MOLECULAR AND MUTATIONAL ANALYSIS OF MAMMALIAN GENES 
CONTROLLING PATTERN FORMATION 
Alexandra L. Joyner, Ph.D., International Research Scholar 
One strategy that has been very effective in identi- 
fying mammalian genes required for pattern forma- 
tion has been to use cross-species sequence conser- 
vation to clone related genes from vertebrates and 
invertebrates. A number of families of mammalian 
genes have been identified in this manner, based on 
their homology to Drosophila genes that control 
the process of segmentation. These families include 
the Wnt genes that are homologues of the Drosoph- 
ila wingless {wg) gene, the En genes that are similar 
in structure to the Drosophila engrailed (en) gene, 
the Pax genes that contain a conserved paired box 
domain that is found in the Drosophila paired 
(prd) and gooseberry (gsb) genes, and the Gli 
genes that have homology to the Drosophila cubi- 
tus interruptus (Ci) gene. Analysis of gene expres- 
sion patterns and limited mutant analysis have indi- 
cated that these mammalian genes play important 
roles in pattern formation during gastrulation and 
organogenesis. In Drosophila, the wg, en, prd, gsb, 
and Ci genes have been shown to interact during 
segmentation in common genetic pathways. 
The expression patterns of the mouse homo- 
logues of these genes have shown that they may also 
interact in a similar manner in mammals. Thus it is 
possible that not only the genes but also the genetic 
pathways have been conserved through evolution. 
Dr. Joyner and her colleagues have concentrated on 
determining the roles of the En genes in develop- 
ment and, more recently, on examining whether 
they interact with the Wnt, Pax, and Gli genes. 
Function of Mouse En Genes 
Mice, humans, and chickens all contain two en- 
grailed genes, En- 1 and En- 2, that are expressed in a 
very similar band of cells across the developing 
midbrain-hindbrain border, beginning shortly after 
formation of the neural ectoderm. In the adult the 
En genes are expressed in specific neurons in the 
cerebellum and pons region, rather than in a spa- 
tially restricted domain. An En-2 mouse mutant was 
made by gene targeting in embryonic stem (ES) 
cells, and mice homozygous for this mutation, En- 
2*"*, show an abnormality in patterning of the folds 
of the cerebellum, although they are viable, fertile, 
and show no obvious behavioral defects. Recently 
ES cells containing a mutation in En-1 have been 
made and chimeras have been produced from the 
cells. If mice heterozygous for this mutation are ob- 
tained from the chimeras, they will be bred to test 
whether En-1, unlike En-2, is essential for develop- 
ment and/or whether there is a level of redundancy 
of function between En-1 and -2. 
In order to characterize further the En-^^ mutant 
phenotype, the developmental profile of presenta- 
tion of the defect was characterized. By analyzing 
the cerebellums of mutant and wild-type mice dur- 
ing the first two weeks after birth, the time at which 
the cerebellar folds are produced, the adult pheno- 
type was shown to arise from abnormalities that oc- 
cur during the first week of postnatal development. 
The defects include fusion of folds, abnormal place- 
ment of folds, and transformations of one fold to 
another. These types of defects are reminiscent of 
those that occur in Drosophila en mutants. In an 
attempt to understand the mechanism underlying 
this patterning defect. Dr. Joyner and her colleagues 
are analyzing the expression of En- 1 and En-2 in the 
postnatal cerebellum. 
The En Genetic Pathways 
To identify the upstream gene(s) that regulates 
the spatially restricted expression of the En genes, 
the laboratory is carrying out a promoter analysis in 
transgenic mice. By making reporter constructs 
(containing DNA fragments 5' and 3' to En-2, a mini- 
mal promoter, and the lacZ gene) and analyzing /3- 
galactosidase expression in transgenic embryos, a 
1-kb fragment that contains a mid-hindbrain en- 
hancer has been identified. A similar DNA sequence 
has been identified upstream of the human En-2 
gene based on DNA sequence similarities. A bio- 
chemical analysis of the embryonic brain proteins 
that bind the mouse En-2 enhancer fragment will be 
undertaken, with the aim of cloning the genes re- 
sponsible for regulating En-2, and possibly En-1, 
expression in the embryo. 
To determine whether the Wnt, Gli, and Pax 
genes interact with the En genes. Dr. Joyner and her 
colleagues are analyzing comparative expression in 
wild-type and mutant embryos and characterizing 
double mutants. The initial studies have concen- 
trated on the developing cerebellum. To facilitate 
this analysis, the mouse Gli, Gli-2, and Gli-3 genes 
were cloned and their expression patterns deter- 
mined in the embryo, in collaboration with Dr. Rob- 
ert Holmgren (Northwestern University). In addi- 
tion, an existing mouse developmental mutant. 
Extra toes, was shown to contain a deletion of Gli- 5. 
The phenotype of this mutant and double mutants 
INTERNATIONAL RESEARCH SCHOLARS 
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