Download Anterior pituitary cells defective in the cell

Development 122, 151-160 (1996)
Printed in Great Britain © The Company of Biologists Limited 1996
DEV4599
151
Anterior pituitary cells defective in the cell-autonomous factor, df, undergo
cell lineage specification but not expansion
Philip J. Gage, Michelle L. Roller, Thomas L. Saunders, Lori M. Scarlett and Sally A. Camper*
Department of Human Genetics, University of Michigan Medical School, Medical Science II M3816, Ann Arbor, MI 48109-0618,
USA
*Author for correspondence
SUMMARY
The Ames dwarf mouse transmits a recessive mutation (df)
resulting in a profound anterior pituitary hypocellularity
due to a general lack of thyrotropes, somatotropes and lactotropes. These cell types are also dependent on the
pituitary-specific transcription factor, Pit-1. We present
evidence that expression of Pit-1 and limited commitment
to these cell lineages occurs in df/df pituitaries. Thus, the
crucial role of df may be in lineage-specific proliferation,
rather than cytodifferentiation. The presence of all three
Pit-1-dependent cell types in clonally derived clusters
provides compelling evidence that these three lineages
share a common, pluripotent precursor cell. Clusters con-
taining different combinations of Pit-1-dependent cell types
suggests that the Pit-1+ precursor cells choose from
multiple developmental options during ontogeny. Characterization of df/df↔+/+ chimeric mice demonstrated that df
functions by a cell-autonomous mechanism. Therefore, df
and Pit-1 are both cell-autonomous factors required for
thyrotrope, somatotrope and lactotrope ontogeny, but their
relative roles are different.
INTRODUCTION
adrenocorticotrophpic hormone (ACTH), appear on embryonic
day 12.5, followed by thyrotropes, producing thyroid stimulating hormone (TSH), at e14.5; somatotropes, producing
growth hormone (GH), at e15.5; gonadotropes, producing
luteinizing and follicle stimulating hormones (LH and FSH,
respectively), at e16.5-17.5; and lactotropes, producing
prolactin (PRL), at e18.5 (Japon et al., 1994). The pituitaryspecific transcription factor, Pit-1 (GHF-1), is first expressed
on e14.5 (Lin et al., 1994) subsequent to the early period of
intense cell proliferation within Rathke’s pouch.
Analysis of classical mouse mutants, ablation experiments
and human pituitary tumors have each provided important
insights into the developmental relationships that exist between
the five anterior pituitary cell types. Anterior pituitary glands
from Ames (df) and Pit-1-defective Snell (dw) dwarf mice are
profoundly hypocellular due to a general absence of thyrotropes, somatotropes and lactotropes (Bartke, 1965; Cheng
et al., 1983; Gage et al., 1995; Phelps et al., 1993). The
existence of these two nonallelic mutations that both impact
the same three pituitary cell types is consistent with the hypothesis that these three lineages are developmentally related. In
addition, transgene ablation experiments have shown that most
lactotropes derive initially from somatotropes (Behringer et al.,
1988; Borrelli et al., 1989), via a GH+, PRL+ intermediate cell
(Hoeffler et al., 1985; Lloyd et al., 1988). Detection of TSH+,
GH+ cells following chemical ablation of the thyroid is consistent with a close relationship between TSH and GH cells
(Horvath et al., 1990). Finally, many human thyrotrope
The specification and expansion of diverse cell lineages from
one or a few stem cell populations are fundamental processes
required for the generation of complex, multicellular tissues
and organisms. The mammalian anterior pituitary gland
provides an excellent model for the study of cell lineage
specification and organogenesis. At embryonic day 8.5 (e8.5)
in the mouse, the anterior and intermediate lobe anlagen begin
to arise from an invagination of oral ectoderm, termed
Rathke’s Pouch, posterior to the anterior neuropore (Schwind,
1928). The posterior lobe arises simultaneously from an evagination of neuroectoderm. Thickening of Rathke’s pouch occurs
concomitantly with separation of the pouch from the oral
ectoderm at e12.5 (Schwind, 1928). Intense cell proliferation
within the ventral wall of Rathke’s pouch from e12.5-14
initiates formation of the nascent anterior pituitary gland (Ikeda
and Yoshimoto, 1991). A second round of increased cell proliferation occurs within the developing anterior pituitary gland
from e16.5-17.5 (Ikeda and Yoshimoto, 1991). Cell proliferation and subsequent cytodifferentiation within the nascent
anterior lobe depend on contact with the overlying neuroectoderm, implying the existence of local inductive influences
(Daikoku et al., 1982, 1983).
The ontogeny of the five anterior pituitary cell types, defined
by activation of their respective polypeptide hormone gene(s),
is temporally and spatially regulated (Japon et al., 1994;
Simmons et al., 1990). In mouse, corticotropes, producing
Key words: cell differentiation, cell autonomous, pituitary gland,
anterior, dwarfism, somatostatin, thyrotropin, prolactin
152
P. J. Gage and others
adenomas also produce growth hormone or prolactin, further
supporting a relationship between the three cell types (Kannan,
1987).
The similarity between the pituitary phenotypes of Ames
and Snell dwarf mice demonstrates that the normal products at
the df and dw loci participate in a common regulatory hierarchy
that is essential for thyrotrope, somatotrope and lactotrope
ontogeny. The df mutation has been localized on mouse chromosome 11 but the nature of the defective gene is unknown
(Buckwalter et al., 1991). The Snell phenotype results from a
mutation within the Pit-1 gene that functionally inactivates the
Pit-1 protein (Camper et al., 1990; Li et al., 1990). A member
of the POU-homeodomain family of transcription factors
(Ruvkun and Finney, 1991), Pit-1 binds to, and transcriptionally activates, the promoter-regulatory elements from several
pituitary genes, including those for TSHβ, GH (Rhodes et al.,
1994), PRL, GRFR and Pit-1 (Chen et al., 1990; Rhodes et al.,
1993). The absence of detectable TSH, GH, PRL, GRFR and
Pit-1 transcripts in pituitaries from adult dw/dw mice establishes that functional Pit-1 is essential for expression of these
genes. The hypocellularity of dw/dw pituitaries is likely to
result from the role of Pit-1 in cell proliferation (Castrillo et
al., 1991).
Pituitary development is regulated by both cell-autonomous
and non-cell-autonomous factors. The product of the dw locus
was shown to be cell autonomous by pituitary transplantation
experiments, prior to its identification as Pit-1 (Carsner and
Rennels, 1960). Non-cell-autonomous factors are likely to be
responsible for the inductive interactions that influence early
pituitary development (Daikoku et al., 1982, 1983; Kawamura
and Kikuyama, 1995; Watanabe, 1982a,b). In addition,
numerous extrinsic factors have been shown to affect proliferation of individual pituitary cell types in adult animals,
including thyroid hormone (Horvath et al., 1990a), growth
hormone releasing factor (Guillemin et al., 1982; Mayo et al.,
1988) and estrogen (Lieberman et al., 1983). However, no
extrinsic factors affecting all three cell types deficient in Ames
dwarf mice have been described. Determining the mechanism
of df action is crucial for understanding the function of this
factor in pituitary ontogeny.
In the present report, we critically assess the effect of the df
mutation on pituitary development, particularly with respect to
the three Pit-1-dependent cell types. We also use chimeric mice
to test whether df functions as a cell-autonomous or non-cellautonomous factor.
MATERIALS AND METHODS
Mice
DF/B-df/df mice were kindly provided by Dr A. Bartke (Southern
Illinois University; Carbondale, IL) in 1988 and have been bred and
maintained at the University of Michigan. Stain 83 mice carry 1000
copies of a β-globin transgene (Tg) (Lo, 1986). The Tg marker was
bred onto the DF/B-df/+ background to generate Tg/Tg,df/df mice.
Homozygous dwarf males (df/df or Tg/Tg,df/df) used for mating were
injected with 2 µg thyroid hormone (T4, Sigma T-0397) and 50 µg
ovine growth hormone (National Hormone Pituitary Program) three
times a week for three weeks to enhance fertility (O’Hara et al., 1988).
Dwarf mice were also fed a diet containing 25 mg thyroid powder per
kg chow (AIN-76A, US Biochemicals), (Eicher and Beamer, 1980).
CD-1 and FVB females were purchased from Charles River Labs.
These experiments were approved by the University of Michigan
Committee on Use and Care of Animals (AAALAC accredited,
Animal Welfare Assistance no. A3114-01) and all mice were housed
and cared for according to NIH guidelines.
Genotyping
The genotype at the df locus was deduced from the dwarf phenotype
(df/df) or by test mating (+/+ or df/+). To assay for the transgene
marker, genomic DNA (250 ng) isolated from tail biopsies (Miller et
al., 1988) was amplified by polymerase chain reaction (PCR) using
the primer sequences (5′-CCAATCTGCTCACACAGGATAGAGAGGGC AGG-3′ and 5′-CCTTGAGGCTGTCCAAGTGATTCAGGCCATCG-3′). In samples containing 1000/2000 copies of the
transgene, 20 amplification cycles were sufficient to visualize the
specific product; in non-transgenic animals 25 cycles were required
to generate a signal from the endogenous β-globin gene.
Generation of aggregation chimeras
Morulae were obtained from Tg/Tg,df/df × Tg/Tg,df/df matings and
matings of albino, non-transgenic CD1 or FVB/N mice. The fertility
of Tg/Tg,df/df mice was enhanced with hormone replacement therapy
(O’Hara et al., 1988). Females were superovulated by injection with
5 I.U. pregnant mare serum gonadotropin, followed 46 hours later
with 5 I.U. human chorionic gonadotropin (Hogan et al., 1986).
Morulae were isolated 2.5 days post coitum (dpc) and Tg/Tg,df/df and
normal CD-1 or FVB/N embryos were aggregated (Hogan et al.,
1986). Based on the proportion of chimeric mice produced, CD-1 and
FVB/N morulae fused with df/df morulae at equal efficiency.
Generation of df/df morulae proved to be the significant ratelimiting step in these experiments due to the infertility of both males
and females. Although the hormone-treated Tg/Tg,df/df males were
prescreened for fertility by breeding, only 55% of superovulated
Tg/Tg,df/df females mated with Tg/Tg,df/df males. In contrast, CD-1
and FVB/N females plugged at a rate of 75% and 98%, respectively.
In situ hybridization histochemistry
Pituitaries from adult chimeric and control mice were fixed with 4%
paraformaldehyde and embedded into TissuePrep 2 (Fisher Scientific). Embedded pituitaries were sectioned at a thickness of 3 µm
(immunohistochemistry only) or 1.5 µm (combined in situ and
immunohistochemistry).
In situ hybridization histochemistry to detect the Tg marker was
performed using elements from several protocols (Lo, 1986; Thomson
and Solter, 1988). A biotinylated β-globin probe was generated by
nick-translation of pMBd2 (Lo, 1986) using a BioNick Translation kit
(BRL). Sections were deparaffinized in Histoclear (National Diagnostics, Inc.) and xylene, rehydrated through graded alcohols, deproteinated in 0.2 N HCl and acetylated in 0.1 M triethanolamine/0.25%
(v/v) acetic anhydride. Target DNA sequences were denatured by
incubating sections at 70°C. Sections were incubated with an empirically determined amount of biotinylated probe. Specifically bound
probe was detected by incubation of slides with strepavidin-horse
radish peroxidase (Detek-HRP, Enzo Diagnostics), followed by a
chromagen solution containing diaminobenzidine (0.0125%), NiCl2
(0.015%) and H2O2 (0.06%), which yielded a blue-black reaction
product.
The percent of labeled nuclei in each of the three pituitary lobes
was determined after scoring the numbers of labeled and total nuclei
in prints from randomly photographed fields of the pituitary sections.
Labeled nuclei were never observed in pituitaries from non-transgenic
negative control animals. Labeling was observed in approximately
80% of the nuclei from non-chimeric Tg/Tg-positive control pituitaries, consistent with previously reported results (Thomson and
Solter, 1988). Therefore, the percent contribution of df/df cells was
calculated by dividing the fraction of labeled nuclei in experimental
sample by the fraction of labeled nuclei in Tg/Tg control samples
processed in parallel.
Intrinsic block in pituitary cell expansion
153
Immunohistochemistry
Tissues for immunohistochemistry were fixed and processed as
described above. Immunohistochemistry with primary antisera
directed against human ACTH (Dako Corp.), rat TSHβ (AFP1274789), rat GH (AFP 4112), rat PRL (AFP425-10-91), rat LHβ
(AFP-222387) or rat Pit-1 (Howard and Maurer, 1994) was performed
as described previously (Kendall et al., 1991). In experiments
designed to detect Pit-1, sections were pretreated with 5 µg pepsin;
0.01 N HCl (pH 8.0) followed by 5 µg saponin in H2O. Bound primary
antibody was visualized using the ABC method (Vector) with
diaminobenzidine as the chromogen. Sections were counterstained
with hematoxylin. Staining for each cell type could be blocked by
preincubation of the primary antiserum with the relevant hormone
(data not shown).
(Jackson and Bennett, 1990; Kwon et al., 1988)). Reaction products
were labeled by substituting α-[32P]-dATP for dATP in the PCR
amplification and were digested overnight with DdeI to distinguish
the albino (130 and 30 bp) and normal (165 bp) Tyr alleles (Fig. 6).
The fragments were fractionated through 7% polyacrylamide and
quantitated using a phosphorimager (Molecular Dynamics) and
ImageQuant analysis software. To account for the difference in the
number of adenosine sites between the two allele-specific fragments,
the raw quantitation for the 130 bp mutant-specific band was multiplied by a factor of 1.18. The contribution of df/df cells to a tissue
was calculated as: cts/minute in 165 bp product divided by cts/minute
in 130 bp product × 1.18. Each tissue sample was measured in triplicate PCR experiments.
Quantitative polymerase chain reaction
Brain, kidney, liver, lung and spleen isolated from chimeric and
control animals were homogenized briefly at low speed with a
Polytron (Brinkman) to disrupt the tissues and the homogenates were
digested overnight at 37°C in 400 µg/ml proteinase K; 50 mM
Tris•HCl, (pH 8.0); 2 mM EDTA and 0.5% Tween 20 (Ramakrishnan et al., 1994). Samples were heated at 95°C for 10 minutes to
denature residual proteinase K activity and immediately added to
polymerase chain reaction (PCR) amplifications. PCR (primers: 5′TCAAAGGGGTGGATGACCG-OH and 5′-GACACATAGTAATGCATCC) was used to amplify a 340 bp region from within the
coding region for the tyrosinase gene (Tyr) (nucleotides +212 to +552,
RESULTS
TSH
Immunodetection of Pit-1-dependent cell lineages in
df/df pituitaries
While df/df pituitaries are hypocellular and generally devoid of
the three Pit-1-dependent cell types, the demonstration of rare
somatotropes in df/df mice suggested that thyrotropes and lactotropes might also be present (Gage et al., 1995). Normal
anterior pituitary glands immunostained for the presence of
TSH, GH and PRL contain 2-5% thyrotropes, 30-40% somatotropes and 20-40% lactotropes (Fig. 1a-c) ((Kendall et al.,
GH
PRL
wt
df
df
Fig. 1. Identification of rare thyrotropes, somatotropes and lactotropes in df/df pituitaries. (a-c) Normal and (d-i) df/df pituitaries were
immunostained to detect the presence of the pituitary hormones TSH (a,d,g), GH (b,e,h), PRL (c,f,i), or an individual hormone and Pit-1 (g-i).
In experiments designed to detect a single hormone (a-f), DAB was used as chromogen (brown). For double immunostaining experiments (g-i),
DAB and VectorVIP (violet) were used as chromagens to detect Pit-1 and hormone, respectively. Although the normal and mutant pituitary
sections were photographed at the same magnification, only a portion of the anterior lobe is visible in the normal pituitary, but all three lobes of
the hypocellular df/df pituitary are depicted. Cells staining for TSH, GH or PRL were consistently observed in df/df pituitaries (arrowheads, df). However, the frequency of these cells was rare compared to wild-type pituitaries (compare a-c versus d-f). All TSH+, GH+, or PRL+ cells
having an identifiable nucleus present in the section also expressed Pit-1. Bar, 100 µm (a-f) or 10 µm (g-i).
154
P. J. Gage and others
Table 1. Content of hormone-positive clusters suggests
multiple terminal differentiation pathways
Positive Cells Per 1/3 Pituitary
140
120
100
80
60
1
2
3
Total
clusters
TSH
GH
PRL
TSH, GH
GH, PRL
TSH, PRL
TSH, GH, PRL
7
1
4
1
2
2
1
3
3
3
0
1
1
2
5
3
0
0
1
1
1
15
7
7
1
4
4
4
*Hormones present within cells of an individual cluster. No cells staining
for multiple hormones were observed.
†Number of clusters for each category in three different individuals.
40
20
0
df/df pituitary†
Hormones
present*
1
2
3
4
Fig. 2. Quantitation of thyrotropes, somatotropes and lactotropes in
dwarf pituitaries. Pituitaries from Tg/Tg,df/df (1-4) mice were
sectioned in their entirety and immunostained with antisera specific
for TSH, GH or PRL. For each animal, 1/3 of the sections, taken
from throughout the organ, were immunostained for each hormone.
The total number of hormone-expressing cells for each lineage was
then scored. Key: black, TSH; gray, GH; white, PRL.
1991) and S. Kendall, unpublished results). Pituitary sections
from four Ames dwarf animals revealed the presence of
occasional cells immunostained for TSH, GH or PRL in each
of the animals (Fig. 1d-f). These rare cells occurred either individually or in small clusters of 2 to 20 cells per section. Quantitation of the rare hormone-positive cells revealed that each
df/df pituitary contains several hundred thyrotropes and somatotropes, as well as a few lactotropes (Fig. 2). These numbers
represent less than 1% of the normal component of these cells
((Kendall et al., 1991; Sasaki and Sano, 1982), P. Gage, data
not shown). Thus, commitment to all three Pit-1-dependent cell
lineages can occur in df/df pituitaries. Consistent with previous
reports (Lin et al., 1994; Slabaugh et al., 1981), these cells were
never observed dw/dw pituitaries (data not shown).
The previous demonstration that Pit-1 is required for
hormone-positive thyrotropes, somatotropes and lactotropes in
adults prompted us to determine whether Pit-1 was present in
these rare cells in df/df animals. Sections were immunostained
for both Pit-1 and the hormones TSH, GH and PRL. Cells
expressing both Pit-1 and each of these hormones were
observed in pituitary sections from multiple df/df animals
(n=3) (Fig. 1g-i). Although not a quantitative assay, the
intensity of staining for Pit-1 and the various hormones
appeared to be equivalent, on a per cell basis, between df/df
and normal cells. There were no examples of hormone-positive
cells that lacked Pit-1. Thus, the df/df thyrotropes, somatotropes and lactotropes appear to be Pit-1-dependent and arise
via normal developmental pathways.
Clusters of Pit-1+ cells staining positively for an individual
hormone frequently contained Pit-1+ cells that were negative
for that hormone (Fig. 1g-i), suggesting that clusters of Pit-1+
cells might consist of more than one differentiated cell lineage.
This hypothesis was confirmed by examining df/df pituitary
sections immunostained for the presence of all three hormones,
using a different chromogen to detect each. Clusters contained
one, two or three of the Pit-1-dependent cell lineages (Table
1). In clusters containing two of the cell lineages, each possible
pairwise combination was observed.
Generation of chimeric mice
We tested whether df is cell-autonomous or non-cell
autonomous by assaying for the behavior of df/df cells in
aggregation chimeric mice consisting of df/df and normal cells
(+/+). If df is non-cell autonomous, the number of df/df cells
that populate the thyrotrope, somatotrope and lactotrope
lineages should be enhanced by the presence of normal cells
in the chimera. If df is cell autonomous, the intrinsic defect
would prohibit any increase in df/df cell differentiation.
Chimeric mice were constructed such that the two cell types
could be distinguished and quantitated both histologically and
genetically (Table 2). A 1000-copy β-globin transgene (Tg)
detectable by in situ hybridization (Lo, 1986) was introduced
onto the DF/B-df/df background from which the df/df-cell
donor embryos were isolated. The utility of this transgene as a
histological marker for cell lineage analyses in chimeric
animals has been established previously (Thomson and Solter,
1988). All known albino mouse strains carry a G to C transversion in codon 103 of the tyrosinase (Tyr) gene (Jackson and
Bennett, 1990; Yokoyama et al., 1990) that also leads to a Dde
I restriction fragment length polymorphism (RFLP). Since the
normal cells were derived from albino mice this RFLP was
exploited to discriminate between the wild-type and mutant tyr
alleles (Jackson and Bennett, 1990) present in df/df and normal
cells, respectively (Table 2).
Chimeric mice were constructed by morulae aggregation. A
total of 16 chimeric mice were obtained. Coat color chimerism
is a good indication of the relative contribution of cells from
Table 2. Cell types used to construct chimeric mice
Cell type
Locus
df
tyrosinase*
Marker transgene†
Dwarf
Normal
df/df
Pigmented (C/C)
Tg/Tg
+/+
Albino (c/c)
nontransgenic
*Used for phenotypic and genotypic quantitation of df/df cell contribution
to chimeric tissues.
†1000-copy β-globin transgene present in df/df cells.
Intrinsic block in pituitary cell expansion
155
Fig. 3. Underrepresentation of df/df
cells in the anterior lobe
(ant) relative to the
posterior (post) and
intermediate (int) lobes.
Paraffin-embedded
chimeric pituitaries were
sectioned at approximately
the thickness of one cell
(1.5-3 µm) and processed
by in situ hybridization
histochemistry to detect
the transgene tag present
in df/df cells. Specifically
bound probe was detected
by incubation of sections
with a strepavidin-horse
radish peroxidase
conjugate (Enzo
Diagnostics) followed by DAB and NiCl2. Nuclei of df/df cells contained two dark, black foci of reaction product corresponding to the two Tgmarked chromosomes (Tg/Tg) (arrowheads). Results shown are from chimera 1.
each parent to the tissues of the chimeric animal (Vogt et al.,
1987). Based on this criterion, several mice contained high
df/df cell contributions, demonstrating that df/df cells were able
to contribute to chimeric mice with high efficiency. All
chimeras displayed normal growth and adult body size, regardless of the df/df cell contribution. Six chimeric mice with
medium to high df/df cell contributions were examined in
detail. The anterior pituitary glands from these mice were
normal in size and appearance. Therefore, the overall contribution of even an apparently small number of normal cells to
the chimeric mice could rescue both the somatic growth defect
and the hypopituitarism characteristic of non-chimeric df/df
mice.
Histology of chimeric pituitaries
Pituitary glands were examined by in situ hybridization histochemistry designed to detect the transgene marker present in
df/df cells. Consistent with previously reported results
(Kusakabe et al., 1988), the two parental cell types were
dispersed essentially randomly throughout the pituitary glands
of the chimeras. The number of Tg-marked df/df cells in the
anterior pituitary gland was dramatically reduced relative to the
posterior and intermediate lobes (Fig. 3). The percent contribution of df/df cells to each lobe of these pituitaries was quantified by in situ analysis (Fig. 4). In chimera 1, df/df cells represented only 30% of the anterior lobe versus 90% for the
posterior and intermediate lobes. Similar results were obtained
in each of the other chimeras except number 5. The df/df cell
contribution was reduced in both the anterior and intermediate
lobes of chimera 5, possibly reflecting the common origin of
these lobes from Rathke’s pouch. The anterior pituitary gland
of all six chimeras was composed primarily of wild-type cells.
These results demonstrate that df/df pituitary cells have an
intrinsic defect in cell proliferation or survival, consistent with
the profound hypocellularity of non-chimeric df/df anterior
pituitary glands.
In order to classify the Tg-marked df/df cells into one of the
five anterior pituitary cell lineages, in situ-labeled sections
were immunostained with antisera specific for the individual
anterior pituitary hormones. Most df/df cells were corticotropes
or gonadotropes (data not shown). Thyrotropes, somatotropes,
and lactotropes originating from df/df cells were only rarely
observed, even in chimeras with exceptionally high overall
df/df cell contributions (Fig. 5). There was no increase in the
number of these cells per section relative to that observed in
non-chimeric df/df mice. Therefore, the normal cells within the
chimeric animals did not enhance the contribution of df/df cells
to the thyrotrope, somatotrope and lactotrope lineages.
Quantitation of df/df cell contribution to peripheral
tissues
Quantitative PCR was used to confirm that the under representation of df/df cells was specific to the anterior pituitary
gland (Fig. 6). This approach exploited the DdeI RFLP
between the wild-type and mutant tyrosinase alleles present in
the df/df and normal cells, respectively. The df/df cell contributions to the brain, kidney, liver, lung and spleen from the six
mice were quantitated. The df/df cell contribution to the five
peripheral organs, as well as to the posterior and intermediate
lobes of the pituitary gland, were all approximately equivalent
(Fig. 4). These quantitative results also matched estimates of
df/df cell contribution to each mouse based on coat color
chimerism (data not shown). However, the df/df cell contribution to an animal’s anterior pituitary gland was significantly
reduced relative to the other organs. For example, the df/df cell
contribution to the anterior pituitary gland of chimera 1 was
only 32% but the df/df cell contribution to all of the other
organs ranged from 75-98% and averaged 90% (Fig. 4). This
established that the lack of expansion by df/df cells was specific
to the anterior pituitary gland.
DISCUSSION
Specification and expansion of differentiated cell lineages
within the mammalian anterior pituitary gland is controlled by
a hierarchy of genes encoding both cell-autonomous and noncell-autonomous factors. The study of classical mouse mutants
P. J. Gage and others
156
% Contribution by df/df-Derived Cells
Chimera 1
Chimera 2
100
90
80
70
60
50
40
30
20
10
0
Br Ki Li Lu Sp
P
I
A
Chimera 4
% Contribution by df/df-Derived Cells
Chimera 3
Br Ki Li Lu Sp
P
I
A
Chimera 5
Br Ki Li Lu Sp
P
I
A
P
I
A
Chimera 6
100
90
80
70
60
50
40
30
20
10
0
Br Ki Li Lu Sp
GH
P
I
A
Br Ki Li Lu Sp
P
I
A
TSH
Br Ki Li Lu Sp
Fig. 4. The df/df cell
contribution to the
anterior pituitary is
low relative to the
posterior and
intermediate
pituitaries, and five
peripheral tissues
Results for PCR (open
columns) and in situ
(shaded columns).
Quantitation of df/df
cell contributions to
the 6 chimeric mice
studied are shown.
Results from each
data set were derived
from triplicate PCR
experiments. Bars
represent the standard
deviation of the mean.
Br, brain; Ki, Kidney;
Li, liver; Lu, lung; Sp,
spleen; P, posterior
pituitary; I,
intermediate pituitary;
A, anterior pituitary.
PRL
Fig. 5. Demonstration that df/df thyrotropes, somatotropes and lactotropes are rare in chimeric pituitaries. Sections of chimeric pituitaries
previously in situ-stained to detect the Tg marker in df/df cells were subsequently immunostained for the presence of GH, TSH or PRL using
DAB as the chromogen. Doubly-stained cells (arrowheads) containing both the blue-black nuclear in situ signal and the brown immunostaining
signal were rare, but detectable, in all 6 chimeric pituitaries. Results shown are from chimera 5. Most df/df cells observed were corticotropes
and gonadotropes (data not shown).
has not only resulted in the molecular identification of several
factors within this hierarchy but has also established their functional relevance in vivo. For example, the functional role of the
cell-autonomous factor, Pit-1, in pituitary cell lineage specification and proliferation was demonstrated through analysis of
the Snell dwarf mouse (Camper et al., 1990; Li et al., 1990).
Intrinsic block in pituitary cell expansion
Fig. 6. PCR assay used to quantitate df/df cell contribution to
peripheral tissues. The df/df cell contribution to five peripheral
tissues from the chimeric mice was quantitated. A DdeI
polymorphism between the wild-type (C/C) and mutant (c/c)
tyrosinase alleles (Yokoyama et al., 1990) present in df/df and
normal cells, respectively, was exploited to quantitate the
contribution of each cell type to peripheral tissues. PCR
amplification of a 340 bp target sequence spanning the polymorphic
site and digestion of the resulting products with DdeI (arrows)
generates fragments diagnostic for both alleles. An autoradiograph
demonstrating results from experiments programmed with genomic
DNA from control animals (lanes 1-9) and tissues from chimera 5
(lanes 10-15) is shown. Quantification of the wild-type (165 bp) and
mutant (130 bp) tyrosinase allele-specific bands in experiments
programmed with control DNAs at varying ratios (df/df:+/+) (lanes
4-9) yielded the expected ratios for the two cell types and established
that the assay was quantitative over the range used. The
predominance of the wild-type-allele-specific 165 bp band
demonstrates that the brain (Br), liver (Li), lung (Lu), spleen (Sp)
and kidney (Ki) were derived largely from df/df cells.
Analysis of the less severe murine little mutation established
the importance of an extrinsic factor, the hypothalamic neuropeptide growth hormone-releasing factor, and its receptor for
proliferation of committed somatotropes (Godfrey et al., 1993;
Lin et al., 1992). The Ames dwarf mouse offers another opportunity for insight into anterior pituitary ontogeny since the df
mutation defines a factor that is crucial in the development of
the three Pit-1-dependent cell lineages. The near identity of the
Ames and Snell dwarf phenotypes has provided compelling
evidence that df and Pit-1 participate in a common developmental program during pituitary ontogeny. We have demonstrated that df is a cell-autonomous factor that is required for
proliferation, but not commitment, of cells within the thyrotrope, somatotrope and lactotrope lineages.
The detection of rare Pit-1+ cells expressing TSH, GH or
PRL in df/df pituitaries represents a fundamental distinction
between the effects of the df and dw mutations on thyrotrope,
somatotrope and lactotrope ontogeny. If the df mutation is a
complete loss-of-function allele, then these data demonstrate
that df is not absolutely required for lineage specification,
including activation of Pit1 or the relevant hormone genes.
Rather, its major role must be in the expansion or survival of
157
these three lineages. Ectopic programmed cell death does not
appear to account for the hypocellularity of df/df anterior pituitaries since we have observed no evidence of apoptotic nuclei.
Thus, df likely plays a central role in efficient cell proliferation within the thyrotrope, somatotrope and lactotrope
lineages. Alternatively, if the df mutation is a partial loss-offunction mutation, then df may have a role in lineage specification and proliferation. An example of such a partial loss-offunction allele is provided by the point mutation within the Pit1
gene that destroys the ability of Pit-1 to transactivate the GH
and PRL genes, but leaves the TSHβ activation and cell proliferation functions intact (Pfaffle et al., 1992).
We considered the possibility that the rare committed cell
types in df/df mice result from genetic mechanisms such as
somatic reversion or suppression, phenomena observed for
several types of mutations, including retroviral insertion
(Copeland et al., 1983), DNA duplication (Brilliant et al.,
1992), small insertions and deletions, and point mutations
(Greenspan et al., 1988). However, the frequencies of these
events are at least an order of magnitude less than the frequencies of commitment that we have documented in Ames
dwarf mice. Moreover, we have never observed df/df animals
with near normal growth as would be expected if reversion
occurred early in gestation (Melvold, 1971). Thus these explanations seem less likely than the idea that the clusters represent
committed cells arrested in development due to the lack of a
lineage-specific cell proliferation factor, df.
The Pit-1+ cells in df/df pituitaries occurred within small,
defined clusters, suggestive of a clonal origin. Some clusters
were homogeneous, consisting of a single Pit-1-dependent cell
type. Heterogeneous clusters containing each possible pairwise
combination or all three cell types were also detected. Over
20% of the clusters contained thyrotropes together with somatotropes, lactotropes, or both. This is consistent with the
hypothesis that the three Pit-1-dependent cell lineages share a
common progenitor (Fig. 7). The heterogeneity of the clusters
also suggests that each Pit-1+ progenitor did not follow the
same, rigidly defined developmental program. Rather, distinct
clusters, and the individual cells within them, must have the
potential to complete different developmental program(s). The
numerous examples of Pit-1+ clusters composed of only thyrotropes, somatotropes or lactotropes indicates that each cell
type has the capacity to differentiate directly from the Pit-1+
progenitor independently of the others (Fig. 7). The direct
differentiation to lactotropes without a GH+ intermediate may
not be the primary mechanism for generation of lactotropes.
The potential for more than one route could explain the
inability to completely eliminate lactotropes via somatotrope
ablation (Behringer et al., 1988; Borrelli et al., 1989).
The hypothesis that somatotropes, lactotropes and thyrotropes derive from a common progenitor is consistent with
examples in other systems (Anderson, 1989). Mammalian
neural crest cell differentiation and hematopoesis, and
Drosophila neurogenesis each involve the derivation of
multiple cell lineages from a common precursor. However
other relationships between the Pit-1+ precursor and the three
differentiated cell types have been proposed. These range from
the strictly linear (see e.g. (Rosenfeld, 1991)) to the complex
(see e.g., Karin et al., 1990). The most attractive alternative
hypothesis to the common progenitor model is one in which
thyrotropes arise from a set of Pit-1+ precursors that are distinct
158
P. J. Gage and others
e12
e14.5
df
Pit-1
Unknown
Factor(s)
e16.5
df
Pit-1
e18.5
df
Pit-1
TSH
TRH
df
Pit-1
GH
GRF
df, Pit-1
GH, PRL
df
Pit-1
PRL
E2
Fig. 7. Model for ontogeny of thyrotropes, somatotropes and
lactotropes. Two periods of intensified cell proliferation occur during
anterior pituitary ontogeny (bars). df is crucial for the later
proliferative phase and may impact the earlier phase as well. Because
df is cell autonomous, it must be expressed within the proliferating
cells. A progenitor cell expresses Pit-1 and at least one additional,
unknown factor in order to generate the fully differentiated
thyrotropes, somatotropes and lactotropes (Lew et al., 1993). Each of
the three differentiated cell types can be generated directly from a
common progenitor by stochastic and/or extrinsically determined
processes. However, most lactotropes are likely to derive from
somatotropes via a GH+, PRL+ intermediate cell. Proliferation and
function of thyrotropes, somatotropes and lactotropes are stimulated
by thyrotropin releasing hormone (TRH) (Horvath et al., 1990),
growth hormone-releasing factor (GRF) (Guillemin et al., 1982;
Hammer et al., 1985) and estrogen (E2) (Lieberman et al., 1983;
Lloyd, 1983), respectively. The df mutation renders the cells unable
to respond to these extrinsic cues.
from those that produce somatotropes and lactotropes
(Borrelli, 1994; Karin et al., 1990). If this is the case, the
frequent appearance of thyrotropes with somatotropes and lactotropes in df/df pituitaries may suggest local interactions
which are facilitated by aggregation of the two lineages. Nevertheless, the high frequency with which somatotrope and/or
lactotropes appear independently of thyrotropes implies that
these interactions are either not essential or transient.
The interdependence of thyrotrope, somatotrope and lactotrope differentiation is supported by the analysis of mice
generated by gene targeting that are deficient in the pituitary
hormones TSH, LH and FSH. These dwarf mice exhibit
extreme hyperplasia of thyrotropes, apparently at the expense
of somatotrope and lactotrope proliferation (Kendall et al.,
1995). One interpretation of this data is that when Pit-1+
precursor cells are recruited to produce thyrotropes, fewer
somatotropes and lactotropes can be formed (Fig. 7). Alternatively, thyrotropin or thyroid hormone might be required in
order to attain normal numbers of somatotropes and lactotropes. The importance of thyrotropes for somatotrope and
lactotrope differentiation could be resolved by examining the
effects of thyrotrope ablation.
Regardless of whether the differentiation of thyrotropes,
somatotropes and lactotropes involves a single common, or
multiple progenitor(s), it is clear that the pathway involves
more steps than are defined by the current mouse mutants (Fig.
7). The atypical cells detected in df/df and dw/dw pituitaries by
ultrastructural analysis may represent undifferentiatied precursors that accumulate prior to the expression of df and Pit1
(Cheng et al., 1983). Activation of Pit-1 expression is an intermediate step in the cytodifferentiation pathway. A Pit-1+ cell
line that fails to express any of the hormone genes reveals that
other factors in addition to Pit-1 are required for progression
through the pathway and provides a model for the study of one
intervening step (Lew et al., 1993). The next step, diversification into individual differentiated phenotypes, is probably
regulated by lineage-specific transcriptional activation and
repression events downstream of the Pit-1+ precursor cell.
Distinct transcriptional activators are involved in Pit-1dependent activation of the TSHβ, GH and PRL promoters
(Rhodes et al., 1994).
The size of the anterior pituitary gland and proportion of
each of the three differentiated cell types is probably regulated
by extrinsic factors such as TRH and thyroid hormone, growth
hormone releasing factor and estrogen (Fig. 7). Some examples
of factors that regulate the expansion of distinct sublineages by
stimulating lineage-specific proliferation of precursor cells
include the lymphokines in hematopoiesis and both extracellular matrix and the pituitary hormone α-MSH in neural crest
differentiation (Anderson, 1989). A particularly striking
feature of the aggregation chimeric mice was the ability of the
wild-type cells to generate a normal-sized anterior pituitary
gland composed of the appropriate ratios of the five endocrine
cell types. This was achieved even in chimeric mice composed
almost entirely of df/df cells, suggesting that relatively few
progenitors are sufficient to populate the entire lobe and that
the extrinsic cues effectively regulate the proportions of each
cell type. It is important to note that this compensation by
normal cells is restricted to the Pit-1-dependent cell types since
corticotrope and gonadotrope pools in these animals are
composed primarily of df/df cells. The colonization of the liver
by normal cells in c-jun−/−↔wild-type chimeric mice is similar
to the phenomena that we observed in the anterior pituitaries
of df/df↔+/+ chimeras (Hilberg et al., 1993). However, not
all organogenesis defects can be compensated so fully by
normal cells. For example, mice chimeric for the cellautonomous staggerer mutation had underdeveloped cerebellums due to the fact that wild-type cells could only partially
compensate for the Purkinje cell defect (Herrup and Mullen,
1979). Thus, the precise control of anterior pituitary cell
number and cell type composition by extrinsic cues is remarkable. It may reflect the fact that these pituitary cell types retain
their proliferative capacity into adulthood (Bach et al., 1995;
Horvath et al., 1990). In addition, adult mice retain the ability
to respond to changing demands, such as pregnancy and
lactation, by altering the proportion of individual pituitary cell
types. The inability of df/df cells to recognize or respond to
these proliferative signals demonstrates that df plays a central
role in mediating these events, potentially in both fetal and
adult mice.
The limited number of Pit-1+ cells in df/df mice suggests that
Intrinsic block in pituitary cell expansion
df is essential for the second period of intensified pituitary cell
proliferation at e15.5-16.5 that occurs after the activation of
Pit-1 at e14.5 (Ikeda and Yoshimoto, 1991). Given that Pit-1
is important for proliferation in cell culture, the small clusters
observed in df/df mice may result directly from Pit-1
expression (Castrillo et al., 1991), however df may be
important for efficient Pit-1-mediated cell proliferation. Many
important developmental control genes are required at more
than one time during ontogeny. For example genes encoding
the helix-loop-helix proteins that are crucial for initating sex
determination in early Drosophila embryogenesis are also
required later in development for the initiation of neurogenesis (Jan and Jan, 1990). Thus, it is possible that df is also
required for the earlier wave of cell proliferation that occurs
prior to the activation of Pit-1 (<e14.5) (Ikeda and Yoshimoto,
1991). Further molecular analysis of the effects of the df
mutation and molecular identification of the df gene will help
to resolve these questions.
We thank Davor Solter for the strain 83 mice and the pMBd2
plasmid, and Richard A. Maurer the Pit-1 antiserum. We acknowledge Cecelia Lo, who constructed the strain 83 mice, for encouraging discussions, Ricardo V. Lloyd and Long Jin for contributions early
in the project and Shirley Tilghman and David Burke for helpful
advice and critical reading of the manuscript. We acknowledge the
NHPP, the NIDDKD, the NICHHD and the USDA for supplying the
antihormone antisera. This work was supported by The University of
Michigan Developmental Biology Training Grant (P. J. G.), and the
American Cancer Society and the National Institutes of Health (S. A.
C).
REFERENCES
Anderson, D. (1989). The neural crest cell lineage problem: neuropoiesis?
Neuron 3, 1-12.
Bach, I., Rhodes, S., Pearse, R., II, Heinzel, T., Gloss, B., Scully, K.,
Sawchenko, P. and Rosenfeld, M. (1995). P-Lim, a LIM homeodomain
factor, is expressed during pituitary organ and cell commitment and
synergizes with Pit-1. Proc. Natl Acad. Sci. USA 92, 2720-2724.
Bartke, A. (1965). The response of two types of dwarf mice to growth
hormone, thyrotropin, and thyroxine. Gen. Comp. Endocrinol. 5, 418-426.
Behringer, R., Mathews, L., Palmiter, R. and Brinster, R. (1988). Dwarf
mice produced by genetic ablation of growth hormone-expressing cells.
Genes Dev. 2, 453-461.
Borrelli, E. (1994). Pitfalls during development: controlling differentiation of
the pituitary gland. Trends Genetics 10, 222-4.
Borrelli, E., Heyman, R., Arias C, Sawchenko, P. and Evans, R. (1989).
Transgenic mice with inducible dwarfism. Nature 339, 538-541.
Brilliant, M., Gondo, Y. and Eicher, E. (1992). The mouse pink-eyed
unstable mutation: a DNA duplication revealed by genome scanning.
Pigment Cell Res. Suppl 2, 271-4.
Buckwalter, M., Katz, R. and Camper, S. (1991). Localization of the
panhypopituitary dwarf mutation (df) on mouse chromosome 11 in an
intersubspecific backcross. Genomics 10, 515-26.
Camper, S., Saunders, T., Katz, R. and Reeves, R. (1990). The Pit-1
transcription factor gene is a candidate for the murine Snell dwarf mutation.
Genomics 8, 586-90.
Carsner, R. and Rennels, E. (1960). Primary site of gene action in anterior
pituitary dwarf mice. Science 131, 829.
Castrillo, J., Theill, L. and Karin, M. (1991). Function of the homeodomain
protein GHF1 in pituitary cell proliferation. Science 253, 197-9.
Chen, R., Ingraham, H., Treacy, M., Albert, V., Wilson, L. and Rosenfeld,
M. (1990). Autoregulation of pit-1 gene expression mediated by two cisactive promoter elements. Nature 346, 583-6.
Cheng, T., Beamer, W., Phillips, J., Bartke, A., Mallonee, R. and Dowling,
C. (1983). Etiology of growth hormone deficiency in little, Ames, and Snell
dwarf mice. Endocrinology 113, 1669-78.
159
Copeland, N., Hutchison, K. and Jenkins, N. (1983). Excision of the DBA
ecotropic provirus in dilute coat-color revertants of mice occurs by
homologous recombination involving the viral LTRs. Cell 33, 379-387.
Daikoku, S., Chikamori, M., Adachi, T. and Maki, Y. (1982). Effect of the
basal diencephalon on the development of Rathke’s pouch in rats: a study in
combined organ cultures. Dev. Biol. 90, 198-202.
Daikoku, S., Chikamori, M., Adachi, T., Okamura, Y., Nishiyama, T. and
Tsuruo, Y. (1983). Ontogenesis of hypothalamic immunoreactive ACTH
cells in vivo and in vitro: role of Rathke’s pouch. Dev. Biol. 97, 81-8.
Eicher, E. and Beamer, W. (1980). New mouse dw allele: Genetic location
and effects on lifespan and growth hormone levels. J. Hered. 71, 187-190.
Gage, P., Lossie, A., Scarlett, L., Lloyd, R. and Camper, S. (1995). Ames
dwarf mice exhibit somatotrope commitment but lack growth hormonereleasing factor response. Endocrinology 136, 1161-7.
Godfrey, P., Rahal, J., Beamer, W., Copeland, N., Jenkins, N. and Mayo, E.
(1993). GHRH receptor of little mice contains a missense mutation in the
extracellular domain that disrupts receptor function. Nat. Genet. 4, 227-231.
Greenspan, J., Xu, F. and Davidson, R. (1988). Molecular analysis of ethyl
methanesulfonate-induced reversion of a chromosomally integrated mutant
shuttle vector gene integrated into chromosomal DNA. Mol. Cell. Biol. 85,
4185-4189.
Guillemin, R., Brazeau, P., Bohlen, P., Esch, F., Ling, N. and Wehrenberg,
W. (1982). Growth hormone-releasing factor from a human pancreatic tumor
that caused acromegaly. Science 218, 585-587.
Hammer, R., Brinster, R., Rosenfeld, M., Evans, R. and Mayo, K. (1985).
Expression of human growth hormone-releasing factor in transgenic mice
results in increased somatic growth. Nature 315, 413-6.
Herrup, K. and Mullen, R. (1979). Staggerer chimeras: intrinsic nature of
purkinje cell defects and implications for normal cerebellar development.
Brain Research 178, 443-457.
Hilberg, F., Aguzzi, A., Howells, N. and Wagner, E. (1993). c-Jun is essential
for normal mouse development and hepatogenesis. Nature 365, 179-181.
Hoeffler, J., Boockfor, F. and Frawley, L. (1985). Ontogeny of prolactin cells
in neonatal rats: initial prolactin secretors also release growth hormone.
Endocrinology 117, 187-95.
Hogan, B., Costantini, F. and Lacy, E. (1986). Manipulating the Mouse
Embryo. Cold Spring Harbor Laboratory, Cold Spring Harbor.
Horvath, E., Lloyd, R. and Kovacs, K. (1990). Propylthiouracyl-induced
hypothyroidism results in reversible transdifferentiation of somatotrophs
into thyroidectomy cells. Lab. Invest. 63, 511-520.
Howard, P. and Maurer, R. (1994). Thyrotropin releasing hormone
stimulates transient phosphorylation of the tissue-specific transcription
factor, Pit-1. J. Biol. Chem. 269, 28662-9.
Ikeda, H. and Yoshimoto, T. (1991). Developmental changes in proliferative
activity of cells of the murine Rathke’s pouch. Cell Tissue Res. 263, 4147.
Jackson, I. and Bennett, D. (1990). Identification of the albino mutation of
mouse tyrosinase by analysis of an in vitro revertant. Proc. Natl Acad. Sci.
USA 87, 7010-4.
Jan, Y. and Jan, L. (1990). Genes required for specifying cell fates in
Drosophila embryonic sensory nervous system. Trends Neurol. Sci. 13, 4938.
Japon, M., Rubinstein, M. and Low, M. (1994). In situ hybridization analysis
of anterior pituitary hormone gene expression during fetal mouse
development. J Histochem. Cytochem. 42, 1117-25.
Kannan, C. (1987). The Pituitary Gland. New York: Plenum Med. Book Co.,
Karin, M., Castrillo, J.-L. and Theill, L. (1990). Growth hormone gene
regulation: A paradigm for cell-type-specific gene activation. Trends Genet.
6, 92-96.
Kawamura, K. and Kikuyama, S. (1995). Induction from posterior
hypothalamus is essential for the development of the pituitary
proopiomelacortin (POMC) cells of the toad (Bufo japonicus). Cell Tissue
Res. 279, 233-239.
Kendall, S., Saunders, T., Jin, L., Lloyd, R., Glode, L., Nett, T., Keri, R.,
Nilson, J. and Camper, S. (1991). Targeted ablation of pituitary
gonadotropes in transgenic mice. Mol. Endocrinol. 5, 2025-36.
Kendall, S., Samuelson, L., Saunders, T., Wood, R. and Camper, S. (1995).
Targeted disruption of the pituitary glycoprotein α-subunit produces
hypogonadal and hypothyroid mice. Genes Dev. 9, 2007-2019.
Kusakabe, M., Yokoyama, M., Sakakura, T., Nomura, T., Hosick, H. and
Nishizuka, Y. (1988). A novel methodology for analysis of cell distribution
in chimeric mouse organs using a strain specific antibody. J. Cell Biol. 107,
257-65.
Kwon, B., Wakulchik, M., Haq, A., Halaban, R. and Kestler, D. (1988).
160
P. J. Gage and others
Sequence analysis of mouse tyrosinase cDNA and the effect of melanotropin
on its gene expression. Biochem. Biophys. Res. Commun. 153, 1301-9.
Lew, D., Brady, H., Klausing, K., Yaginuma, K., Theill, L., Stauber, C.,
Karin, M. and Mellon, P. (1993). GHF-1-promoter-targeted
immortalization of a somatotropic progenitor cell results in dwarfism in
transgenic mice. Genes Dev. 7, 683-93.
Li, S., Crenshaw, E., Rawson, E., Simmons, D., Swanson, L. and Rosenfeld,
M. (1990). Dwarf locus mutants lacking three pituitary cell types result from
mutations in the POU-domain gene pit-1. Nature 347, 528-33.
Lieberman, M., Slabaugh, M., Rutledge, J. and Gorski, J. (1983). The role
of estrogen in the differentiation of prolactin producing cells. J. Steroid
Biochem. 19, 275-281.
Lin, C., Lin, S., Chang, C. and Rosenfeld, M. (1992). Pit-1-dependent
expression of the receptor for growth hormone releasing factor mediates
pituitary cell growth. Nature 360, 765-8.
Lin, S., Li, S., Drolet, D. and Rosenfeld, M. (1994). Pituitary ontogeny of the
Snell dwarf mouse reveals Pit-1-independent and Pit-1-dependent origins of
the thyrotrope. Development 120, 515-22.
Lloyd, R., Anagnostou, D., Cano, M., Barkan, A. and Chandler, W. (1988).
Analysis of mammosomatotropic cells in normal and neoplastic human
pituitary tissue by the reverse hemolytic plaque assay and
immunocytochemistry. J. Clin. Endocrinol. Metab. 66, 1103-1110.
Lloyd, R. (1983). Estrogen-induced hyperplasia and neoplasia in the rat
anterior pituitary gland: an immunohistochemical study. Am. J. Pathol. 113,
198-206.
Lo, C. (1986). Localization of low abundance DNA sequences in tissue
sections by in situ hybridization. J. Cell Sci. 81, 143-62.
Mayo, K., Hammer, R., Swanson, L., Brinster, R., Rosenfeld, M. and
Evans, R. (1988). Dramatic pituitary hyperplasia in transgenic mice
expressing a human growth hormone-releasing factor gene. Mol. Endocrinol.
2, 606-12.
Melvold, R. (1971). Spontaneous somatic reversion in mice. Effects of parental
genotype on stability at the p-locus. Mutat. Res. 12, 171-4.
Miller, S., Dykes, D. and Polesky, H. (1988). A simple salting out procedure
for extracting DNA from human nucleated cells. Nucleic Acids Res. 16,
1215.
O’Hara, B., Bendotti, C., Reeves, R., Oster-Granite, M., Coyle, J. and
Gearhart, J. (1988). Genetic mapping and analysis of somatostatin
expression in Snell dwarf mice. Mol. Brain Res. 4, 283-292.
Pfaffle, R., DiMattia, G., Parks, J., Brown, M., Wit, J., Jansen, M., Van der
Nat, H., Van den Brande, J., Rosenfeld, M. and Ingraham, H. (1992).
Mutation of the POU-specific domain of Pit-1 and hypopituitarism without
pituitary hypoplasia. Science 257, 1118-21.
Phelps, C., Carlson, S., Vaccarella, M. and Felten, S. (1993). Developmental
assessment of hypothalamic tuberoinfundibular dopamine in prolactindeficient dwarf mice Endocrinology 132, 2715-22.
Ramakrishnan, R., Fink, D., Jiang, G., Desai, P., Glorioso, J. and Levine,
M. (1994). Competitive quantitative PCR analysis of herpes simplex virus
type 1 DNA and latency-associated transcript RNA in latently affected cells
in the rat brain. J. Virology 68, 1864-1873.
Rhodes, S., Chen, R., DiMattia, G., Scully, K., Kalla, K., Lin, S., Yu, V. and
Rosenfeld, M. (1993). A tissue-specific enhancer confers Pit-1-dependent
morphogen inducibility and autoregulation on the pit-1 gene. Genes Dev. 7,
913-32.
Rhodes, S., DiMattia, G. and Rosenfeld, M. (1994). Transcriptional
mechanisms in anterior pituitary cell differentiation. Current Opinion Genet.
Dev. 4, 709-717.
Rosenfeld, M. (1991). POU-domain transcription factors: pou-er-ful
developmental regulators. Genes Dev. 5, 897-907.
Ruvkun, G. and Finney, M. (1991). Regulation of transcription and cell
identity by POU domain proteins. Cell 64, 475-8.
Sasaki, F. and Sano, M. (1982). Role of the ovary in the sexual differentiation
of prolactin and growth hormone cells in the mouse adenohypophysis: a
stereological morphometric study by electron microscopy. J. Endocrinol. 93,
117-21.
Schwind, J. (1928). The development of the hypophysis cerebri of the albino
rat. Amer. J. Anat. 41, 295-315.
Simmons, D., Voss, J., Ingraham, H., Holloway, J., Broide, R., Rosenfeld,
M. and Swanson, L. (1990). Pituitary cell phenotypes involve cell-specific
Pit-1 mRNA translation and synergistic interactions with other classes of
transcription factors. Genes Dev. 4, 695-711.
Slabaugh, M., Lieberman, M., Rutledge, J. and Gorski, J. (1981). Growth
hormone and prolactin synthesis in normal and homozygous Snell and Ames
dwarf mice. Endocrinology 109, 1040-6.
Thomson, J. and Solter, D. (1988). The developmental fate of androgenetic,
parthenogenetic, and gynogenetic cells in chimeric gastrulating mouse
embryos. Genes Dev. 2, 1344-51.
Vogt, T., Solter, D. and Tilghman, S. (1987). Raf, a trans-acting locus,
regulates the alpha-fetoprotein gene in a cell-autonomous manner. Science
236, 301-3.
Watanabe, Y. (1982a). Effects of brain and mesenchyme upon the cytogenesis
of rat adenohypophysis in vitro. I. Differentiation of adrenocorticotropes.
Cell Tissue Res. 227, 257-66.
Watanabe, Y. (1982b). An organ culture study on the site of determination of
ACTH and LH cells in the rat adenohypophysis. Cell Tissue Res. 227, 26775.
Yokoyama, T., Silversides, D., Waymire, K., Kwon, B., Takeuchi, T. and
Overbeek, P. (1990). Conserved cysteine to serine mutation in tyrosinase is
responsible for the classical albino mutation in laboratory mice. Nucleic
Acids Res. 18, 7293-8.
(Accepted 4 October 1995)