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 Previous Article | Next Article 
The Journal of Neuroscience, September 1, 1998, 18(17):6631-6640
Authentic Cell-Specific and Developmentally Regulated Expression
of Pro-Opiomelanocortin Genomic Fragments in Hypothalamic and Hindbrain
Neurons of Transgenic Mice
Juan I.
Young1,
Verónica
Otero1,
Marcelo G.
Cerdán1,
Tomás L.
Falzone1,
E.
Cheng
Chan2,
Malcolm J.
Low2, and
Marcelo
Rubinstein1, 3
1 Instituto de Investigaciones en Ingeniería
Genética y Biología Molecular, Universidad de Buenos
Aires-Consejo Nacional de Investigaciones Científicas y
Técnicas, 1428 Buenos Aires, Argentina, 2 Vollum
Institute, Oregon Health Sciences University, Portland, Oregon 97201, and 3 Departamento de Química Biológica,
Facultad de Ciencias Exactas y Naturales, Universidad de Buenos
Aires, 1428 Buenos Aires, Argentina
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ABSTRACT |
The pro-opiomelanocortin (POMC) gene is expressed in a subset of
hypothalamic and hindbrain neurons and in pituitary melanotrophs and
corticotrophs. POMC neurons release the potent opioid  -endorphin and
several active melanocortins that control homeostasis and feeding
behavior. POMC gene expression in the CNS is believed to be controlled
by distinct cis-acting regulatory sequences. To analyze
the transcriptional regulation of POMC in neuronal and endocrine cells,
we produced transgenic mice carrying POMC27*, a transgene containing
the entire 6 kb of the POMC transcriptional unit together with 13 kb of
5' flanking regions and 8 kb of 3' flanking regions. POMC27* was tagged
with a heterologous 30 bp oligonucleotide in the third exon. In
situ hybridization studies showed an accurate cell-specific
pattern of expression of POMC27* in the arcuate nucleus and the
pituitary. Hypothalamic mRNA-positive neurons colocalized entirely with
-endorphin immunoreactivity. No ectopic transgenic expression was
detected in the brain. Deletional analyses demonstrated that
neuron-specific expression of POMC transgenes required distal 5'
sequences localized upstream of the pituitary-responsive proximal
cis-acting elements that were identified previously.
POMC27* exhibited a spatial and temporal pattern of expression
throughout development that exactly paralleled endogenous POMC. RNase
protection assays revealed that POMC27* expression mimicked that of
POMC in different areas of the CNS and most peripheral organs with no
detectable ectopic expression. Hormonal regulation of POMC27* and POMC
was identical in the hypothalamus and pituitary. These results show
that distal 5' sequences of the POMC gene located between  13 and 2
kb target expression into the CNS of transgenic mice in a precise
neuron-specific, developmentally and hormonally regulated
manner.
Key words:
pro-opiomelanocortin; transgenic mice; gene expression; -endorphin; melanocortin; neuron-specific expression; arcuate
nucleus; hypothalamus; pituitary
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INTRODUCTION |
The pro-opiomelanocortin (POMC) gene
encodes a multipeptide prohormone that gives rise to various active
products after tissue-specific posttranslational processing (Eipper and
Mains, 1980 ). Expression of the POMC gene is limited to a number of
cell types of endocrine and neuronal origin, and the physiological role
of its derived peptides is to orchestrate the mammalian stress
response. Melanotrophs and corticotrophs of the pituitary gland release
two of the best characterized POMC-derived peptides,
 -melanocyte-stimulating hormone and adrenocorticotropic
hormone, respectively (Smith and Funder, 1988). In the CNS, two
discrete groups of neurons located in the arcuate nucleus of the
hypothalamus and in the nucleus of the tractus solitarius also express
POMC and produce -endorphin, a potent opioid peptide that interacts
with µ-opioid receptors to mediate stress-induced analgesia
(Rubinstein et al., 1996) and to regulate reproduction (Seifer and
Collins, 1990) and autonomic functions (Olson et al., 1997). In
addition to -endorphin, POMC-expressing neurons release melanocortin
peptides that participate in the control of homeostasis by binding to
the central melanocortin receptors MC-3 and MC-4 (Cone et al., 1996; Li
et al., 1996). Hypothalamic melanocortins are receiving considerable
attention because they seem to regulate feeding and fasting behavior
(Fan et al., 1997). Leptin receptors are expressed in POMC hypothalamic neurons (Cheung et al., 1997), and leptin-induced fasting is prevented by melanocortin receptor antagonists (Seeley et al., 1997). In addition, MC-4 receptor agonists reduce food intake (Fan et al., 1997),
and MC-4 receptor-deficient mice become obese (Huszar et al.,
1997).
The developmental onset of POMC expression follows a distinct temporal
pattern (Japón et al., 1994). POMC mRNA is first detected at
embryonic day 10.5 (E10.5) in the base of the mouse
diencephalon. This early expression preceding all other hypothalamic
neuropeptides suggests a developmental role for POMC-derived peptides.
In the pituitary, POMC mRNA is detected in melanotrophs at E14.5 and in
corticotrophs 2 d earlier, also preceding the expression of all
other pituitary hormone genes (Japón et al., 1994). Despite the
extensive progress that several laboratories achieved to unravel the
organogenetic program of the hypothalamic-pituitary axis (Sheng et
al., 1997) and to understand the specific commitment of the different
cell lineages (Treier and Rosenfeld, 1996), the mechanisms determining
POMC cell fate still remain elusive.
Appropriate pituitary expression of reporter transgenes carrying up to
4 kb of the POMC promoter has allowed the study of tissue-specific
expression of POMC in this gland (Liu et al., 1992, 1995). However, all
these transgenes failed to recapitulate proper POMC expression in the
brain (Rubinstein et al., 1993) (our unpublished results). To study the
spatial and temporal regulation of POMC gene expression in the CNS, we
produced transgenic mice carrying large fragments of 5' and 3' POMC
gene-flanking regions. Here, we report for the first time the accurate
targeting of transgenes exclusively to POMC-expressing neurons and that
distal 5' sequences of the POMC promoter are required for
neuronal-specific expression in the brain.
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MATERIALS AND METHODS |
Screening of a mouse genomic library. A mouse cosmid
library was constructed by partial MboI digestion of total
genomic DNA obtained from 129/SvEv mouse embryonic stem cells.
Size-fractionated fragments were ligated into the BamHI site
of the cosmid vector SuperCos 1 (Stratagene, La Jolla, CA) and then
packaged in vitro using Gigapack II XL packaging
extracts (Stratagene). Recombinant cosmids were then transfected into
Escherichia coli NM554. The final calculated titer of
this library was 3 × 105 independent clones
with an average fragment size of 29 kb. This library was screened at
high-stringency hybridization in duplicate nylon filters using
simultaneously two 32P-labeled, random-primed probes
corresponding to the terminal sequences of pHAL*, a mouse genomic POMC
clone previously used by us (see Fig. 1) (Rubinstein et al., 1993). We
obtained 23 double-positive clones, and two overlapping clones (1 and
13) were selected for further subcloning.
DNA constructs. Plasmid POMC27* contains a 27 kb fragment
cloned into the multiple cloning site of pBluescript SK+/
(Stratagene). It was designed to carry a 30 bp heterologous sequence
into POMC exon 3 (Rubinstein et al., 1993) and was obtained after
subcloning together pHAL* fragments with flanking sequences taken from
two cosmid clones. The 27 kb fragment was generated by ligating first the ClaI-EcoRI fragment of HAL* together with
the 17.5 kb NotI-ClaI fragment of cosmid clone
13. The final plasmid was completed at the 3' end with a 6 kb
EcoRI-SmaI fragment obtained from cosmid clone 1 (see Fig. 1). Plasmid P27*5' was constructed by deleting the
NotI-EcoRI fragment of POMC27* corresponding to
the 5' region not contained in pHAL*. The resulting fragment was
blunt-ended and religated. Plasmid P27*3' was generated by deleting
the EcoRI-SmaI fragment of POMC27* corresponding
to the 3' region not included in pHAL*. The extremes were blunt-ended
and religated. The plasmid rEx3* used to make antisense riboprobes was
constructed by subcloning the pHAL* BglII-ApaI
exon 3-purified fragment into the multiple cloning site of pBluescript
SK+/ (Stratagene).
Production of transgenic mice. Transgenic mice were
generated by pronuclear microinjection of B6CBF2 zygotes through glass capillary micropipettes attached to a manual micromanipulator (Leica,
Nussloch, Germany). Fertilized eggs isolated between 9 and 10 A.M., as described elsewhere (Low, 1992), were observed under
the differential interference contrast optics of an inverted microscope
(FS Labovert, Leica) and microinjected into the most visible pronucleus
with ~500 molecules of the transgene dissolved in 1 pl of a sterile
solution containing 5 mM Tris-HCl, pH 7.4, and 0.1 mM EDTA. Transgenes were released from the plasmids by an
SalI and NotI double digestion, separated by
agarose electrophoresis, collected by electroelution, and purified
through an ion-exchange Elutip-D (Schleicher & Schuell, Keene, NH)
column. After microinjection, eggs were transferred to the oviduct of
6- to 10-week-old pseudopregnant Swiss Webster females.
Transgenic mouse identification. Screening for positive
transgenic mice was performed by PCR on genomic DNA extracted
from tail biopsies. Primers M329 (5'-GAAGTACGTCATGGGTCACT-3') and M330 (5'-AGCTCCCTCTTGAACTCTAG-3') amplify a 180 bp band for the wild-type POMC allele and a 210 bp band for a transgenic POMC allele. DNA was
amplified into a 1605 Air Thermo Cycler (Idaho Technology, Idaho Falls,
ID) as follows: a first denaturation step at 94°C during 5 min
followed by 35 cycles at 94°C for 0 sec, 60°C for 10 sec, and
72°C for 30 sec, with a final elongation step at 72°C for 10 min.
To confirm transgene integration and to estimate the copy number of
each pedigree, we performed Southern blot hybridization of
XhoI-EcoRI-digested genomic DNA extracted from
tail biopsies. DNA fragments were separated by submarine
electrophoresis in a 0.7% agarose gel and then transferred to a
Zeta-Probe nylon membrane (Bio-Rad, Hercules, CA). Blot hybridization
was performed using a 0.9-kb EcoRI-HindIII
fragment isolated from plasmid pHAL* and radiolabeled by random priming
(Life Technologies, Bethesda, MD) with [-32P]dCTP
(DuPont NEN, Boston, MA). Hybridization was performed at 65°C during
16 hr in a solution containing 6× SSC (1× SSC: 0.15 M
NaCl and 0.015 M Na citrate, pH 7.2), 25 mM
phosphate buffer, pH 7.2, 5× Denhardt's solution, 0.5% SDS, 1 mM EDTA, pH 8.0, and 100 µg/ml denatured salmon sperm
DNA. Blots were then washed in 2× SSC and 0.1% SDS at room
temperature twice for 15 min and then in 0.1× SSC and 0.1% SDS at
60°C and were finally exposed to x-ray film (XAR5; Eastman Kodak,
Rochester, NY) using intensifying screens at 70°C. The transgene
copy number of each line was determined by a scanning densitometric
analysis (Gelworks 1D; UVP). Values were interpolated into a
concentration curve prepared with mouse control DNA carrying known
amounts of the transgenes. F1 mice were analyzed for transgene
expression between 6 and 10 weeks of age.
In situ hybridization. In situ hybridizations
were performed as described previously (Rubinstein et al., 1993). Adult
normal mice or heterozygote transgenic mice were killed by
cervical dislocation, and brains and pituitaries were immediately
removed and frozen in OCT-containing plastic cubes. Serial
coronal sections (16 µm) were cut using a cryostat microtome (IEC
Microtome, Walldorf, Germany) at 20°C. Brain sections included the
entire anterior-posterior limits of the arcuate nucleus of the
hypothalamus. For developmental in situ hybridization, mouse
embryos were collected, and extraembryonic membranes were removed and
then frozen, embedded in OCT, and stored at 70°C. Sections were
thaw-mounted on Vectabond (Vector Laboratories, Burlingame, CA)-coated
slides and stored at 70°C until used. Sections were fixed in
neutral phosphate-buffered 10% formalin (Sigma, St. Louis, MO) at room
temperature for 30 min and washed three times with PBS (in
mM: 140 NaCl, 3 CaCl2, 10 Na2HPO4, and 1.8 KH2PO4) for 10 min at room temperature,
followed by three washes in 2× SSC for 10 min at room temperature.
Slides were drained 5-10 min and then incubated under parafilm
coverslips with 35-50 µl of a solution containing 50% formamide,
4× SSC, 1 × Denhardt's solution, 250 µg/ml yeast tRNA, 500 µg/ml sheared and denatured salmon sperm DNA, 10 mM
dithiothreitol, and 10% dextran sulfate at 37°C for 1 hr. Slides
were drained again and incubated overnight at 37°C with
prehybridization solution containing 11,000-20,000 cpm/µl
oligonucleotide probe. Slides were washed four times with 1× SSC and 1 mM dithithreitol for 15 min at room temperature followed by
four washes with 2× SSC and 50% formamide for 15 min at 37°C. Finally, slides were washed three times with 1× SSC for 30 min at room
temperature. After a quick rinse in water, slides were dehydrated
through 5 min steps in ethanol at 70, 95, and 100%, dried at room
temperature, dipped in Kodak N-TB3 emulsion (diluted 1:1 in distilled
water), and exposed for 14-19 d. Slides were developed, counterstained
with neutral red (Sigma), and coverslipped under Permount (Fisher
Scientific, Pittsburgh, PA). Radiolabeled probes were generated
by incubating 5 pmol of synthetic oligodeoxynucleotides with 50 pmol of
[35S]dATP (1300 Ci/mmol; DuPont NEN) and 50 U
of terminal deoxynucleotidyltransferase (Life Technologies). To detect
both the endogenous POMC mRNA and the transgenic transcripts we used a
30 mer antisense to exon 2 (E2-POMC,
5'-CCCTGAGCGACTGTAGCAGAATCTCCGGCAT-3'), whereas to detect
specifically the transgenic transcripts we used 553-TG, a 30 mer
antisense to the inserted oligonucleotide described above (553-TG,
5'-CGCGATAGCAGACTCGAGAGCGACAGACAG-3'). To detect specifically the
endogenous transcript, we used E3-POMC, a 30 mer antisense to the POMC
region that surrounds the insertion site (E3-POMC, 5'-CGGAAGTGCTCCATGGAGTAGGAGAGCGCTTG-3').
RNA extraction. Cytoplasmic RNA was prepared from individual
mouse hypothalami and pituitaries as follows: 0.5-2 mg of frozen tissue was homogenized using a 1 ml syringe with a 25 ga needle in a
buffer containing 0.01 M Tris-HCl, pH 7.0, 0.15 M NaCl, 2 mM MgCl2, 1.2%
Nonidet P-40 (Sigma), and 10 U of RNasin (Sigma). The extract was
centrifuged at 10,000 × g for 5 min at 4°C. One volume of a solution containing 0.01 mM Tris-HCl, pH 7.6, 0.15 M NaCl, 5 mM EDTA, and 1.2% SDS was added
to the supernatant and mixed by vortexing, and an equal volume of
phenol:isoamyl alcohol:chloroform (50:1:50) was added. The emulsion was
centrifuged at 10,000 × g at 4°C for 10 min.
Cytoplasmic RNA was isolated from the aqueous phase by centrifugation
in 0.3 M sodium acetate and 70% ethanol. After washing and
drying, the precipitate was redissolved in sterile water and stored at
70°C until used. Total RNA was isolated from other tissues by the
acid-guanidinium thiocyanate-acid-phenol method (Chomczynski and
Sacchi, 1987), and RNA concentration was determined by UV absorption at
260 nm.
Ribonuclease protection assay. Antisense cRNA probes were
produced by in vitro transcription of rEx3*, a plasmid
containing a mouse POMC genomic insert that encompasses the entire exon
3, including the heterologous 30 bp inserted originally into the NcoI site (Rubinstein et al., 1993). rEx3* was linearized
with XhoI and then subjected to in vitro
transcription using T7 RNA polymerase as described by the manufacturer
(Gemini; Promega, Madison, WI). Uniformly labeled riboprobes were
generated using [-32P]UTP, and full-length 214 nucleotide transcripts were isolated after running a vertical PAGE. The
radiolabeled riboprobe was eluted, quantified, and incubated with the
totality of extracted RNA in the case of hypothalamus or with fractions
in the case of other tissues. To normalize among samples, a mouse
-actin riboprobe was also included in all reactions and prepared
after in vitro transcription of a linearized plasmid
supplied with the RPA II ribonuclease protection assay kit (Ambion,
Austin, TX). RNA samples and probes were coprecipitated in 0.5 M ammonium acetate and 70% ethanol, and the resulting
pellets were resuspended in the hybridization buffer supplied with the
kit. Reaction mixtures were denatured for 5 min at 90°C and then
incubated overnight at 43°C to allow hybridizations to occur.
Unhybridized single-stranded RNA was digested by a mixture of 4 U/ml
RNase A and 15 U/ml RNase T1 in digestion buffer supplied by the
manufacturer at room temperature for 30 min. Protected fragments were
precipitated, and RNase activities were halted by adding an RNase
inactivation-precipitation mixture. The protected cRNA-mRNA hybrids
containing mRNA transcribed from the endogenous POMC gene (185 bases),
from the transgenes (199 bases), and from the mouse -actin gene (300 bases) were separated by electrophoresis in 5% polyacrylamide
denaturing gels in Tris-borate-EDTA (0.9 M Tris-HCl, 0.9 M boric acid, and 20 mM EDTA) buffer. The experimental design is depicted in Figure 5A. The gels were
exposed via direct contact autoradiography, and individual bands were quantified by determining their total integrated optical density using
an LKB-Wallac densitometer.
Colocalization of transgenic mRNA and -endorphin.
Simultaneous in situ detection of transgenic POMC27* mRNA
and -endorphin was achieved by in situ hybridization
using the deoxyoligonucleotide probe 553-TG followed by
immunocytochemistry using a -endorphin antiserum (Rubinstein et al.,
1996). Slides previously subjected to in situ hybridization,
exposed and developed as described above, were immersed in toluene to
remove the coverslips and then dipped in a freshly prepared 0.2% (w/v)
trypsin solution in PBS, 0.01 M, pH 7.2, for 30 sec. The
slides were washed twice in PBS for 45 min followed by 30 min in
KPBS (0.9% NaCl, 16 mM
K2HPO4, and 3.6 mM
KH2PO4) and then in KPBS and
1% H2O2 for 20 min and finally were rinsed
three times in KPBS for 5 min. Slides were then preincubated in KPBS-0.3% Triton X-100-2% normal goat serum at room temperature for 2 hr and then incubated overnight at 4°C with a -endorphin antiserum (1:1000 in KPBS, 0.3% Triton X-100, and 2% normal goat serum). After washing with KPBS two times for 20 min, antigen-antibody complexes were incubated with a biotinylated goat anti-rabbit IgG
solution (Sigma) (1:200 in KPBS and 0.3% Triton X-100) for 2 hr at
room temperature and then washed twice with KPBS for 20 min. Finally,
slides were incubated with avidin-peroxidase (1:1000 in KPBS, 0.3%
Triton X-100, and 0.1% bovine serum albumin) for 1 hr, rinsed in TBS
(50 mM Tris-HCl and 150 mM NaCl, pH 7.5), and
incubated with diaminobenzidine (25 mg/ml in TBS, 0.05%
H2O2). The reaction was stopped by
dipping the slides in TBS. Slides first dehydrated in ethanol 70, 95, and 100%, followed by xylene, were then coverslipped under
Permount.
Restraint stress paradigm. Stress was applied by a 1 hr
immobilization. Each animal was inserted into a plastic cylindrical device, which could be adapted to the size of the animal, between 10:30
and 11:30 A.M. for 5 consecutive days. Adhesive tape was used to
tightly close the tube. Immediately after the end of the last restraint
session, experimental and control animals were killed, and the tissues
were collected.
Surgical procedures. Bilateral adrenalectomies and
castrations were performed under tribromoethanol anesthesia (300 mg/kg, i.p.). Mice subjected to adrenalectomy were supplemented with 0.9%
NaCl or received a replacement dose of dexamethasone (Sigma; 50 µg/ml
in 0.9% NaCl) in their drinking water for 5 d. At the fifth day,
mice were killed by cervical dislocation, and tissues were removed.
Mice subjected to bilateral orchiectomy were killed by cervical
dislocation 3 weeks after the surgical procedures. For the ontogenic
studies, timed pregnant females checked by a morning vaginal plug were
killed by cervical dislocation at 1 d intervals starting at E9.5,
and the embryos were removed and analyzed macroscopically to confirm
developmental staging.
Drug treatments. POMC27* transgenic mice housed in groups of
five received two daily subcutaneous injections (at 7 A.M. and 7 P.M.)
of 1 mg/kg apomorphine (Laboratoires Meram), 1 mg/kg haloperidol (Janssen Farmaceutica, Buenos Aires, Argentina), or vehicle for 5 d. All animals were killed by cervical dislocation 1 hr after the last
injection, and tissues were immediately removed.
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RESULTS |
Neuron-specific expression of POMC genomic fragments in
transgenic mice
We constructed and screened a 129/SvEv mouse genomic library and
obtained two overlapping cosmid clones that covered 27 kb of the POMC
locus. These fragments were used to generate POMC27*, a transgene that
contained the entire 6 kb of the POMC transcriptional unit together
with 13 kb of 5' flanking regions and 8 kb of 3' flanking regions. To
follow the expression of the transgene unequivocally from that of the
endogenous mouse POMC gene by in situ hybridization, we
inserted a double-stranded 30 mer oligonucleotide at the unique NcoI site present in exon 3 (Fig.
1). Microinjection of POMC27* into the
pronuclei of B6CBF1 mouse zygotes originated three independent transgenic pedigrees. Analysis of the expression of the transgene in
comparison with that of the endogenous POMC gene was performed on 16 µm cryostat coronal sections of brains and pituitaries obtained from
F1 transgenic and nontransgenic littermates from the three lines.
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Figure 1.
Structure of POMC transgenes. POMC27* is a 27 kb
genomic fragment that contains the entire transcriptional unit of the
mouse POMC gene together with 13 kb of 5' flanking sequences and 8 kb
of 3' flanking sequences. P27*3' carries only 2 kb of mouse POMC 3'
flanking sequences, whereas P27*5' carries only 2 kb of 5' flanking
sequences. HAL* is a 10.2 kb genomic fragment that contains the entire
transcriptional unit of the mouse POMC gene together with 2 kb of 5'
and 3' flanking sequences. The asterisk depicted in all
transgenes indicates a 30 bp heterologous sequence inserted into the
unique NcoI site present in exon 3, as described in
Materials and Methods. Black boxes indicate exon
sequences. E2-POMC,
553-TG, and
E3-POMC indicate the positions of the
oligonucleotides used for expression analysis. C,
ClaI; E, EcoRI;
S, SmaI; N,
NotI.
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Detection of the genuine mouse POMC mRNA was performed by using
E3-POMC, an antisense 30 mer oligonucleotide probe designed against the
15 nucleotides that flank both sides of the NcoI site present in exon 3. Preliminary hybridization studies performed under
stringent conditions with plasmid DNA transferred to nylon membranes
showed that this probe hybridized with fragments carrying the wild-type
exon 3 but failed to hybridize to POMC27*, resulting in an efficient
tool to detect only endogenous POMC mRNA (data not shown). E3-POMC
revealed the identical distribution of POMC-expressing neurons in the
arcuate nucleus of the medial basal hypothalamus of transgenic mice
(Fig.
2A,B)
and their nontransgenic siblings (Fig.
2E,F). Detection of
transgenic expression was performed by the use of 553-TG, an antisense
oligonucleotide designed against the heterologous 30 bp sequence
inserted at the NcoI site. In situ hybridization
performed on brain coronal slices with 553-TG showed POMC27* expression
only in the hypothalamus of transgenic mice (Fig. 2, compare
C,G) with a qualitative and quantitative pattern
similar to that found for the endogenous POMC mRNA (Fig. 2B). This result suggested an appropriate expression
of the transgene POMC27* in POMC neurons. A 30 mer probe designed
against exon 2 sequences (E2-POMC) that hybridizes with the endogenous
POMC mRNA as well as with the transgenic POMC27* mRNA showed an
identical spatial pattern of expression, although it exhibited a higher hybridization signal. This quantitative difference reflected, in part,
the additive contribution of the wild-type and the transgenic mRNAs to
this signal (Fig. 2D). Nonetheless, we also noted
that the in situ signal obtained with E2-POMC was
consistently higher than that observed with E3-POMC in serial sections
of nontransgenic mice (Fig. 2, compare
F,H), suggesting a higher
in situ accessibility of E2-POMC for POMC-derived mRNA
despite sharing similar thermodynamic properties with E3-POMC
(Tm, 82.2 and 84.8°C, respectively). The transgene is also
efficiently targeted to endocrine cells of the intermediate and
anterior lobes of the pituitary gland (Fig.
2I,J). The results shown in
Figure 2 correspond to tissue sections taken from male mice. The same
pattern of distribution for the transgenic and endogenous POMC mRNAs
was also observed in female mice.
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Figure 2.
Localization of POMC and POMC27* mRNA in
hypothalamic and pituitary sections by in situ
hybridization. Alternate 16 µm coronal sections taken at the level of
the mediobasal hypothalamus of POMC27* transgenic
(A-D) and nontransgenic
(E-H) mice were hybridized with
-35S-labeled 30 bp oligonucleotides. Similarly,
pituitary sections were taken from a POMC27* transgenic mouse
(I, J). Probe E3-POMC detects only
endogenous POMC mRNA (B, F). Probe
553-TG detects only transgenic POMC27* mRNA (C,
G, I), and probe E2-POMC
recognizes both endogenous and transgenic POMC mRNAs (D,
H, J). Dark-field photomicrographs
show endogenous and transgenic expression in neurons of the arcuate
nucleus of the hypothalamus. Cellular architecture of this brain region
stained with neutral red is shown in the bright-field photographs on
the left (A, E).
Magnification, 40×.
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To further confirm that arcuate neurons labeled with 553-TG
corresponded to genuine POMC-expressing cells, we performed a colocalization experiment by combining in situ hybridization
with 553-TG followed by immunocytochemical labeling with a
-endorphin antiserum. Silver grains deposited on cell bodies were
only detected on neurons that immunoreacted with the -endorphin
antiserum, demonstrating that transgenic expression from POMC27* was
accurate and limited to POMC-producing cells in the hypothalamus (Fig. 3). We have not detected ectopic
expression sites anywhere in the brain in all transgenic lines
analyzed. This result validated the assumption that POMC27* contains
strong neuron-specific sequences to target transgene expression to
neurons of the POMC lineage.
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Figure 3.
Colocalization of transgenic POMC27* mRNA and
-endorphin immunoreactivity on neurons of the arcuate nucleus of the
hypothalamus. Left, Representative 20 µm coronal
section taken from the mediobasal hypothalamus of a POMC27* transgenic
mouse that was subjected first to in situ hybridization
using probe 553-TG and then to immunolabeling with a -endorphin
antiserum. Immunoreactivity was detected using diaminobenzidine as
described in Materials and Methods. Magnification, 100×.
Right, A, B,
Representative areas of the arcuate nucleus section on the
left at a 400× magnification scale.
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The distal 5' flanking region of POMC is required for expression in
the CNS
In a previous report, we showed that a 10.2 kb mouse POMC genomic
fragment that contained 2 kb of 5' flanking sequences together with 2 kb of 3' flanking sequences (Fig. 1, HAL*) failed to direct proper expression to POMC neurons in transgenic mice (Rubinstein et
al., 1993). A comparison of the expression pattern achieved with
POMC27* in relation to that previously observed with HAL* (Fig. 1)
indicated that additional 5' or 3' flanking sequences would contain the
necessary elements to target transgenic expression to POMC-producing
neurons. To evaluate whether these elements would reside on the 5' or
the 3' arm, we generated transgenic mice carrying either a 3' truncated
version of POMC27* named P27*3' or a 5' version of POMC27* named
P27*5' (Fig. 1). Deletion of the 5' flanking sequences resulted in a
complete lack of neuronal expression in the brain of transgenic mice
from three independent lines analyzed (Fig.
4A-D).
Interestingly, appropriate expression of P27*5' in the pituitary
gland was observed in mice from the three lines generated (Fig.
4E-F) with lower levels of the
transgenic mRNA in comparison with POMC mRNA in the anterior lobe. This
result demonstrates directly that the control of brain and pituitary expression of the POMC gene is exerted by independent mechanisms. Conversely, deletion of the 3' flanking sequences of POMC27* did not
affect the efficient targeting of the transgene to POMC-expressing neurons or the pituitary (Fig. 4G-L). The
neuronal expression pattern of the transgene P27*3' was determined
by in situ hybridization in coronal brain slices and shown
to be identical to that observed for the endogenous POMC gene in the
two independent pedigrees generated (Fig.
4G-J). These results indicated that
critical neuron-specific elements are localized within an 11 kb stretch
of distal 5' sequences from the POMC gene.
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Figure 4.
Neuron-specific expression of the POMC gene is
controlled by distal 5' flanking sequences. Expression analysis of
P27*5' (A-F) and P27*3'
(G-L) transgenic mice by in
situ hybridization performed on alternate coronal sections of
the mediobasal hypothalamus (A-D,
G-J) or the pituitary
(E, F,
K, L). Left,
Sections incubated with an -35S-labeled E2-POMC probe.
Right, Sections incubated with an
-35S-labeled 553-TG probe. Bright-field photographs show
neutral red-stained sections (A,
B, E-H,
K, L). The same sections
under dark field allowed a clearer detection of the silver grain signal
(C, D, I,
J). Magnification, 40×.
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Quantitative analysis of POMC27* transgene expression in
different tissues
To analyze both the spatial expression pattern and steady-state
levels of POMC27* mRNA, we developed an RNase protection assay sensitive enough to detect simultaneously endogenous and transgenic POMC mRNAs from single tissue samples. The presence of the
double-stranded 30 bp heterologous sequence inserted in the coding
region of POMC27* exon 3 allowed us to use a unique radiolabeled
214-nucleotide riboprobe to perform a relative quantitative analysis
between the 185-nucleotide band protected by endogenous POMC mRNA and the 199-nucleotide band protected by transgenic POMC27* mRNA (Fig. 5A). A riboprobe directed to
the mouse -actin mRNA was included in all incubations as an internal
control. Tissues obtained from several transgenic and control mice were
monitored for the presence of the two POMC mRNA forms. As expected,
total RNA extracted from tissues known to express the POMC gene such as
the pituitary, the hypothalamus, and testes from wild-type mice
protected a single band of 185 nucleotides (Fig. 5B,
right). When RNA was prepared from tissues obtained from
POMC27* transgenic mice, an additional protected band of 199 nucleotides was evident, revealing the presence of transgenic mRNA. An
analysis across a wide variety of tissues showed an almost complete
overlap of expression for both mRNAs (Fig. 5B). POMC and
POMC27* mRNA were detected in all brain regions analyzed, including
hypothalamus, brain cortex, cerebellum, brainstem, and the rest of
brain (Fig. 5B). Both POMC mRNA forms were also detected in
the testes and the spleen. However, in the ovaries and serum we only
detected the shorter protected band that corresponded to endogenous
POMC (Fig. 5B). A similar result was obtained with skin
samples taken from the flanks of the animals (data not shown). Total
RNA isolated from other tissues such as liver, skeletal muscle, and
heart failed to protect either band (data not shown). In conclusion,
over the complete range of tissues analyzed, the expression of the
transgene POMC27* paralleled almost entirely that of the endogenous
gene with no ectopic expression detected by this sensitive method.
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Figure 5.
Simultaneous detection of POMC and POMC27* mRNA in
different tissues of transgenic mice by an RNase protection strategy.
A, Description of the RNase protection assay developed
to detect simultaneously both POMC mRNA forms isolated from single
tissues as described in Results. The white and
black squares denote the 30 bp heterologous
oligonucleotide inserted in exon 3. The black squares
depict the 15-nucleotide portion that hybridizes to the riboprobe.
B, Cytoplasmatic RNA samples isolated from different
tissues of POMC27* transgenic and wild-type (WT)
mice were subjected to an RNase protection assay using a mouse POMC*
exon 3 riboprobe. A mouse -actin riboprobe was included in all
protection reactions as an internal control. Pit,
Pituitary; AL, anterior lobe; BS,
brainstem; RB, rest of brain; BC, brain
cortex; Hy, hypothalamus; Cer,
cerebellum; Test, testis; Ser, serum;
Ova, ovary; Spl, spleen.
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Developmental expression of POMC27*
The ontogenic program of POMC gene expression is rather complex.
To test whether POMC27* was under the same restricted temporal and
spatial pattern of expression as the mouse POMC gene, we set up timed
pregnancies and collected embryos for consecutive days starting at
E9.5. In situ hybridization of sagittal and coronal slices
of mouse embryos revealed that the expression of POMC27* followed an
identical developmental pattern of expression to the endogenous POMC
mRNA (Fig. 6). As early as E10.5,
transgenic signal was observed in a region of the basal diencephalon
that will give rise to the developing arcuate nucleus, in agreement
with what has been previously observed for the onset of POMC gene
expression (Japón et al., 1994). In addition, POMC27* mRNA was
also clearly detected in the base of the fourth ventricle (Fig.
6A-C) and in the neuroepithelium of the
neural tube (Fig. 6D-F), regions
that will develop into the brainstem and the lower spinal cord,
respectively. At E13.5 POMC27* expression was observed in the primitive
anterior lobe of the pituitary gland (Fig.
6G-I), and 1 d later cells of the
intermediate lobe were also evident (Fig.
6J-L). Coronal sections of the pituitary
at E17.5 showed the ventromedial to dorsolateral migration of the
transgene after the typical recruitment of this cell type within the
developing anterior lobe (Fig. 6M-O).
POMC expression in neurons of the nucleus of the tractus solitarius was
detected at E17.5 in coronal sections, and that of the transgene was
identical (Fig. 6P-R). No ectopic
expression of the transgene was detected throughout development.
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Figure 6.
Developmental analysis of POMC and POMC27*
expression in transgenic mice. Dark-field photomicrographs of
endogenous POMC and transgenic POMC27* mRNA detected by in
situ hybridization using []35S-labeled
oligonucleotide probes on sections of mouse embryos collected at E10.5,
E13.5, E14.5, and E17.5. Probe 553-TG (right column)
detects only transgenic POMC27* expression, and probe E2-POMC
(middle column) detects the endogenous and transgenic
mRNA simultaneously. A-L were cut using
a sagittal orientation, M-O were
cut in a coronal plane, and P-R
were taken on a horizontal plane. Bright-field photographs of embryo
sections stained with neutral red are shown on the left
column for anatomical reference. III, Third
ventricle; IV, fourth ventricle; BD,
basal diencephalon; RP, Rathke's pouch;
NL, neural lumen; nNT, neuroepithelium of
neural tube; NTS, nucleus of the tractus solitarius.
Arrows in H and I identify
anterior lobe corticotrophic cells, whereas arrows in
K and L indicate intermediate lobe
melanotrophic cells. Arrows in Q and
R are pointing at cells from the nucleus of the tractus
solitarius. Magnifications: A-F,
M-R, 40×; G-
L, 100×.
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Hormonal regulation of POMC27* expression in the pituitary and
brain of transgenic mice
POMC gene expression is highly regulated by neurotransmitters and
hormones. Melanotrophs of the intermediate lobe are negatively regulated by tuberoinfundibular dopamine release, whereas
glucocorticoids inhibit POMC transcription in corticotrophs of the
anterior lobe. In the hypothalamus, POMC gene expression has been
reported to be regulated by neurotransmitters and hormones that
participate in the stress response (Baubet et al., 1994) and in
reproductive function (Matera and Wardlaw, 1994). To determine whether
the cis-acting elements present in POMC27* would account for
the hormonal regulation of the gene in the different pituitary cell
types and in the hypothalamus, we performed the following series of
experiments on transgenic mice from one of the POMC27* pedigrees. Mice
were killed after pharmacological or surgical manipulations, and total RNA was obtained from single neurointermediate and anterior pituitary lobes as well as from single hypothalami for analysis with the RNase
protection strategy described above.
The dopamine receptor blocker haloperidol administered twice a day for
5 consecutive days induced a twofold increase in the levels of both
POMC mRNA forms isolated from the neurointermediate pituitary lobe
(Fig. 7A). However, the
dopamine receptor agonist apomorphine given for 5 consecutive days
failed to modify the expression levels of melanotrophic POMC or
POMC27*. Interestingly, a densitometric scanning analysis determined
that the relative levels of the bands protected by POMC and POMC27*
mRNA were almost identical in all treatments, including the control
animals receiving saline (Fig. 7A). These results suggest
that the transcriptional strength of the transgene in melanotrophs
mirrors that of the endogenous gene. The treatment with dopaminergic
agents did not modify the mRNA content for either POMC form in the
anterior lobe (data not shown) or in the hypothalamus (Fig.
7C). In the anterior lobe, we found that the levels of
transgenic POMC27* mRNA were significantly lower that those seen for
POMC in all lines analyzed. Nonetheless, bilateral adrenalectomy
induced an almost threefold increase in the levels of POMC in this lobe
for both mRNAs (Fig. 7B). Mice receiving dexamethasone
replacement experienced a significant reduction in the mRNA contents of
both POMC products (Fig. 7B). We then studied the regulation
of POMC gene expression in the arcuate nucleus of the hypothalamus.
Gonadal steroids mediate the negative control of hypothalamic
gonadotropin-releasing hormone secretion through a pathway that
involves the participation of the POMC product -endorphin (Matera
and Wardlaw, 1994; Olson et al., 1997). Steroid modulation of POMC gene
expression has been implicated in such a mechanism (Yang and Lim,
1995). To this end, bilateral orchiectomies were performed in
transgenic and nontransgenic mice that were killed 21 d after
surgery. Contrary to what has been reported in rats, castration failed
to produce a significant effect on the contents of either POMC mRNA
species (Fig. 7C). We then evaluated whether repetitive
stressful stimuli would modify hypothalamic POMC mRNA levels in mice as
it had been reported in rats (Baubet et al., 1994). Mice were stressed
by immobilization for 1 hr during 5 consecutive days and killed
immediately after receiving the 10th session. We found that the
expression levels of POMC and POMC27* were not significantly modified
following this paradigm (Fig. 7C). Interestingly enough, the
relative levels of POMC and POMC27* mRNA in the hypothalamus of
transgenic mice were identical, suggesting that expression from the
transgene paralleled that of POMC in a quantitative as well as in a
qualitative manner, similar to what we observed in the
neurointermediate lobe.
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Figure 7.
Hormonal regulation of POMC and POMC27* in
transgenic mice. Detection and quantification of POMC and POMC27* mRNA
by RNase protection followed by densitometric scanning of single tissue
samples isolated from POMC27* transgenic mice receiving different drug
or surgical treatments. A, Neurointermediate lobe
samples taken from mice receiving saline (SAL), 1 mg/kg
apomorphine subcutaneously (APO), or 1 mg/kg haloperidol
subcutaneously (HAL) twice daily during 5 consecutive
days. B, Anterior lobe samples taken from sham-operated
mice receiving daily injections of saline (SAL) during
the following 5 d from bilaterally adrenalectomized mice
(ADX) that received saline in their drinking
bottles on the 5 consecutive days or 50 µg/ml dexamethasone
(DEX) dissolved in saline in their drinking
bottles during the 5 consecutive days after surgery. C,
Hypothalamic samples taken from mice receiving during 5 d saline
(SAL), 1 mg/kg haloperidol subcutaneously
(HAL), a bilateral orchiectomy treatment
(ORX), or a restraint stress treatment
(STR). In all cases mRNA levels are indicated as a
relative percentage of endogenous POMC mRNA content normalized to
-actin content from mice receiving saline. Bars indicate mean ± SEM of samples of 6-10 mice per group. Statistical analysis was
performed by ANOVA followed by a Mann-Whitney U test.
*p < 0.05 versus same mRNA species from
saline-treated mice; **p < 0.01 versus same mRNA
species from saline-treated mice; §p < 0.01 versus endogenous POMC receiving the same treatment.
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DISCUSSION |
For the first time we show targeted expression of transgenes
exclusively to POMC neurons of the CNS under an accurate spatial and
temporal control. Expression of transgenes carrying POMC gene-flanking sequences was clearly detected in POMC-producing cells of the arcuate
nucleus of the hypothalamus as well in the developing diencephalon,
spinal cord, and nucleus of the tractus solitarius. The
cis-acting elements necessary for this efficient targeting were localized within 11 kb of distal sequences of the 5' flanking region upstream of the POMC promoter. This distal fragment was not
required for the expression of POMC derived in pituitary cells of
endocrine origin such as melanotrophs and corticotrophs. Previous studies using transgenic mice demonstrated that cell-specific expression of the POMC gene in the pituitary was controlled by the
combinatorial presence of cis-acting elements localized
within 300 nucleotides proximal to the transcriptional start site (Liu et al., 1992, 1995). We now show that the neuronal expression of the
POMC gene requires additional elements localized further upstream. We
cannot rule out the possible participation of DNA sequences present
within the introns or the 2 kb fragment of 3' flanking sequences that
were included in all transgenes studied, although it has been shown
that pituitary-specific expression of the POMC gene does not require
intronic or 3' flanking sequences (Liu et al., 1992, 1995). Because
neurons collectively express a higher number of genes than all other
cell types, it is conceivable that cell-specific gene expression in the
CNS follows more sophisticated hierarchical mechanisms of
transcriptional control that may include additional transcription
factors to orchestrate an active enhanceosome (Carey, 1998). There are
very few examples in the literature reporting flanking sequences
capable of directing the expression of transgenes in a neuron-specific
and developmentally regulated manner within the CNS, and they include
the vasopressin gene (Ang et al., 1993), the tyrosine hydroxylase gene
(Min et al., 1994), and the choline acetyltransferase gene (Lonnerberg
et al., 1995).
All independent transgenic pedigrees produced with POMC27* or with
P27*3' exhibited a high-level accurate expression in the CNS. This
100% penetrance suggests that the POMC genomic fragment used in both
transgenes has full transcriptional strength to recruit an active
enhanceosome, making expression independent from chromosomal influences
that could silence transgene expression. Although all transgenic mice
generated in this study exhibited a correct spatial expression in the
pituitary gland, the levels of expression in corticotrophs of the
anterior lobe were always significantly lower than those observed with
the endogenous POMC gene. This characteristic has been reported
previously for other POMC-derived transgenes (Liu et al., 1992;
Rubinstein et al., 1993) and may suggest that a corticotrophic enhacer
is localized even further out from the genomic fragments used in this
study. Interestingly, pituitary POMC-expressing cells seem to be the
only cell lineage that still develops in all null allele mutant mice
lacking master genes that control pituitary organogenesis or pituitary
differentiation such as Lhx3 (Sheng et al., 1996), Lhx4 (Sheng et al.,
1997), Pit-1 (Li et al., 1990), and Prophet-1 (Sornson et al., 1996).
The key master gene for pituitary POMC differentiation still remains
elusive, although the recently cloned homeodomain transcription factor Ptx1/P-OTX is a possible candidate because it controls POMC expression in the corticotrophic cell line AtT20 (Lamonerie et al., 1996; Szeto et
al., 1996).
Tracking POMC expression throughout development showed that the
temporal pattern of transgenic ontogeny in the different embryonic areas was identical to that observed for the mouse POMC gene, suggesting that the transgene carries the cis-acting
elements required for the early stages of cell lineage determination
and phenotype commitment. POMC and POMC27* expression were detected as
early as E10.5 in the base of the diencephalon and in the base of the
fourth ventricle, preceding all other neuropeptide gene expression in
the developing brain. This early expression suggests that POMC peptides
may play a developmental role in hypothalamic organogenesis or in
establishing synaptic pathways to different brain regions. However,
this hypothesis has been challenged by the fact that mutant mice
lacking -endorphin (Rubinstein et al., 1996) or the melanocortin
MC-4 receptor (Huszar et al., 1997) did not show any developmental
abnormality. The ontogenic analysis performed in this study allowed us
to clearly detect POMC gene expression in two CNS regions in which POMC
mRNA is not readily observed in the adult rodent, such as the nucleus
of the tractus solitarius (Bronstein et al., 1992) and the lumbar
spinal cord. Normally, POMC mRNA is not detected in the spinal cord,
but under particular traumatic conditions, its expression is rapidly
induced (Gutstein et al., 1992; Hughes and Smith, 1993). We observed in sagittal sections taken from E10.5 embryos an intense signal in a
region of the neural epithelium that corresponds to the primitive lower
spinal cord. POMC expression is also negligible in the adult nucleus
tractus solitarii (NTS). Although -endorphin containing cell
bodies are readily visible in this area, detection of the mRNA has been
questionable. We observed, however, a definitive signal in the
primitive brainstem at E10.5 and in the developing NTS at E17.5. It
still remains unclear why POMC expression in these areas is higher
during development than in the adult.
Regulation of arcuate POMC gene expression has been reported in the rat
after repetitive inescapable stress by in situ hybridization analysis of coronal sections at different levels of the arcuate nucleus
(Baubet et al., 1994). However, in our laboratory we were not able to
reproduce those results in the mouse. It is conceivable that in the
mouse only a limited subset of hypothalamic POMC neurons is regulated
after this treatment and that these differences are masked when we
analyze the whole population of hypothalamic POMC mRNA content by an
RNase protection assay that used total RNA from single entire
hypothalami.
In conclusion, the achievement of an accurate spatial and temporal
targeting of transgenes to POMC neurons of the CNS opens the
possibility to further explore the physiological function of these
neurons and the mechanisms underlying POMC cell fate during
organogenesis of the hypothalamus. For example, targeting fluorescent
proteins will allow electrophysiological recording of easily identified
POMC neurons in whole basal hypothalamic slices. POMC arcuate neurons
are also an ideal target to study feeding and fasting physiology and
particularly to assess the interplay between leptin and POMC-derived
melanocortins, an issue that is now under controversy (Seeley et al.,
1997; Boston et al., 1997). Oncogene targeting to these cells in
transgenic mice (Alarid et al., 1996) may serve to produce a neuronal
cell line with a -endorphinergic phenotype useful for studying the
properties of this neuron population in vitro and as an
invaluable source for transcriptional regulation studies. Finally, we
believe that having the capacity to reliably direct transgenes to these
cells early in development will contribute to understanding the role of
the POMC lineage in hypothalamic organogenesis and lineage commitment
for each neuropeptide.
| |
FOOTNOTES |
Received March 11, 1998; revised June 8, 1998; accepted June 9, 1998.
This research has been supported by grants from the National Institutes
of Health/Fogarty International Research Collaborative Award
(M.J.L., M.R.), Universidad de Buenos Aires (M.R.), Fundación Antorchas (M.R.), and the International Scholar Program of the Howard
Hughes Medical Institute (M.R.). J.I.Y. and M.G.C. received doctoral
fellowships from Consejo Nacional de Investigaciones Científicas y Técnicas, Argentina. E.C.C. received a
fellowship from the Wollongong Government Employee's Medical Research
Fund. We thank the Transgenic Core Facility at Oregon Health Sciences University for valuable assistance with one of the constructs.
Correspondence should be addressed to Dr. Marcelo Rubinstein, Instituto
de Investigaciones en Ingeniería Genética y
Biología Molecular, Consejo Nacional de Investigaciones
Científicas y Técnicas, Vuelta de Obligado 2490, 1428 Buenos Aires, Argentina.
Dr. Chan's present address: Endocrine Unit, John Hunter Hospital,
Newcastle, Australia.
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V. F. Bumaschny, F. S. J. de Souza, R. A. Lopez Leal, A. M. Santangelo, M. Baetscher, D. H. Levi, M. J. Low, and M. Rubinstein
Transcriptional Regulation of Pituitary POMC Is Conserved at the Vertebrate Extremes Despite Great Promoter Sequence Divergence
Mol. Endocrinol.,
November 1, 2007;
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[Abstract]
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H. Shimizu, K. Inoue, and M. Mori
The leptin-dependent and -independent melanocortin signaling system: regulation of feeding and energy expenditure
J. Endocrinol.,
April 1, 2007;
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J. L. Smart, V. Tolle, V. Otero-Corchon, and M. J. Low
Central Dysregulation of the Hypothalamic-Pituitary-Adrenal Axis in Neuron-Specific Proopiomelanocortin-Deficient Mice
Endocrinology,
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L. S. Overstreet-Wadiche, D. A. Bromberg, A. L. Bensen, and G. L. Westbrook
Seizures Accelerate Functional Integration of Adult-Generated Granule Cells
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M. Nudi, J.-F. Ouimette, and J. Drouin
Bone Morphogenic Protein (Smad)-Mediated Repression of Proopiomelanocortin Transcription by Interference with Pitx/Tpit Activity
Mol. Endocrinol.,
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F. S. J. de Souza, A. M. Santangelo, V. Bumaschny, M. E. Avale, J. L. Smart, M. J. Low, and M. Rubinstein
Identification of Neuronal Enhancers of the Proopiomelanocortin Gene by Transgenic Mouse Analysis and Phylogenetic Footprinting
Mol. Cell. Biol.,
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W. Herzog, C. Sonntag, B. Walderich, J. Odenthal, H.-M. Maischein, and M. Hammerschmidt
Genetic Analysis of Adenohypophysis Formation in Zebrafish
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May 1, 2004;
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L. S. Overstreet, S. T. Hentges, V. F. Bumaschny, F. S. J. de Souza, J. L. Smart, A. M. Santangelo, M. J. Low, G. L. Westbrook, and M. Rubinstein
A Transgenic Marker for Newly Born Granule Cells in Dentate Gyrus
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S. M. Appleyard, M. Hayward, J. I. Young, A. A. Butler, R. D. Cone, M. Rubinstein, and M. J. Low
A Role for the Endogenous Opioid {beta}-Endorphin in Energy Homeostasis
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N.-A. Liu, H. Huang, Z. Yang, W. Herzog, M. Hammerschmidt, S. Lin, and S. Melmed
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Mol. Endocrinol.,
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T. Moriguchi, T. Sakurai, S. Takahashi, K. Goto, and M. Yamamoto
The Human Prepro-orexin Gene Regulatory Region That Activates Gene Expression in the Lateral Region and Represses It in the Medial Regions of the Hypothalamus
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J. Liu, C. Lin, A. Gleiberman, K. A. Ohgi, T. Herman, H.-P. Huang, M.-J. Tsai, and M. G. Rosenfeld
Tbx19, a tissue-selective regulator of POMC gene expression
PNAS,
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E. J. Lee, F. Martinson, T. Kotlar, B. Thimmapaya, and J. L. Jameson
Adenovirus-Mediated Targeted Expression of Toxic Genes to Adrenocorticotropin-Producing Pituitary Tumors Using the Proopiomelanocortin Promoter
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S. Beimesche, A. Neubauer, S. Herzig, R. Grzeskowiak, T. Diedrich, I. Cierny, D. Scholz, T. Alejel, and W. Knepel
Tissue-Specific Transcriptional Activity of a Pancreatic Islet Cell-Specific Enhancer Sequence/Pax6-Binding Site Determined in Normal Adult Tissues in Vivo Using Transgenic Mice
Mol. Endocrinol.,
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J. Liu, C. Lin, A. Gleiberman, K. A. Ohgi, T. Herman, H.-P. Huang, M.-J. Tsai, and M. G. Rosenfeld
Tbx19, a tissue-selective regulator of POMC gene expression
PNAS,
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Top
Abstract
Introduction
Substance via MeSH
