Analysis of Cx43alpha1 promoter function in the developing zebrafish embryo.
The Cx43alpha1 gap junctions play an important role in cardiovascular development. Studies using transgenic mouse models have indicated that this involves an essential role for Cx43alpha1 in modulating neural crest cell motility. We previously showed that a 6.8 kb mouse genomic sequence containing the promoter and upstream regulatory sequences of the Cx43alpha1 gene can drive lacZ reporter gene expression in all neural crest cell lineages in the mouse embryo. To obtain further insights into the sequence motifs and regulatory pathways involved in targeting Cx43alpha1 gene expression in neural crest cells, we assayed the activity of the mouse Cx43alpha1 promoter in evolutionarily distantly related zebrafish embryos. For these studies, the 6.8kb Cx43alpha1 genomic sequence and various deletion derivatives were used to generate GFP or lacZ expression vectors. The transcriptional activities of these constructs were analyzed in vivo after microinjection into one- or two- cell stage zebrafish embryos. These studies indicated that the mouse Cx43alpha1 promoter can drive lacZ expression in neural crest cells in the zebrafish embryos. Analysis by whole mount in situ hybridization showed that the endogenous zebrafish Cx43alpha1 gene is expressed maternally and zygotically, and expression is observed in regions where neural crest cells are found. To further elucidate the developmental regulation of Cx43alpha1 gene expression, we screened a zebrafish BAC library and identified a clone containing the entire zebrafish Cx43alpha1 gene and flanking upstream and downstream sequences. The upstrean Cx43alpha1 promoter sequences from zebrafish, mouse, and human were analyzed for evolutionarily conserved DNA motifs. Overall these studies suggest that the sequence motifs and transcriptional regulation involved in the targeting Cx43alpha1 expression to neural crest cells are evolutionarily conserved in zebrafish and mouse embryos.
Chatterjee
B
B
Biology Dept., University of Pennsylvania, Philadelphia, USA.
Li
Y X
YX
Zdanowicz
M
M
Sonntag
J M
JM
Chin
A J
AJ
Kozlowski
D J
DJ
Valdimarsson
G
G
Kirby
M L
ML
Lo
C W
CW
D000818:Animals; D018031:Connexin 43 / Q000235:genetics* / Q000378:metabolism; D017630:Connexins / Q000235:genetics* / Q000378:metabolism; D017629:Gap Junctions / Q000378:metabolism*; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins; D006321:Heart / Q000196:embryology / Q000254:growth & development; D007202:Indicators and Reagents / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D018345:Mice, Knockout; D009432:Neural Crest / Q000166:cytology / Q000502:physiology; D011401:Promoter Regions, Genetic; D017422:Sequence Analysis, DNA; D015027:Zebrafish / Q000196:embryology* / Q000235:genetics*; D029961:Zebrafish Proteins / Q000235:genetics* / Q000378:metabolism
D018031:Connexin 43; D017630:Connexins; D007202:Indicators and Reagents; D008164:Luminescent Proteins; D029961:Zebrafish Proteins; C471698:connexin 43alpha1, mouse; D049452:Green Fluorescent Proteins