MOLECULAR MECHANISMS OF DEVELOPMENTAL AND REGULATED 
EXPRESSION OF NEUROENDOCRINE GENES 
Michael G. Rosenfeld, M.D., Investigator 
I . Molecular Mechanisms of Anterior Pituitary Gland 
Development. 
Dr. Rosenfeld's central research focus has been 
to define further the molecular mechanisms that 
dictate the developmental and regulated expres- 
sion of neuroendocrine genes and to apply these 
principles to analysis of neuronal gene expression. 
The extensive characterization of the expression of 
the growth hormone and prolactin genes in related 
cell types within the anterior pituitary gland pro- 
vides an excellent model system for study of devel- 
opmental regulation of gene expression. The ante- 
rior pituitary gland is derived from a common 
primordium originating in Rathke's pouch, an area 
of mesoderm discontinuity that makes the precise 
identification of its ectodermal origins uncertain; 
after proliferation, the five classical specific cell 
types appear in a stereotypical order during ontog- 
eny, defined on the basis of the trophic hormone 
that they elaborate. The structurally related prolac- 
tin and growth hormone genes are expressed in 
discrete cell types— lactotrophs and somatotrophs, 
respectively— with their expression virtually limited 
to the pituitary gland. The lactotrophs and 
somatotrophs are the last cell types to appear in 
the developing rat anterior pituitary. The co-expres- 
sion of these two genes within the single cells prior 
to appearance of mature lactotrophs in a sub- 
population of mature anterior pituitary cells and in 
many pituitary adenomas suggested that the prolac- 
tin and growth hormone genes are developmen- 
tally regulated by related factors. Data from gene-di- 
rected ablation studies in transgenic animals are 
consistent with the notion that lactotroph lineage 
is derived from presomatotrophs. The molecular 
basis of pituitary cell phenotype has been ap- 
proached by characterizing the cis-active elements 
that are necessary for tissue-specific expression and 
then isolating the tissue-specific transcription fac- 
tors that selectively bind to these elements. High 
levels of cell-specific expression of the rat prolactin 
gene are dictated by two separate regions, a distal 
enhancer (-1,830 to -1,530) and a proximal region. 
Although both regions alone are capable of direct- 
ing lactotroph-specific physiological levels of ex- 
pression of fusion genes in transgenic mice, both 
regions acting in concert are required to give full 
physiological levels of expression. 
DNase I footprinting experiments reveal that the 
distal and proximal regulating regions contain mul- 
tiple, related sequences that appear to bind tissue- 
specific nuclear protein(s); two similar binding se- 
quences are present in the 180 bp of rat growth 
hormone 5 '-flanking sequences that are sufficient 
to target somatotroph expression in transgenic 
mice. Mutation of even a single cis-active element in 
either gene can reduce gene expression by 80-98%. 
Binding sites for the tissue-specific transcription 
factor Pit-1 are A/T-rich sequences related to the se- 
quences that bind octamer transcription factors 
and homeodomain proteins. Binding of the Pit-1 
factor to its cognate sequences provided the basis 
for its purification, using affinity chromatography 
and isolation of a Pit-1 clone from rat pituitary and 
G/C cell cDNA expression libraries. The coding se- 
quence of Pit-1 predicted a 291-amino acid 32,800 
Da protein that contained a 60-amino acid se- 
quence with homology to the homeodomains of 
Drosophila regulatory proteins and a second 76- to 
78-amino acid region of homology with several 
other transcriptional or developmental factors, re- 
ferred to as the POU-specific domain (Pit-1, Oct-1, 
Oct-2, and unc-86). Expression of Pit-1 in a non- 
pituitary cell line activates expression of both pro- 
lactin and growth hormone fusion genes, even at 
levels of Pit-1 expression < 10-fold those in pituitary 
(G/C) cells. Expression of the cloned Pit-1 struc- 
tural gene in bacteria generates a 32,900 Da pro- 
tein that specifically binds to the tissue-specific ele- 
ments in the distal and proximal regions of the 
prolactin gene, as well as the growth hormone pro- 
moter, and activates in vitro transcription of both 
prolactin and growth hormone genes at equivalent 
concentrations. These observations raise the ques- 
tion of the roles of Pit-1 and additional factors in 
dictating pituitary phenotype and cell-specific pitu- 
itary gene expression, which is being evaluated 
using biochemical and genetic approaches. 
II. A Large Family of POU Domain Proteins in Mam- 
malian Brain Development. 
Five additional mammalian and two Drosophila 
members of the POU domain gene family have been 
cloned; all of the known POU domain genes are ex- 
pressed during neural development and exhibit 
precisely restricted temporal and spatial patterns of 
Continued 
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