The Regulation of Mammalian Development 
Shirley M. Tilghman, Ph.D. — Investigator 
Dr. Tilghman is also Howard A. Prior Professor of the Life Sciences in the Molecular Biology Department at 
Princeton University and Adjunct Professor of Biochemistry at the University of Medicine and Dentistry 
of New Jersey, Robert Wood Johnson Medical School. She obtained a B.Sc. degree at Queen's University in 
Kingston, Ontario, Canada. Following two years in Sierra Leone, West Africa, where she taught secondary 
school, she attended graduate school at Temple University in Philadelphia, where she received her Ph.D. 
degree in biochemistry. Her postdoctoral work was done with Philip Leder at NLH Before joining the 
faculty at Princeton, Dr. Tilghman held positions at Temple University and the Institute for Cancer 
Research, Philadelphia. 
ORDERLY development of the mammalian 
embryo requires the appropriate activation 
and subsequent modulation of genes in a spatial 
and temporal manner. For the vast majority of 
genes, both the mother's and father's copies are 
activated and modulated identically, but for a 
small class of genes, only the mother's or father's 
copy is expressed. Such genes are parentally im- 
printed. That is, during the process that generates 
eggs or sperm, these genes are marked in such a 
way that the resulting embryo can distinguish the 
parental origin and express it accordingly. Our 
laboratory is studying a locus that encodes at least 
two imprinted genes on the distal end of mouse 
chromosome 7. 
One, the insulin-like growth factor II gene 
(Igf2) encodes a fetal-specific growth factor that 
is exclusively expressed from the paternal chro- 
mosome. The other gene is HI 9, which encodes 
an RNA, evolutionarily conserved, that is only 
found in high abundance during fetal develop- 
ment in tissues originating in endoderm and me- 
soderm. Unlike Igf2, HI 9 is exclusively ex- 
pressed from the maternal chromosome. These 
genes lie in tandem about 75 kilobases (kb) of 
DNA apart and are expressed in a very similar 
manner during mouse embryogenesis. 
We are investigating the activation and role of 
these two differentially imprinted genes. The 
function of the H19 RNA is unknown. Its pattern 
of sequence conservation in mammals is reminis- 
cent of other functional RNAs, such as those asso- 
ciated with telomerases and RNase P. We are us- 
ing both genetic and biochemical approaches to 
understand its role during development. 
Over the past 50 years a large number of muta- 
tions have been described that affect all aspects of 
mouse physiology. However, molecular access to 
the genes, which would allow us to identify those 
of developmental importance, has been difficult 
because the mouse genome is so large. Advances 
in DNA analysis and cloning methods have effec- 
tively reduced the barriers to studying these 
genes at the molecular level. We have generated a 
yeast artificial chromosome library that now con- 
tains 2.5 copies of the mouse genome in over 
28,000 yeast strains, each of which harbors a seg- 
ment of mouse genomic DNA averaging 275 kb. 
The library has been constructed to serve the 
mouse genome community, and over 60 labo- 
ratories worldwide have so far screened it suc- 
cessfully. This work is supported by a grant from 
the National Institutes of Health. 
We have used the mouse Fused (Fu) locus to 
test the utility of the library for isolating large 
chromosomal DNA segments. Fu, a dominant mu- 
tation on mouse chromosome 17, generates 
kinky tails in heterozygotes and early embryonic 
lethality in homozygotes. The lethality is asso- 
ciated with overgrowth of neuroectoderm and 
duplications of the body axis. To localize Fu pre- 
cisely, 1,000 progeny were generated by back- 
crossing mice carrying the Kinky allele of Fu 
with a distantly related wild mouse, Mus spretus. 
Because these mice are genetically very distinct, 
it is easy to follow the segregation of genes in 
their progeny. 
The offspring were scored for seven molecular 
markers that map in a small interval around Fu, 
and a high-density genetic map gives us molecu- 
lar landmarks every 100-200 kb of DNA. One 
marker, a pseudogene of the a-globin gene fam- 
ily, cannot be separated from Fu in this cross, 
suggesting that it is very close to the gene — 
within 100-200 kb. We have used this close 
proximity to isolate approximately 650 kb of 
DNA around the marker. By comparing the segre- 
gation of this DNA with that of Fu, we should be 
able to pinpoint Fu's location. 
A similar approach has been adopted for the 
piebald (5) locus on mouse chromosome 14. 
Mice carrying the original 5 mutation have spot- 
ted coats, a result of the absence of melanocytes 
in genetically specified regions of the midsec- 
tion. Mice carrying more-severe s mutations are 
almost entirely white. In addition, they develop 
419 
