Pituitary-Directed Leukemia Inhibitory Factor Transgene Causes Cushings Syndrome: Neuro-Immune-Endocrine Modulation of Pituitary Development
Hiroki Yano,
Carol Readhead,
Masahiro Nakashima,
Song-Guang Ren and
Shlomo Melmed
Department of Medicine Cedars-Sinai Research Institute
University of California Los Angeles School of Medicine Los
Angeles, California 90048
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ABSTRACT
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Leukemia inhibitory factor (LIF) regulates the
mature hypothalamic-pituitary-adrenal axis in vivo.
In vitro, LIF determines corticotroph cell proliferation
and induces POMC transcription. To explore LIF action on pituitary
development, transgenic mice expressing LIF driven by the pituitary
glycoprotein hormone
-subunit (
GSU) promoter were generated.
Transgenic mice exhibited dwarfism with low IGF-I (29 ± 9 ng/ml
vs. wild type (WT) 137 ± 16 ng/ml; P
< 0.001), hypogonadism with low FSH (0.04 ± 0.023 ng/ml
vs. WT 0.63 ± 0.18 ng/ml; P <
0.001), and Cushingoid features of thin skin and truncal obesity with
elevated cortisol levels (86 ± 22 ng/ml vs. WT
50 ± 14 ng/ml; P = 0.002). Their pituitary
glands showed corticotroph hyperplasia, striking somatotroph and
gonadotroph hypoplasia, and multiple Rathke-like cysts lined by
ciliated cells. LIF, overexpressed in Rathkes pouch at embryonal day
10, diverts the differentiation stream of hormone-secreting cells
toward the corticotroph lineage and ciliated nasopharyngeal-like
epithelium. Thus, inappropriate expression of LIF, a neuro-immune
interfacing cytokine, plays a key role in the terminal differentiation
events of pituitary development and mature pituitary function.
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INTRODUCTION
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The pituitary is an essential organ for mammalian homeostasis
after birth. During pituitary ontogeny, distinct cell types
differentiate in a time-dependent serial pattern involving a precise
balance between proliferation and differentiation of individual cells
orchestrated by cell-cell and/or epithelial-neuroectodermal-mesenchymal
interactions mediated by soluble factors and/or extracellular matrix
(1, 2, 3, 4, 5). During this process, pituitary cells express a variety of genes
in spatial and temporal patterns and differentiate to different
phenotypes by critically turning on or off specific genes through
several transcriptional factors, such as Pit-1 (6, 7), Lhx3
(pLim/mLim-3) 98100, Rpx (Hesx1) (11, 12), P-OTX (Ptx-1) (13, 14),
and Prop-1 (15), to produce cytological and functionally distinct
pituitary cells. These individual committed cells synthesize and
secrete unique hormones in utero, including ACTH,
somatotropin (GH), TSH, PRL, and gonadotropin (FSH and LH) (5, 16).
These tropic hormones coordinate central regulation of metabolism,
growth, reproduction, and behavior and also play crucial roles in
peripheral organ development and function.
Leukemia inhibitory factor (LIF), a pleiotropic cytokine, inhibits
embryonic stem cell differentiation and primitive ectoderm formation
in vitro (17, 18, 19). LIF transgenic expression directed by a
tissue-specific promoter switched pancreatic noradrenergic sympathetic
to cholinergic innervation (20) and also caused thymic and lymph node
morphological interconversion (21). In the pituitary, LIF and its
receptors are recognized in human fetal corticotrophs and somatotrophs
as early as 14 weeks of gestation (22). LIF also potently synergizes
with CRH to enhance POMC transcription and ACTH secretion in
vitro (23, 24). In vivo, intraperitoneal injection of
lipopolysaccharide or interleukin-1 induces both LIF and LIF-receptor
gene expression in the murine hypothalamus and pituitary, concomitantly
with ACTH induction (25, 26, 27). This immune-neuro-endocrine interface
is abrogated in mice harboring a disrupted LIF gene (28, 29).
Pituitary-directed LIF transgenic mice, utilizing a rat GH promoter,
exhibit dwarfism and persistent Rathkes cysts (30). These findings
lead us to hypothesize that LIF plays a key role in pituitary
development, subserving both adenohypophyseal ontogeny and mature
pituitary function.
In this paper, we demonstrate that early pituitary overexpression
driven by the
GSU promoter (31, 32) causes thin skin, truncal
obesity, corticotroph hyperplasia, and hypercortisolism, consistent
with Cushings disease. These mice also exhibited central
hypogonadism, dwarfism, and mild hypothyroidism, with gonadotroph,
somatotroph, lactotroph, and thyrotroph hypoplasia. These findings
strongly imply that neuro-immune-endocrine interfacing molecules act as
key players in terminal pituitary differentiating events, in addition
to the known transcription factors.
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RESULTS
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Cushingoid Features and Growth Retardation of LIF Transgenic
Mice
Eleven LIF transgenic mice, obtained from a total of 85 weaned
pups, were indistinguishable from siblings at birth, but all except one
exhibited severe growth retardation by 2 weeks after birth, and truncal
obesity was evident by 1 month (Fig. 1c
).
Although wild-type (WT) males usually grow larger than females (33), no
intergender weight differences were observed in the rotund LIF
transgenic mice. Plasma IGF-I levels were extremely low in transgenic
animals [transgenic (n = 6) 29 ± 9 (±SD) ng/ml
vs. WT (n = 9) 137 ± 16 ng/ml, P
< 0.001] (Fig. 1d
). These mice had thin skin, and basal
corticosterone levels were higher (n = 5) than in
transgene-negative littermates (n = 10) (86 ± 22 ng/ml
vs. 50 ± 14 ng/ml; P = 0.002).
Corticosterone levels were incompletely suppressed by dexamethasone
administration, indicating autonomous hypercortisolism with a
Cushingoid phenotype (Fig. 2
). Although
subdermal fat appeared similar in WT and transgenic mice, more
intraperitoneal fat was observed in LIF transgenic animals.
Histologically, fatty tissue in transgenic mice consisted of both
mature and brown fat. Adrenal glands of the rotund transgenic mice were
similarly sized to WT littermates in spite of differences in body size.
At necropsy, however, some adrenals obtained from transgenic mice were
intensely hyperemic and larger than WT, supporting the biochemical
observation of hypercortisolism.

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Figure 1. LIF Transgenic Mice Exhibited Dwarfism
a, Diagrammatic representation of genomic murine LIF and the transgene
construct. b, Tail DNA was digested with XbaI and run on
a 0.8% agarose gel. After transfer, the membrane was hybridized with a
random-radiolabeled probe from the 670-bp
EcoRI-BamHI mLIF cDNA fragment. Expected
sizes of the genomic and transgenic fragments were 2.7 and 1.5 kb,
respectively. M, marker; P, injected plasmid digested with
XbaI as control. c, Pituitary-directed LIF transgene
attenuates growth. LIF transgenic mice (10 weeks old) were rotund with
symmetrical dwarfism and truncal obesity. Ten of 11 transgenic mice
showed weight gain less than half of control weight gain, and
male and female LIF transgenic mice were not different. Solid
and dotted lines indicate linear regression of respective
weights of male and female WT animals. d, Plasma IGF-I levels were
attenuated in LIF transgenic mice (P < 0.001).
Values shown are mean ± SD.
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Figure 2. LIF Transgenic Mice Exhibited Morphological and
Biochemical Features of Hypercortisolism
a, Twelve-week-old transgenic male skin appendages were not affected,
but transgenic dermis was thin and only attained the depth of the
appendage layer. Collagen fibers were atrophic. LIF transgenic mice
also showed increased brown and mature intraperitoneal fat. b, Basal
plasma corticosterone levels were higher in the transgenic mice, and
these levels were incompletely suppressed 4 h after dexamethasone
administration (0.1 mg/kg body wt). Histological figures are
hematoxylin and eosin stains of paraffin sections. Bars,
100 µm. Values shown are mean ± SD.
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LIF Transgene Attenuates Gonadal Maturation and Thyroid
Function
Although breeding was attempted between LIF transgenic and
WT mice, no pregnancies were obtained. Reproductive organs of both
dwarf male and female animals were poorly developed (Fig. 3a
). Testes and ovaries were markedly
reduced in size and the uterus was thread-like. Transgenic testes
exhibited arrested spermatogenesis at the first meiotic division and
greatly reduced number of Leydig cells in the sparse interstitium. The
transgene was not expressed in testis. In the ovary, no antral stage
follicles nor ovarian corpora lutea were observed. Plasma FSH levels
(n = 6) were less than 10% of transgene-negative littermates
(n = 9) (0.04 ± 0.023 ng/ml vs. 0.63 ± 0.18
ng/ml, P < 0.001) (Fig. 3b
). FSH is required for
ovarian follicle maturation but not male fertility (34), suggesting
that plasma LH levels must also be reduced in the mice (33). In the
thyroid gland, some transgenic animals had tiny disorganized follicles
whose interstitium was filled with brown fat, while others had a more
normal thyroid (Fig. 4
). Although total
T4 levels were reduced (2.63 ± 0.99 µg/dl (n =
6) vs. 4.54 ± 1.40 µg/dl (n = 9),
P = 0.021), wide variations of T4 were
observed in transgenic mice.

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Figure 3. LIF Transgene Blocks Gonadal Maturation
a, Transgenic testes, ovaries, and uterus (12 weeks old).
Although transgenic mice exhibited primary sexual differentiation, they
had severely reduced gonadal development after birth. b, Circulating
FSH levels in transgenic mice were markedly suppressed
(P < 0.001). Histological figures are hematoxylin
and eosin stains of paraffin sections. Bars, 100 µm.
Values shown are mean ± SD.
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Figure 4. LIF Transgene Impairs Thyroid Development
a, Thyroid follicles of some transgenic mice were hypoplastic and
disorganized while some appeared normal histologically. b, Total
T4 was reduced in transgenic mice. Histological figures are
hematoxylin and eosin stains of paraffin sections. Bars,
100 µm. Values shown are mean ± SD.
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Cell Composition of LIF Transgenic Pituitary
The LIF transgenic pituitary was less than half the size of WT
(Fig. 5a
).
Histologically, both posterior and intermediate lobes of the LIF
transgenic pituitary were not altered; however adenohypophyseal
hypoplasia was prominent in the anterior lobe, and many cysts with cell
debris were observed. Cells lining these cysts were ciliated, and their
contents were periodic acid- Schiff positive (data not shown),
resembling upper airway epithelium. The original Rathkes cleft showed
little invagination as was previously seen in the GH promoter-directed
LIF transgenic mice (30), and these multiple cysts were less contiguous
with each other. All pituitaries from the 10 rotund transgenic animals
appeared identical histologically. In the transgenic pituitary anterior
lobe, somatotrophs producing GH and lactotrophs producing PRL were
strikingly reduced in number, but individual GH-producing cells stained
more intensely (Fig. 5c
). The hypoplastic transgenic pituitaries could
be attributed to the extreme hypoplasia of these two lineages, which
normally represent 70% of the adenohypophyseal cell population.
Gonadotrophs were also markedly decreased in all transgenic
pituitaries. Interestingly, although TSH producing cells were reduced
in number, there were some differences among transgenic pituitaries
(data not shown), similar to the inconsistent findings in thyroid
tissue and plasma T4 levels. The anterior pituitary lobe of
transgenic mice comprised mainly clustered ACTH-producing cells.
Corticotroph hyperplasia was absolute and not a reflection of
hypoplasia of the other cell lineages. In transgenic animals,
ACTH-immunopositive cells accounted for
65% of anterior pituitary
cells, while WT animals harbored
13% ACTH-positive cells. In
transgenic pituitaries, double immunostaining for LIF and
GSU showed
many LIF immunostaining cells colocalized with
GSU-positive cells in
the anterior lobe, although less LIF staining was observed in the WT
anterior pituitary (Fig. 5d
). Single LIF-positive immunostaining in the
posterior lobe indicated endogenous LIF expression in both WT and
transgenic pars posterior. The LIF and
GSU double-positive cells
implied transgene expression driven by the
GSU promoter. Thus, in
the transgenic pituitary, corticotroph hyperplasia, hypoplasia of other
lineages, and multiple cysts lined by ciliated epithelium were
associated with LIF overexpression.



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Figure 5. Morphology and Histology of the LIF Transgenic
Pituitary
a, LIF transgenic pituitary (14 weeks) was hypoplastic and weighed less
than half of WT gland. b, Hematoxylin and eosin staining. Although
posterior and intermediate lobes were not affected by LIF transgene,
the anterior lobe was dramatically altered with cellular hypoplasia and
multiple cystic changes. Epithelium lining the cysts was ciliated, and
cystic cavities were filled with PAS-positive content and occasional
cell debris. c, Immunohistochemistry for GH, PRL, bLH, and ACTH of
frontally sectioned pituitaries. Somatotrophs, lactotrophs, and
gonadotrophs in the transgenic pituitary were markedly reduced in
number. However, the LIF-transgenic anterior lobe was mainly occupied
by ACTH-immunopositive cells. d, Double immunostaining of LIF
(alkaline-phosphatase staining, blue) and GSU
(peroxidase staining, brown). Only posterior lobe cells,
which served as a positive control for LIF immunostaining, were
immunopositive for LIF in the WT, but many LIF-positive cells were
recognized in the anterior and posterior lobes of the transgenic
pituitary. P, Posterior lobe; I, intermediate lobe; A, anterior
lobe.
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Sensitivity and Selectivity of the
GSU Promoter Activity
Green fluorescent protein (EGFP) (35, 36, 37) cDNA was employed
as a reporter by replacing mouse (m)LIF cDNA in the
LIF-transgenic construct to confirm the
GSU promoter activity. Nine
Southern-positive mice were obtained from a total of 43 pups. Both
genders of these EGFP transgenic animals showed normal growth and
reproduction patterns (data not shown). The distribution of EGFP
expression was reproducible and identical in all lines, indicating
specificity of the 4.6-kb
GSU promoter for transgenic expression. In
the EGFP transgenic pituitary (Fig. 6a
), green fluorescence was recognized in
ectodermal cells lining Rathkes pouch as early as embryonal day E10,
reflecting the sensitivity of EGFP (32). With development of pars
anterior, increased fluorescence was observed around areas of active
cellular proliferation in the anterior wall of Rathkes pouch and weak
fluorescence in the wall of the infundibular recess (E12.5). Later in
embryonal development (after E14.5), strong fluorescence was noted in
the anterior lobe and rostral tip in a speckled pattern, and cell
fluorescence of both Rathkes cleft lining and the posterior lobe
became weaker. Interestingly, almost all cells in the adult anterior
lobe expressed EGFP, but no expression was evident in either posterior
or intermediate lobes (Fig. 6b
). Although high-intensity cells must be
considered thyrotrophs or gonadotrophs because of their respective
GSU hormonal structural components, other lineages also expressed
this exogenous fluorescent protein driven by the transgenic 4.6-kb
GSU promoter, suggesting that most anterior pituitary cell types
might have a potential to express
GSU. As expected, no EGFP
fluorescence was observed in skin, thyroid, adrenal, liver, adipose
tissue, or testis.

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Figure 6. Transgene Expression by the Mouse 4.6-kb GSU
Promoter
Fluorescent microscopy of frozen sections showing pituitary ontogeny
(a, saggital section). Fluorescence was identified in cells lining
Rathkes pouch as early as E10.5. Later, fluorescence spread in the
anterior lobe, and Rathkes pouch expression weakened. In the
12-week-old adult pituitary (b, frontal section), all anterior lobe
cells exhibited EGFP fluorescence with no fluorescence in either
posterior or intermediate lobes. R, Rathkes pouch; If, infundiblum;
P, posterior lobe; I, intermediate lobe; A, anterior lobe.
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Comparison between LIF Transgenic and WT Pituitary Assessed by
Tandem Transgene Expression
To compare development of LIF transgenic to WT pituitary, as well
as to monitor transgene expression and penetration, LIF and EGFP tandem
transgenic mice were also generated. As expected, these mice showed the
same phenotype, including pituitary histology, as the LIF-alone
transgenic animals. Bright EGFP fluorescence was recognized in the
anterior lobe of the adult tandem transgenic mice (Fig. 7a
) and distributed more widely than the
LIF immunoreactivity observed in the LIF transgenic pituitary (Fig. 5d
). The tandem transgenic pituitary also showed similar corticotroph
hyperplasia as observed in the LIF transgenic (Fig. 7b
), and many of
these cells also exhibited EGFP fluorescence. EGFP fluorescence of the
tandem transgenic pituitary at E14.5 was only recognized in scattered
cells, while almost all cells expressed EGFP in EGFP-alone transgenic
pituitary (Fig. 7c
). Lhx3 and Pit-1 expression was suppressed at E14.5
in the tandem transgenic pituitary. In contrast, in the LIF transgenic
pituitary, POMC-expressing cells were more abundant than in the
EGFP-alone transgenic pituitary.

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Figure 7. Developmental Effect of GSU-Directed LIF
Transgene
a, Fluorescent microscopy of the tandem transgenic pituitary (12 weeks
old). Many cells exhibited bright fluorescence, indicating transgenic
LIF expression. b, POMC expression in the 12-week-old EGFP-alone and
tandem transgenic pituitary. The tandem transgenic pituitary exhibited
corticotroph hyperplasia by in situ hybridization,
similar to the ACTH immunohistochemical results in the LIF-alone
transgenic pituitary. c, LIF transgene altered embryonic pituitary
transcription factor expression at E14.5. The tandem-transgenic
pituitary exhibited decreased Lhx3 and Pit-1 transcripts, in addition
to GSU itself (EGFP fluorescence). In contrast, the LIF transgene
manifested abundant POMC accumulation.
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DISCUSSION
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During pituitary ontogeny, five phenotypically distinct
hormone-producing cell types are believed to arise from a common
lineage (5, 13, 16, 32, 38, 39). The common progenitor cells originate
in Rathkes pouch, an invagination of oral ectoderm (16, 40). In the
mouse, the initial hormonal evidence of pituitary organ commitment
commences with expression of
GSU. Subsequently, POMC transcripts
appear at E12, bovine (b)TSH at E1213, bLH and bFSH at E15, and GH
and PRL finally emerge at E1617 (40, 41). In the LIF transgenic
pituitary, the LIF product could divert progenitor cells to
differentiate from Lhx3-dependent cell lineages (gonadotroph,
thyrotroph, somatotroph, and lactotroph) to a Lhx3-independent cell
lineage (42) (Fig. 8
). Embryonic
pituitary Lhx3 expression was, in fact, markedly attenuated in
transgenic animals, supporting this hypothesis. From this cytological
turning point, corticotrophs appear to develop and flourish in response
to LIF translated from the transgene.

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Figure 8. Schematic Illustration of Divergent Pituitary
Development of LIF Transgenic Mice
In the pituitary, LIF blocks the stream to Lhx3 and subsequent
Pit-1-dependent cell lineages. Directly or indirectly, LIF stimulates
the corticotroph lineage and also diverts pituitary progenitor cells to
nasal epithelial cells. Solid arrows depict normal
pituitary ontogeny (5 13 15 32 38 39 ).
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CRH is mitogenic for corticotroph cells both in vitro and
in vivo (43, 44), and LIF potently synergizes with CRH to
enhance POMC transcription and ACTH secretion (23, 24, 27). LIF, in
fact, induces a switch of proliferating corticotroph tumor cells
(AtT20) toward a more differentiated phenotype (44). In the stressed
adult pituitary, abundant LIF was recognized in the hypothalamus and
pituitary (25, 26). In fact, several of the hormonal features of these
transgenic mice resemble a neuro-endocrine stress response, whereby
ACTH is elevated and gonadotropin and TSH are suppressed. Thus,
LIF-induced hypercortisolemia may also have secondary inhibitory
effects on the reproductive and thyroid axes.
Interestingly, although somatotroph, lactotroph, and gonadotroph
hypoplasia was manifest, the thyrotroph was variably affected in the
LIF transgenic lines. This observation might support the notion of a
Pit-1-independent pathway for thyrotroph ontogeny (45). Pit-1 mRNA
expression was, in fact, virtually undetectable in embryonic transgenic
animals. Paradoxically, in the embryonal period, however,
GSU
promoter activity in LIF transgenics appeared weaker than WT (Fig. 7c
),
possibly caused by the LIF construct itself. Even in this circumstance
of a delicate balance between embryonal LIF and
GSU promoter
activity, LIF driven by
GSU causes striking pituitary
maldevelopment. However, the adult tandem transgenic pituitary showed
abundant EGFP fluorescence, suggesting that the adult anterior
hypophysis appears to manifest a different expression pattern than
embryonic pituitary. In the transgenic animals, the widespread but low
adenohypophyseal expression of
GSU is consistent with the
observation that most pituitary tumor types express a low abundance of
immunoreactive
-subunits regardless of their cell type.
Alternatively, the presence of a GH-polyadenylation site within the
transgene may also have altered the tissue-specific pattern of
pituitary
-subunit expression (46).
While LIF stimulates the corticotroph lineage, it also causes
progenitor cells to differentiate into ciliated cells, similar to the
embryological differentiation of original ectodermal cells to ciliated
columnar cells in the nasal cavity. Furthermore, invaginated ciliated
cysts, rather than multiple cysts, were also observed in the GH
promoter-driven LIF transgenic (30), supporting the notion that LIF
causes some pituitary progenitor cells to follow an abnormal
differentiation pathway. In the
GSU promoter-driven transgenic mice
reported here, however, LIF overexpression occurred earlier (E 9.5) and
showed more diffuse multiple cysts in the pituitary anterior lobe than
the GH promoter-driven transgenic mice (E17.5), implying that
progenitor cells are distributed diffusely at an early stage of
pituitary development and are contiguous with Rathkes cleft at later
stages. Several additional differences in the two LIF-transgenic
models, including cell-specific expression (GH vs.
GSU) and the intrapituitary concentrations of local LIF achieved,
may also determine their differing phenotypes.
The observed failure of gonadal development presumably results from
gonadotroph failure as evidenced by gonadotroph hypoplasia and
suppressed FSH levels. Dwarfism is due to the lower number of pituitary
somatotrophs as also evidenced by low IGF-I levels, but could also be
partially attributed to the lack of thyroid hormone effect on GH
transcription (47, 48, 49) or the hypercortisolism (50). The thin skin may
be attributed to persistently high levels of corticosteroids, and fat
deposition may be a result of both hypercortisolism and hypothyroidism.
Thus, early pituitary overexpression of LIF inhibits development of
gonadotrophs, lactotrophs, and somatotrophs, but favors differentiation
and over-proliferation of corticotrophs. LIF is known to be a powerful
cytokine mediating both the differentiation or dedifferentiation of
committed cells (17, 18, 19, 20, 21) as well as regulating POMC transcription
(22, 23, 24, 25, 26, 27, 28, 29). In the developing pituitary, LIF inhibits gonadotroph,
thyrotroph, lactotroph, and somatotroph lineages, induces corticotroph
development, and directly or indirectly converts some pituitary cells
to ciliated epithelium. These findings strongly imply that, in addition
to transcription factors, a soluble neuro-immune interfacing factor,
such as inappropriately expressed LIF, regulates terminal
differentiating events during pituitary ontogeny.
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MATERIALS AND METHODS
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Plasmid Construction and Creation of Transgenic Mice
The plasmid containing -5000 to +43 kbp of the mouse
GSU
promoter (a kind gift from Dr. E. C. Ridgway, University of
Colorado) was digested with KpnI and HindIII. For
LIF transgenic mice, this 4.6-kbp fragment was ligated directly into
pBluescriptII SK(-) containing mouse LIF cDNA and bovine GH
polyadenylation signal between the EcoRI and NotI
sites. For EGFP transgenic mice, mLIF cDNA was replaced by EGFP cDNA
(pEGFP-1, Clontech Laboratories, Inc., Palo Alto, CA). For tandem
transgenic mice, the insert for EGFP transgenic mice including the
GSU promoter, EGFP cDNA, and poly-A signal was ligated into the
NotI-SacII site just after the poly-A signal of
the plasmid for LIF-alone transgenic mice, using blunt-end and ligation
(KpnI site of insert of EGFP transgenic; NotI
site of plasmid for LIF transgenic) with SacII ligation.
After being cut with KpnI and NotI or
SacII, DNA inserts were separated from vector sequences by a
sucrose-gradient. The fractions containing the inserts were dialyzed.
Purified DNAs were microinjected into fertilized egg pronucleus, and
the eggs were transplanted to oviducts of pseudopregnant mice to obtain
transgenic mice. Genomic DNA digested with XbaI from tail
biopsy of pups was screened by Southern blot analysis using a probe of
EcoRI-XbaI fragment of mLIF cDNA or
BamHI-NotI of EGFP.
Histology
After fixation in 4% paraformaldehyde, each tissue was
dehydrated, embedded in paraffin, and serially sectioned at 4 µm.
After deparaffinization, specimens were stained with hematoxylin-eosin
and used for immunohistochemistry. The specimens were immersed in 0.3%
H2O2/methanol and preincubated with 1% skim
milk/PBS. Sections were incubated with primary antibodies (antihuman
ACTH, 1:500 dilution (Dako Corp., Carpinteria, CA), antihuman
GH, 1:500; antirat PRL, 1:500; antirat
GSU, 1:200; antirat bLH,
1:200; antirat bTSH 1:200 (National Hormone and Pituitary Program,
National Institute of Diabetes and Digestive and Kidney Diseases,
Rockville, MD); antihuman LIF, 1:100 (Biodesign Int., Kennebunk, ME)
for 6 h at 4 C. After washing with 0.05% Tween 20/PBS, slides
were incubated with polymerized secondary antibodies (Dako Envision
system, Dako) or peroxidase-conjugated anti-guinea pig antibody (Dako)
for bLH, and AEC (Dako) was used as the chromogen. Hematoxylin
counter stainings were then performed, except when used for double
staining. For double immunostaining, slides were washed in 0.1
M glycine HCl, pH 2.2, at 37 C overnight before the
secondary reaction, and BCIP/nitroblue tetrazolium was used for
the colorimetric reaction. Negative controls were prepared by replacing
the primary antibody with nonimmunized rabbit serum.
Dexamethasone Suppresses Test and Hormone Measurements
Five LIF transgene positive mice and 10 negative litters were
kept in unstressed conditions overnight and treated with water-soluble
dexamethasone (Sigma, St. Louis, MO) ip at a dose of 0.1 mg/kg body wt
immediately after collection of a basal blood sample (0900 h). Blood
sampling was then performed after 4 and 24 h. Plasma
corticosterone levels were measured in 10 µl plasma by RIA (ICN
Biomedicals, Inc. Costa Messa, CA). The T4 and IGF-I RIAs
were performed using Coat-A-Count Kit (Diagnostic Products Corp., Los
Angeles, CA) and DSL 5600 (Diagnostic Systems Laboratories, Inc.,
Webster, TX), respectively, according to the manufacturers
instructions. FSH RIAs were performed using kit components provided by
the National Hormone and Pituitary Program after rat FSH iodination by
iodogen.
Fluorescent Microscopy and in Situ Hybridization
Tissue was fixed in 4% paraformaldehyde, frozen in OCT
compound (Sakura Finetechnical Co., Tokyo, Japan), sectioned at 8 µm,
and dried overnight. After immersion in PBS, specimens were evaluated
under fluorescent microscopy according to the manufacturers
instruction (CLONTECH, Palo Alto, CA). Mouse Lhx3 cDNAs were
created using pGEM-T vector systems (Promega Co., Madison, WI) by
RT-PCR with WT mouse pituitary. Forward primer,
5'-TGCAAGGC-GGACTACGAAAC-3' and reverse primer,
5'-CAAGGCTCAAGTTGGTGTCT-3'. Mouse Pit-1 cDNA (51) was obtained
from the ATCC. 35S-labeled cRNA probes for Lhx3, Pit-1, and
POMC 26, were generated using RNA labeling kit (Amersham Co., Arlington
Heights, IL). In situ hybridization was performed, as
previously described (52), on 8 µm-sectioned frozen specimens, which
showed EGFP fluorescence.
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ACKNOWLEDGMENTS
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We are grateful to Dr. D. Chen for assistance with the
transgenic mice.
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FOOTNOTES
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Address requests for reprints to: Shlomo Melmed, M.D., Division of Endocrinology and Metabolism, Cedars-Sinai Medical Center, 8700 Beverly Boulevard, Suite B-131, Los Angeles, California 90048-1865. E-mail:
Melmd{at}CSHS.org
This work was supported by NIH Grants DK-50238 (to S.M.) and RR-12406
(to C.R.), and by the Doris Factor Molecular Endocrinology
Laboratory.
Received for publication May 28, 1998.
Revision received August 12, 1998.
Accepted for publication August 14, 1998.
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