Two Pax-binding sites are required for early embryonic brain expression of an Engrailed-2 transgene.
The temporally and spatially restricted expression of the mouse Engrailed (En) genes is essential for development of the midbrain and cerebellum. The regulation of En-2 expression was studied using in vitro protein-DNA binding assays and in vivo expression analysis in transgenic mice to gain insight into the genetic events that lead to regionalization of the developing brain. A minimum En-2 1.0 kb enhancer fragment was defined and found to contain multiple positive and negative regulatory elements that function in concert to establish the early embryonic mid-hindbrain expression. Furthermore, the mid-hindbrain regulatory sequences were shown to be structurally and functionally conserved in humans. The mouse paired-box-containing genes Pax-2, Pax-5 and Pax-8 show overlapping expression with the En genes in the developing brain. Significantly, two DNA-binding sites for Pax-2, Pax-5 and Pax-8 proteins were identified in the 1.0 kb En-2 regulatory sequences, and mutation of the binding sites disrupted initiation and maintenance of expression in transgenic mice. These results present strong molecular evidence that the Pax genes are direct upstream regulators of En-2 in the genetic cascade controlling mid-hindbrain development. These mouse studies, taken together with others in Drosophila and zebrafish on the role of Pax genes in controlling expression of En family members, indicate that a Pax-En genetic pathway has been conserved during evolution.
Song
D L
DL
Department of Molecular and Medical Genetics, University of Toronto, Canada.
Chalepakis
G
G
Gruss
P
P
Joyner
A L
AL
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D005075:Biological Evolution; D001921:Brain / Q000196:embryology* / Q000378:metabolism; D002531:Cerebellum / Q000196:embryology; D017124:Conserved Sequence; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000378:metabolism*; D005314:Embryonic and Fetal Development; D015870:Gene Expression; D005801:Genes, Homeobox; D018398:Homeodomain Proteins / Q000096:biosynthesis* / Q000235:genetics*; D006801:Humans; D008636:Mesencephalon / Q000196:embryology; D051379:Mice; D008822:Mice, Transgenic; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009419:Nerve Tissue Proteins / Q000096:biosynthesis* / Q000235:genetics*; D009687:Nuclear Proteins / Q000378:metabolism*; D051762:PAX2 Transcription Factor; D051757:PAX5 Transcription Factor; D000072636:PAX8 Transcription Factor; D051761:Paired Box Transcription Factors; D016133:Polymerase Chain Reaction; D012045:Regulatory Sequences, Nucleic Acid; D015183:Restriction Mapping; D017384:Sequence Deletion; D015534:Trans-Activators / Q000378:metabolism*; D014157:Transcription Factors / Q000378:metabolism*; D029961:Zebrafish Proteins; D000099266:Engrailed 2 Protein
D017931:DNA Primers; D004268:DNA-Binding Proteins; D018398:Homeodomain Proteins; D009419:Nerve Tissue Proteins; D009687:Nuclear Proteins; D051762:PAX2 Transcription Factor; D051757:PAX5 Transcription Factor; D000072636:PAX8 Transcription Factor; D051761:Paired Box Transcription Factors; D015534:Trans-Activators; D014157:Transcription Factors; D029961:Zebrafish Proteins; D000099266:Engrailed 2 Protein; C497318:PAX2 protein, human; C497281:PAX5 protein, human; C083325:PAX8 protein, human; C497319:Pax2 protein, mouse; C497282:Pax5 protein, mouse; C499496:Pax8 protein, mouse; C494467:Pax8 protein, zebrafish