Hormone-inducible expression of secreted factors in zebrafish embryos.
The study of gene function at later stages of embryonic development by overexpression experiments is often complicated by genes exerting different functions at multiple stages of development, which renders analysis of stage-specific effects difficult. To address this problem an inducible expression system that supports timed expression of essentially any protein, including secreted proteins was designed. The system is based on a two step mechanism. A glucocorticoid inducible, Gal4-site binding chimeric transcription factor is expressed ubiquitously, whereas a gene of interest is placed under the control of a Gal4-site driven promoter. Treatment of zebrafish embryos injected with such constructs with the synthetic glucocorticoid dexamethasone results in readily detectable reporter activity within 3 h. The system was tested with induced expression of Xactivin(beta)B and X(wnt), which both were shown to induce morphological abnormalities, as well as alterations in the expression patterns of goosecoid and otx2, respectively. Coinjection of an inducible lacZ reporter vector served as an indicator for expressing cells in embryos. The present results demonstrate that this is a versatile inducible expression system for use in vertebrate embryos, that also supports expression of secreted proteins.
de Graaf
M
M
Hubrecht Laboratory, the Netherlands Institute for Developmental Biology, Utrecht.
Zivkovic
D
D
Joore
J
J
D028341:Activins; D000818:Animals; D003907:Dexamethasone / Q000494:pharmacology*; D004625:Embryo, Nonmammalian / Q000378:metabolism; D018507:Gene Expression Regulation, Developmental; D017930:Genes, Reporter; D005821:Genetic Techniques; D005938:Glucocorticoids / Q000494:pharmacology; D051956:Goosecoid Protein; D018398:Homeodomain Proteins / Q000235:genetics; D017403:In Situ Hybridization; D007763:Lac Operon; D008156:Luciferases / Q000032:analysis; D009419:Nerve Tissue Proteins / Q000235:genetics; D009842:Oligopeptides; D051857:Otx Transcription Factors; D010455:Peptides / Q000235:genetics*; D011518:Proto-Oncogene Proteins / Q000235:genetics*; D011994:Recombinant Proteins / Q000378:metabolism; D012097:Repressor Proteins; D015534:Trans-Activators / Q000235:genetics; D014157:Transcription Factors; D051153:Wnt Proteins; D015027:Zebrafish / Q000196:embryology* / Q000235:genetics*; D029961:Zebrafish Proteins; D001616:beta-Galactosidase / Q000032:analysis
D005938:Glucocorticoids; D051956:Goosecoid Protein; D018398:Homeodomain Proteins; D009419:Nerve Tissue Proteins; D009842:Oligopeptides; D051857:Otx Transcription Factors; D010455:Peptides; D011518:Proto-Oncogene Proteins; D011994:Recombinant Proteins; D012097:Repressor Proteins; D015534:Trans-Activators; D014157:Transcription Factors; D051153:Wnt Proteins; D029961:Zebrafish Proteins; C044147:activin B; C497995:gsc protein, zebrafish; C497662:otx2b protein, zebrafish; C094522:wnt8b protein, zebrafish; D028341:Activins; C048617:myelopeptides; D003907:Dexamethasone; D008156:Luciferases; D001616:beta-Galactosidase