The Regulation of 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 the B.Sc. degree at Queen's University 
in Kingston, Ontario, Canada. Following two years in Sierra Leone, West Africa, where she taught sec- 
ondary school, she attended graduate school at Temple University in Philadelphia, where she received her 
Ph.D. degree in biochemistry. Her postdoctoral work at the NLH was done with Philip Leder. Before joining 
the faculty at Princeton, Dr. Tilghman held positions at Temple University and the Institute for Cancer 
Research, Philadelphia. 
THE orderly development of the mammalian 
embryo requires the appropriate activation 
and subsequent modulation of genes in a tissue- 
specific manner. The mechanisms by which this 
is achieved are the subject of our investigations. 
One classical approach to this problem is to study 
in depth the elements responsible for a specific 
gene's expression in a limited subset of tissues. 
We have taken this approach, using the mouse 
a-fetoprotein (AFP) gene as the model. 
Over several years we have identified the cis- 
acting regulatory elements that activate tran- 
scription of this gene in only three cell types in 
the mouse — the fetal liver, fetal gut, and visceral 
endoderm of the yolk sac. These include three 
200-base-long segments of DNA that exhibit prop- 
erties of tissue-specific cellular enhancers and lie 
substantially upstream of the gene. The fourth 
positive element lies within the first 125 bp up- 
stream of the gene and participates in the tissue- 
specific activity of the gene's promoter by inter- 
acting with a liver-specific transcription factor, 
HNF-1. 
The AFP gene undergoes a dramatic decline in 
transcription after birth; this decline is mediated 
by a fifth cis-acting element that lies between the 
most proximal enhancer and the promoter of the 
gene. We have used germline transformation to 
show that this negatively acting element is acti- 
vated at birth and interferes with the continued 
presence of positive transcription factors, not 
only in the context of the AFP gene, but when it is 
placed in the same position within the albumin 
gene, which is not normally repressed after birth. 
The demonstration that the negative repressor 
must lie between these strong positive elements 
to function explains why only the AFP, and not 
the nearby albumin gene, is negatively regulated 
in neonatal liver. 
The decline in hepatic AFP mRNA after birth is 
influenced by raf, an unlinked gene. Mice that 
carry a mutant allele of m/exhibit 10- to 20-fold 
higher levels of AFP mRNA in the adult. We used 
transgenic mice to establish that the raf gene 
product does not act through the negative tran- 
scriptional apparatus but rather affects the con- 
centration of AFP mRNA post-transcriptionally, 
by changing the stability and/or the processing of 
its transcript. Thus the 10,000-fold decline in 
AFP RNA after birth is achieved by changes in 
both transcription and stability of the AFP 
transcripts. 
Our long-standing interest in liver transcrip- 
tional repression led us to examine another fetal- 
specific gene, the H19 gene. Initially we were 
surprised to see that this gene contains no long 
open reading frame, normally found in genes that 
encode protein products. We have subsequently 
shown that the RNA is not associated with polyri- 
bosomes in the cell but is sequestered in a parti- 
cle in the cytoplasm. This leaves us with the chal- 
lenge of understanding the role of a gene 
transcribed and processed as a classical RNA poly- 
merase II gene, whose product is developmen- 
tally regulated in both endoderm and mesoderm, 
but which does not encode a protein. 
One means of understanding the function of 
any gene is to generate both dominant and reces- 
sive mutations in it. To obtain dominant mutants 
in the mouse, we have introduced excess copies 
of the H19 gene into the mouse germline, using 
microinjection into zygotes. No transgenic mice 
that express the gene have been recovered at 
birth. The lethality occurs abruptly, after 14 days 
into the normal 20-day gestation period of the 
mouse. 
Insight into the basis of the lethality caused by 
the extra copies of the H19 gene came from its 
location on the distal tip of chromosome 7. This 
region has been shown to undergo genomic im- 
printing — when both copies of this region are 
inherited from the mother, the animals die late in 
gestation. By taking advantage of differences in 
the H19 gene between different species of 
mouse, we showed that only the mother's copy of 
the H19 gene is active in a normal mouse. By 
introducing extra copies of the gene into the 
germline of mice, we had altered the carefully 
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