2 ENHANCER REGIONS IN THE MOUSE EN-2 LOCUS DIRECT EXPRESSION TO THE MID HINDBRAIN REGION AND MANDIBULAR MYOBLASTS

An En-2/lacZ gene fusion containing 9.5 kb of En-2 genomic DNA was cap able of directing lacZ expression -in an En-2-specific manner both tem porally and spatially during embryogenesis and in the adult. lacZ expr ession was confined in the embryo to cells within the mid/hindbrain an d mandibular arch regions and in the adult to cells of the molecular a nd granular layers of the cerebellum, and within the pons and collicul i regions. Interestingly, in the adult, transgene expression patterns within the cerebellum in two lines appeared to mark distinct anterior- posterior compartments. Analysis of the expression pattern of this tra nsgene, in fetal and adult mice lacking a functional En-2 protein, pro vided evidence that the En-2 gene in mouse is not autoregulated. Delet ion analysis of the En-2 genomic region and the use of a heterologous promoter identified two enhancer-containing regions of 1.5 and 1.0 kb in length, 5' of the transcribed sequences, which independently direct ed expression in the embryo to either the mid/hindbrain region or mand ibular myoblasts, respectively. The 1.5 kb fragment contains the most anterior neural enhancer and the 1.0 kb fragment, the earliest myogeni c enhancer thus far characterized. These En-2-specific regulatory regi ons can now be used in a biochemical analysis to identify proteins imp ortant in anterior-posterior patterning of the vertebrate CNS and in t he specification of muscle identity as well as in a mutational analysi s to direct expression of other developmentally important genes to the se regions.