Induction of Neuropeptide Y Gene Expression in the Dorsal Medial Hypothalamic Nucleus in Two Models of the Agouti Obesity Syndrome
Robert A. Kesterson,
Dennis Huszar,
Catherine A. Lynch,
Richard B. Simerly and
Roger D. Cone
Vollum Institute for Advanced Biomedical Research (R.A.K.,
R.D.C.) Oregon Health Sciences University Portland,
Oregon 97201-3098
Millennium Pharmaceuticals, Inc.
(D.H., C.A.L.) Cambridge, Massachusetts 02139
Oregon Regional Primate Research Center (R.B.S.)
Beaverton, Oregon 97006
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ABSTRACT
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Dominant mutations at the agouti locus
induce several phenotypic changes in the mouse including yellow
pigmentation (phaeomelanization) of the coat and adult-onset obesity.
Nonpigmentary phenotypic changes associated with the agouti
locus are due to ectopic expression of the agouti-signaling protein
(ASP), and the pheomelanizing effects on coat color are due to ASP
antagonism of
-MSH binding to the melanocyte MC1 receptor. Recently
it has been demonstrated that pharmacological antagonism of
hypothalamic melanocortin receptors or genetic deletion of the
melanocortin 4 receptor (MC4-R) recapitulates aspects of the
agouti obesity syndrome, thus establishing that chronic
disruption of central melanocortinergic signaling is the cause of
agouti-induced obesity. To learn more about potential
downstream effectors involved in these melanocortinergic obesity
syndromes, we have examined expression of the orexigenic peptides
galanin and neuropeptide Y (NPY), as well as the anorexigenic POMC in
lethal yellow (Ay), MC4-R knockout
(MC4-RKO), and leptin-deficient (ob/ob) mice. No
significant changes in galanin or POMC gene expression were seen in any
of the obese models. In situ hybridizations using an
antisense NPY probe demonstrated that in obese
Ay mice, arcuate nucleus NPY mRNA levels
were equivalent to that of their C57BL/6J littermates. However, NPY was
expressed at high levels in a new site, the dorsal medial hypothalamic
nucleus (DMH). Expression of NPY in the DMH was also seen in obese
MC4-RKO homozygous (-/-) mice, but not in lean heterozygous (±) or
wild type (+/+) control mice. This identifies the DMH as a brain region
that is functionally altered by the disruption of melanocortinergic
signaling and suggests that this nucleus, possibly via elevated NPY
expression, may have an etiological role in the melanocortinergic
obesity syndrome.
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INTRODUCTION
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Dominant alleles of the agouti locus induce most
notably a yellow coat color in the mouse, whereas the most dominant
mutations (e.g. Ay or
Avy) produce additional phenotypic changes
including adult-onset obesity, increased linear growth, and
hyperinsulinemia (for review, see Refs. 1 and 2). The cloning of the
murine agouti gene (3) predicts a 108-amino acid secreted
protein that is normally expressed in skin during postnatal days 3 to
5, corresponding to the time during which pheomelanin deposition occurs
in the developing hair shaft. In contrast, the dominant lethal
yellow (Ay) allele of agouti
results in constitutive and ubiquitous expression of agouti-signaling
protein (ASP) as a consequence of a promoter rearrangement (4). The
obesity and coat color phenotypes of the Ay
animal are recapitulated by transgenic mice having a ß-actin promoter
driving ubiquitous ASP expression (5). However, transgenic mice using a
keratin-14 promoter to direct skin-specific expression of ASP have only
the yellow coat color phenotype, demonstrating that agouti acts locally
in a paracrine manner (6) and that expression outside of the skin is
required for the induction of obesity.
Experiments by Lu et al. (7) demonstrated that ASP is a
competitive antagonist of the melanocortin-1 receptor expressed in skin
melanocytes (8, 9). The melanocortin-1 receptor is normally stimulated
by
-MSH to increase intracellular cAMP levels, inducing tyrosinase
activity, and eumelanin or brown/black pigment synthesis. One
hypothesis for the nonpigmentary phenotypes seen in
Ay mice is that aberrant expression of ASP in
the central nervous system antagonizes related hypothalamic
melanocortin receptors (7). This model was originally proposed based
upon evidence that ASP is also a competitive antagonist of the highly
related melanocortin-4 receptor (MC4-R) (7), which is expressed in
regions of the hypothalamus important for the control of feeding
behavior (10). Strong support for this model comes from two recent
studies. First, central administration of the
-MSH analog SHU9119, a
high-affinity antagonist of the neural MC3 and MC4 receptors,
significantly stimulates feeding in mice, whereas an agonist potently
inhibited feeding in several murine models of hyperphagia (11). Second,
ablation of functional MC4 receptors by targeted disruption of the
MC4-R gene in mice recapitulates several aspects of the agouti obesity
syndrome (12).
One possible effect of losing MC4-R activity, either by genetic
deletion or chronic antagonism by ASP, may be perturbation of normal
hypothalamic signaling. Neuropeptide Y (NPY) (13) and galanin (14) are
two neuropeptides present in high concentrations in the hypothalamus
that, when injected into the brain, stimulate feeding. Recent
experiments have shown that hypothalamic NPY levels are elevated in
other models of rodent obesity, including the ob/ob mouse
(15) and the Zucker fatty rat (16), as well as in streptozocin-induced
diabetic rats (17) and in mice given hypothalamic lesions via
gold-thioglucose treatment (18). Therefore it seemed possible that NPY
or possibly galanin synthesis may be stimulated in
Ay or MC4-R deficient mice. Alternatively,
melanocortinergic neurons may respond to loss of normal MC4-R function
by compensatory changes in the synthesis of the melanocortin ligands
encoded by the preprohormone gene POMC. In this report, NPY, galanin,
and POMC gene expression were examined as a function of the
Ay or MC4-RKO obesity syndromes.
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RESULTS
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NPY Gene Expression but Not Galanin or POMC Is Altered in
Lethal Yellow (Ay) Mice
Representative autoradiograms of anatomically matched
coronal brain sections [at the level of the dorsal medial hypothalamic
nucleus (DMH)] obtained after in situ hybridizations with
NPY, galanin, and POMC antisense probes are shown in Fig. 1
. The upper panel shows the hypothalamic NPY
mRNA expression for C57BL/6J (C57) mice (left),
C57Bl/6J-Ay (Ay) mice
(middle), and C57Bl/6J-Lepob
(ob/ob) mice (right). Although the hybridization
intensity for NPY gene expression in the hypothalamic arcuate nucleus
(ARC) of the ob/ob mouse was clearly elevated when compared
with the control C57 mouse, there was no visible difference in NPY gene
expression in the ARC of the Ay mouse.
Furthermore, NPY expression in the cerebral cortex, not associated with
feeding behavior, was also similar in all three animal models. However,
in the Ay mouse a population of intensely
labeled neurons in the DMH was identified (with minor labeling of the
lateral hypothalamic area), whereas hypothalamic expression of NPY in
the ob/ob mouse was restricted to the ARC only. No
comparable differences in either galanin (middle) or POMC
(bottom) mRNA expression levels were observed between the
C57, Ay, and ob/ob mice (Fig. 1
).

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Figure 1. NPY, Galanin, and POMC mRNA Expression in C57BL/6J,
Obese C57BL/6J-Ay, and Obese
C57BL/6J-Lepob Mice
Representative autoradiograms of coronal brain sections through the DMH
are shown after hybridizations with prepro-NPY (upper),
galanin (middle), and POMC (lower)
antisense probes. NPY expression in the control C57BL/6J animal
(left) was limited to the ARC and the cerebral cortex
(CTX). Hypothalamic NPY expression is elevated in the ARC of the
C57BL/6J-Lepob animal (right), whereas the
C57BL/6J-Ay animal (middle)
has normal ARC expression but elevated expression in the DMH.
Hybridization signals for galanin and POMC mRNA indicate similar
expression patterns in each mouse strain, with galanin in both the ARC
and DMH, whereas POMC is restricted to the ARC.
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Elevated DMH NPY Gene Expression Is Associated with the Obese State
in Lethal Yellow (Ay)
Mice
To determine whether expression of NPY in the DMH is constitutive
or coincident with the development of obesity in the
Ay mouse, developmental expression of NPY was
examined. Figure 2
displays representative dark-field
photomicrographs of coronal hypothalamic sections (mid-DMH level) of
young nonobese (3 wk, 15 g), adult nonobese (12 wk, 28 g),
and adult obese (6 mo, 55 g) Ay mice
(panels A, B, and C, respectively) after in situ
hybridizations with a NPY probe. Note that in the adult nonobese
Ay animal (12 wk, 28 g), NPY expression is
restricted primarily to the ARC, whereas the obese (55 g)
Ay animal expresses NPY in both the ARC and the
DMH. In contrast, ARC neurons in the obese ob/ob (58 g)
animal (Fig. 2D
) have a readily discernible greater density of silver
grains per cell as well as a greater number of cells expressing NPY
when compared with the Ay animals (Fig. 2
, A-C).
However, virtually no cells in the DMH express NPY in the
ob/ob animal, but numerous cells in this brain region
express high levels of NPY mRNA in the obese Ay
mouse.

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Figure 2. Elevated DMH NPY Gene Expression Is Uniquely
Associated with the Obese State in
C57BL/6J-Ay Mice
Shown are dark-field photomicrographs of coronal hypothalamic sections
(mid- DMH level) of young nonobese (3 wk, 15 g), adult nonobese
(12 wk, 28 g), and obese (6 mo, 55 g)
C57BL/6J-Ay mice (panels A, B, and C,
respectively) in addition to an obese (58 g)
C57BL/6J-Lepob animal (panel D) after
in situ hybridizations with a prepro-NPY probe.
Arrowheads indicate DMH neurons highly expressing NPY
mRNA in the obese C57BL/6J-Ay animal (panel
C).
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NPY expression in the ARC and DMH was evaluated semiquantitatively in
C57, Ay, and ob/ob mice.
Hybridization signals over the ARC and DMH in these animals indicate
that although there is no measurable difference in NPY expression in
the ARC of Ay animals, NPY mRNA levels are
elevated more than 50% in ob/ob mice, when compared with
C57 control animals (Fig. 3
, left). In the
DMH, only obese Ay animals display elevated NPY
mRNA expression levels, when compared with that seen in C57 controls,
ob/ob, or nonobese Ay mice (Fig. 3
, right).

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Figure 3. NPY mRNA Levels in the ARC (left)
and DMH (right) of C57BL/6J,
C57BL/6J-Lepob,
C57BL/6J-Ay Nonobese, and
C57BL/6J-Ay Obese Mice
In the ARC, NPY mRNA levels of leptin-deficient (C57BL/6J-Lepob) mice,
but not C57BL/6J-Ay animals, were
significantly elevated as compared with C57BL/6J control mice
(P < 0.03). In the DMH, measurable NPY mRNA levels
above background were found only in obese
C57BL/6J-Ay mice, which is highly
significant when compared with C57BL/6J control mice
(P < 0.0001). Absorbancies (relative NPY signal
intensity) of the autoradiographic images on Cronex x-ray film were
measured as described (see Materials and Methods) and
analyzed using GraphPad Prism software.
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MC4 Receptor-Deficient Mice Display Elevated NPY Gene Expression in
the DMH
As seen in the autoradiogram in Fig. 4
, in
situ hybridizations of coronal brain sections from MC4-R-deficient
mice using a NPY antisense probe demonstrated that elimination of this
melanocortin receptor by targeted disruption of the MC4-R gene (12)
leads to increased expression of NPY in the DMH. Female homozygous
(-/-) MC4-R deficient mice (bottom), display intense
hybridization of the NPY probe over the DMH, whereas heterozygous (±)
mice (middle) with one functional MC4-R allele, and wild
type (+/+) control mice (upper), display labeling for NPY
over the ARC only. Similar results were seen in comparable male animals
(data not shown).

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Figure 4. Obese Melanocortin-4 Receptor (MC4-R) Deficient
Mice Express NPY in the DMH
Representative autoradiograms of coronal brain sections through the DMH
are shown after in situ hybridization with prepro-NPY
for wild type (+/+), heterozygous MC4-R deficient (±), and homozygous
MC4-R deficient (-/-) mice in panels A, B, and C, respectively.
Hypothalamic expression of NPY is restricted to the ARC in the nonobese
wild type and heterozygous MC4-R-deficient mice.
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DISCUSSION
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The results obtained in these experiments demonstrated no major
changes in hypothalamic gene expression of galanin or POMC in the three
genetic models of murine obesity examined (Ay,
MC4-R knockout, and ob/ob) when compared with controls. In
contrast to the similar expression patterns seen for galanin and POMC,
the distribution of hypothalamic NPY mRNA expression in mice with
melanocortinergic obesity syndrome was dramatically different from
control and leptin-deficient mice. Previous experiments have
demonstrated that ARC NPY levels are elevated in several models of
rodent obesity. In rats, for instance, the genetically obese Zucker
fatty strain has elevated ARC NPY mRNA levels when compared with lean
littermates (16), and, similarly, streptozocin-induced diabetic rats
display increased ARC NPY gene expression (17). In mice, hypothalamic
lesions induced by goldthioglucose treatment result in nearly a 50%
increase in ARC NPY expression (18), whereas in the genetically obese
ob/ob mouse, Northern analysis demonstrated hypothalamic NPY
mRNA to be elevated more than 3-fold (15). In contrast, NPY mRNA levels
in the ARC of both Ay and MC4-R deficient mice
were normal when compared with wild type mice. Surprisingly, however,
obese Ay mice as well as MC4-R-deficient mice
displayed a large induction of NPY gene expression in another
hypothalamic nucleus, the DMH.
In the present experiments, both male and female
Ay mice weighing more than 55 g exhibited
elevated NPY mRNA expression in DMH neurons; younger
Ay mice weighing less than 30 g displayed
normal hypothalamic NPY expression in ARC neurons and no expression in
the DMH (Fig. 2
). Furthermore, nonobese mice that have a single
functional MC4-R allele (heterozygous MC4-R knockout) do not express
NPY aberrantly. The expression of NPY in the DMH is not associated with
all models of obesity, however, because obese ob/ob mice did
not display any detectable NPY expression in the DMH. Therefore,
activation of NPY expression in DMH neurons is correlated specifically
with the melanocortinergic obesity syndromes that derive from a loss of
MC4-R activity.
Expression of NPY mRNA in the DMH has not been reported previously in
other mouse or rat models of obesity; however, there are some reports
of altered NPY peptide levels in this nucleus associated with feeding
status (19, 20, 21). In these experiments, fluctuations in NPY peptide
levels in the DMH are probably reflecting the activation of NPY-ergic
neurons that originate in the ARC and project to the DMH (22).
Interestingly, the only reported activation of NPY-ergic neurons
originating in the DMH is in lactating rats that also display a
characteristic elevation of NPY mRNA in the DMH nucleus (23) in
response to suckling, which may reflect an activation of a feeding
pathway in response to an increased nutritional need.
A simplified model proposing a role for NPY in both the
ob/ob and melanocortinergic obesity models is shown
schematically in Fig. 5
. The disruption of leptin
signaling in the ob/ob or db/db mouse results in
an elevation of NPY gene expression in the ARC, perhaps directly via
leptin receptors in the ARC. This enhanced release of NPY is
responsible for a significant portion of the phenotype in the
ob/ob animal, as supported by recent observations on
attenuation of the obesity syndrome by deletion of the NPY gene from
the ob/ob mouse (24).

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Figure 5. Model of Neuroendocrine Circuitry in the
Melanocortinergic Obesity Syndrome
Normal POMC signaling from ARC neurons to the DMH via -MSH is
disrupted by either 1) ectopic expression of ASP, a competitive
antagonist of -MSH, or 2) genetic deletion of the melanocortin-4
receptor (MC4-R). Loss of MC4-R activity results in the aberrant
expression of NPY in DMH neurons that project to the PVN. Excess NPY in
the PVN may then mediate component(s) of the agouti obesity syndrome.
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It is further proposed that des-acetyl-
-MSH, released at
MC4-R-containing synapses in the DMH, is normally inhibitory of NPY
gene expression in this nucleus. However, when MC4-R activity is
suppressed or absent, due to either chronic antagonism by ectopically
expressed agouti (4) or genetic deletion of the MC4-R (12),
NPY synthesis eventually becomes elevated in DMH neurons. In the
melanocortinergic obesity syndrome, the presumed enhanced release of
NPY by DMH neurons projecting to the paraventricular hypothalamic
nucleus (PVN) then provides a stimulatory input to feeding behavior.
The absence of NPY gene expression in the DMH in young
AY animals indicates that abrogation of
melanocortinergic signaling alone is not sufficient for the induction
of this change. The identification of this discrete gene expression
change in the Ay and MC4-RKO obesity models also
provides further support for the idea that ectopic expression of ASP
and deletion of the MC4-R cause an obesity syndrome via the same
central mechanism. Although this model proposes the most simplistic
view by which NPY gene expression is altered, it is of course possible
that many intermediate steps remain to be discovered.
Induction of NPY in the DMH is clearly an informative marker of the
melanocortinergic obesity syndromes and identifies the DMH as a
possible downstream target of POMC neurons in their effects on feeding
and metabolism. Although NPY gene induction does not appear to precede
obesity in the Ay model, it may be an
etiological factor in melanocortinergic obesity models. Support for
this comes from several lines of research. First, although POMC neurons
within the ARC project throughout the brain, immunostaining with an
antibody directed to
-MSH shows that a major projection of arcuate
POMC neurons is to the DMH. Second, in situ hybridization
studies have demonstrated that one of the major sites of MC4-R
expression is in the anterior part of the DMH (10). Moreover, recent
experiments by Thompson et al. (25) indicate that DMH
neurons project primarily within the hypothalamus, with direct
innervation of the PVN, known to express NPY5 receptors (26). However,
whether the DMH neurons that project to the PVN and release NPY also
express MC4 receptors is unknown.
While deletion of the NPY gene does not produce an overt feeding
behavior disorder (27), genetic crossing of NPY deficiency into
leptin-deficient mice attenuates the hyperphagia of ob/ob
mice approximately 50%, demonstrating a role for elevated NPY in the
pathophysiology of obesity resulting from the absence of leptin (24).
We propose that a component of the hyperphagia, altered metabolism,
increased linear growth, or reduced fertility seen in melanocortinergic
obesity syndromes may be due to altered function of the DMH, and
perhaps specifically due to elevated release of DMH-derived NPY at the
PVN. Ultimately, deletion of the NPY gene from the
AY mouse may be used to determine the role of
elevated NPY expression in the melanocortinergic obesity syndrome.
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MATERIALS AND METHODS
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Animals
Control C57Bl/6J mice, C57Bl/6J-Ay
heterozygous mice, and C57Bl/6J-Lepob mice were
maintained under a 12-h light, 12-h dark cycle at constant temperature,
and provided food (Purina mouse chow) and water ad libitum.
C57Bl/6J-Ay mice in groups of three animals each
consisting of young nonobese (3 wk, 1517 g), adult nonobese (12 wk,
2528 g), and adult obese (6 mo, 5562 g) mice were compared with
aged matched control C57Bl/6J mice (1545 g, one to three animals
each), and three obese C57Bl/6J-Lepob mice (6
mo, 5860 g). Anesthetized (avertin) animals were killed between 1500
and 1700 before lights out via cardiac puncture and perfused with
saline (20 ml) and then 50 ml of ice-cold fixation buffer (4%
paraformaldehyde in borate buffer, pH 9.5). Whole brains were rapidly
removed and then postfixed overnight in 10% sucrose/fixative buffer.
Blocked hypothalamic sections were frozen in powdered dry ice and then
stored at -80 C until sectioned. A detailed description of the MC4-R
gene deletion mouse is described by Huszar et al. (12). For
the MC4-R deletion studies, littermates consisting of a male and a
female mouse from each genotype, homozygous (-/-) MC4-R-deficient
(5560 g), heterozygous (±), and homozygous (+/+) wild type (2530
g) were treated as above.
In Situ Hybridization
Probe Synthesis.
Antisense NPY was prepared by linearizing the plasmid pBLNPY-1, which
contains 511 bp of the rat NPY gene (kindly provided by Dr. Susan
Smith) with FspI. Antisense POMC was prepared by linearizing
the plasmid mPOMCE3ribo with NcoI, which contains exon 3 of
the mouse POMC gene (kindly provided by Dr. Malcolm Low). Antisense
galanin was prepared from a 680-bp rat galanin cDNA plasmid provided by
Dr. Robert Steiner, after HindIII linearization.
[35S]cRNA probes were prepared by transcribing 1 µg of
each linearized DNA with T3 polymerase (NPY), or T7 polymerase (POMC
and galanin) for 1 h at 37 C in a reaction containing
[35S]UTP (NEN, Boston, MA) using a commercially available
in vitro transcription kit (Promega, Madison, WI).
Hypothalamic brain blocks were mounted on a frozen stage and serially
sectioned into four series of 20-µm slices with a sliding microtome.
Sections were prepared and processed for in situ
hybridization as previously described (18). Sections were hybridized
for 20 h at 58 C with 35S-labeled probes (5 x
106 cpm/ml in 65% formamide, 0.26 M NaCl,
1.3x Denhardts solution, 1.3 mM EDTA, 13% dextran
sulfate, 13 mM Tris, pH 8). Sections were then digested
with RNase (20 µg/ml) for 30 min at 37 C and then desalted in a
series of washes from 4x SSC (0.15 M NaCl, 0.1
M
Na3C6H5O7·2
H2O) 1 mM dithiothreitol to a final stringency
of 0.1x SSC/1 mM dithiothreitol at 65 C for 30 min.
Sections were dehydrated in ascending ethanol, vacuum dried at room
temperature for 30 min, and then exposed to Dupont Cronex film (Dupont,
Wilmington, DE) for several days. Dried slides were then dipped in
NTB-2 emulsion (Kodak, Rochester, NY) and developed after 6 days.
Quantification of Autoradiograms.
The absorbancies of the autoradiographic images on Cronex x-ray film
were measured by using a Macintosh-based image analysis system and NIH
Image software obtained from NIH. The film was illuminated with a
ChromaPro 45 light source, which provided even illumination, and the
image was captured with a Dage MTI 70 series video camera equipped with
a Newvicon tube and interfaced with a Scion image capture board. The
mean absorbancies of the autoradiographic images on Cronex x-ray film
over the ARC or DMH (12 h exposure) at the same level from each brain
were measured. The mean absorbance over a large irregularly-shaped
region adjacent to the ARC or DMH, that did not contain specific
hybridization, was also measured on each section and used to calculate
mean background density, which was subtracted from the absorbance
measurement of signals over the ARC or DMH. Commercially available
14[C] autoradiographic standards were exposed to each
x-ray film along with experimental material. The mean absorbance of an
interactively defined region over each standard was measured; these
measurements confirmed the linearity of the films responsiveness, as
well as the consistency of signal detection across films. The mean
absorbancies of the auroradiographic images recorded over the ARC or
DMH all fell within the linear range of the standard values. A two-way
ANOVA test was used to test for significant differences in levels of
POMC, galanin, or NPY mRNA hybridization among the treatment groups in
each experiment. A P value of less than 0.05 was defined as
significant.
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ACKNOWLEDGMENTS
|
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The authors would like to thank Malcolm Low and Guibao Gu for
helpful discussions; Meigan Crabtree, Michele Zee, and Denise OLeary
for technical assistance; and June Shiigi for graphic support.
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FOOTNOTES
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Address requests for reprints to: Roger D. Cone, Vollum Institute for Advanced Biomedical Research, Oregon Health Sciences University, 3181 Southwest Sam Jackson Park Road, Portland, Oregon 97201-3098.
This work is supported by NIH Grants [NIDDK and NICHD to R.D.C., NIDDK
to R.A.K., and NICHD to R.B.S.], and by Millenium Pharmaceuticals,
Inc., (to D.H. and C.A.L.).
Received for publication December 11, 1996.
Revision received January 31, 1997.
Accepted for publication February 4, 1997.
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