1-kb DNA sequence is critical for expression of GATA-2 in neurons. Fine mapping revealed that a 31-bp region is required for neuron enhancer activity, and mutagenesis showed that the DNA motif CCCTCCT is essential for GATA-2 promoter activity in the central nervous system of zebrafish. Our use of zebrafish embryos can be exploited as a whole animal system for the dissection of any developmentally regulated vertebrate promoter.
Meng
A
A
Institute of Molecular Medicine and Genetics, Department of Biochemistry and Molecular Biology, Medical College of Georgia, Augusta, GA 30912, USA.
Tang
H
H
Ong
B A
BA
Farrell
M J
MJ
Lin
S
S
D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000096:biosynthesis*; D004475:Ectoderm / Q000502:physiology; D004625:Embryo, Nonmammalian / Q000502:physiology*; D004742:Enhancer Elements, Genetic; D050989:GATA2 Transcription Factor; D005775:Gastrula / Q000502:physiology; D018507:Gene Expression Regulation, Developmental; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000096:biosynthesis; D016297:Mutagenesis, Site-Directed; D009474:Neurons / Q000502:physiology*; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D014157:Transcription Factors / Q000096:biosynthesis*; D015027:Zebrafish / Q000196:embryology* / Q000235:genetics
D017931:DNA Primers; D004268:DNA-Binding Proteins; D050989:GATA2 Transcription Factor; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D049452:Green Fluorescent Proteins