Gene Pattern Expression in Early Embryogenesis 
In addition, we are using these DNA transcrip- 
tion regulators to express other pattern-formation 
genes aberrantly in the mid- and hindbrain re- 
gion. In this way we can test, for example, 
whether other homeodomain-containing genes, 
expressed in more-posterior regions than En, can 
switch the developmental program of mid- and 
hindbrain cells to more-posterior regions. 
We are also interested in identifying genes that 
are regulated by En. One approach we are taking 
is to analyze mouse homologues of Drosophila 
genes that are regulated by en. To this end, we 
have cloned homologues of the Drosophila cubi- 
tus interruptus (Ci) gene. The mammalian genes 
are called Gli, since one member of the family 
was found to be overexpressed in gliomas. We 
can now analyze the expression of the Gli and 
other potential £n-regulated genes in mice lack- 
ing En- 2. 
Screening for Genes Expressed in Spatially 
Defined Patterns 
With a view to identifying other types of genes 
involved in pattern formation, we devised a 
screen that takes advantage of mammalian gene 
structure and mouse embryonic stem (ES) cells. 
The screen involves randomly integrating a vec- 
tor we refer to as a gene trap into the ES genome. 
The vector has a splice acceptor site upstream of 
the reporter gene lacZ. We reasoned that if the 
vector integrated in the correct orientation into 
the intron, then splicing of the gene would be 
directed to the reporter gene. We have now con- 
firmed this by cloning and characterizing a num- 
ber of transcripts for /flcZ-endogenous gene 
fusions. 
Cells expressing lacZ can be revealed with a 
histochemical stain. ES cells expressing the re- 
porter are then reintroduced into a host embryo 
for lacZ expression pattern analysis. We have 
now analyzed the expression pattern of the 
cloned endogenous genes from two lacZ inser- 
tions and have shown that the lacZ pattern faith- 
fully mimics that of the endogenous gene. 
Finally, lacZ insertions that show a spatially 
defined expression pattern can be transmitted 
into transgenic mice and the insertions analyzed 
for mutant phenotypes. Two out of three such 
mice showed mutant defects. Thus we have dem- 
onstrated the feasibility of the gene trap approach 
and are now carrying out a larger screen for can- 
didate pattern-formation genes. 
All of this work is also funded by grants from 
the Medical Research Council and the National 
Cancer Institute of Canada, the National Insti- 
tutes of Health, and Bristol-Myers Squibb. 
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