Developmental Regulation of
Somatostatin Gene Expression in the
Brain is Region Specific
William L. Lowe, Jr., Anne E. Schaffner, Charles T. Roberts, Jr.,
and Derek LeRoith
Diabetes Branch
National Institute of Diabetes, Digestive and Kidney Diseases
Laboratory of Neurophysiology (A.E.S.)
National Institute of Neurological and Communicative Disorders and
Stroke
National Institutes of Health
Bethesda, Maryland 20892
Developmental regulation of somatostatin (SRIF)
gene expression was studied in five regions of rat
brain and in rat stomach. Total RNA was isolated
from hypothalamus, cortex, brainstem, cerebellum,
and olfactory bulb, as well as stomach at eight
stages of development from prenatal day 16 to postnatal day 82. Hybridization of a 32P-labeled rat SRIF
cDNA probe to Northern blots of total RNA from the
above tissues during development demonstrated a
single hybridizing band approximately 670 base
pairs in length. When SRIF mRNA levels from each
stage of development were quantified and normalized by the amount of poly (A) + RNA present at that
stage of development, a unique pattern of SRIF gene
expression was seen in each region. In brainstem
and cerebellum, SRIF mRNA levels peaked early in
development between prenatal day 21 and postnatal
day 8 and then declined until postnatal day 82.
Hypothalamus and cortex, on the other hand,
showed a progressive increase during development
with peak levels occurring between postnatal days
13 and 82. In contrast, stomach and olfactory bulb
showed SRIF mRNA levels which were low during
early development and which rose late in development (postnatal days 13 to 82). Marked differences
in the amount of SRIF mRNA within each region were
present as well. These data suggest that there is
differential expression of the SRIF gene in different
regions of the brain and in the stomach during development. Further study of this phenomenon may
provide insight into the in vivo control of SRIF gene
expression and the role of SRIF in the developing
brain. (Molecular Endocrinology 1: 181-187, 1987)
in the brain and peripheral nervous system (1, 2). Hypothalamic SRIF functions as an inhibitor of the secretion of pituitary GH and TSH (1). In other regions of the
brain, SRIF presumably functions as a neurotransmitter
(1, 2), although its exact role remains undefined. SRIF
is also produced by the endocrine D cells in the stomach
and pancreas where it inhibits the secretion of multiple
hormones such as insulin, glucagon, secretin, gastrin,
and cholecystokinin (1, 3).
SRIF is translated as a 116 amino acid preprohormone which is processed primarily into three forms,
SRIF-14, SRIF-28, and SRIF-28 [1-12] (4-7). SRIF-14
and SRIF-28 contain the carboxyterminal 14 and 28
amino acids of the preprohormone respectively (4-6).
SRIF-28 is an N-terminal extension of SRIF-14 (4-6),
whereas SRIF-28 [1-12] contains the N-terminal 12
amino acids of SRIF-28 (7). The tissue distributions of
SRIF-14 and SRIF-28 are different, but the functional
differences between them and the function of SRIF-28
[1-12] remain undefined (8). In addition to these three
peptides, other peptides resulting from the processing
of the preprohormone have been isolated, although
their physiological significance is unknown (9,10).
Determination of both the cDNA (4-6) and genomic
(11, 12) sequence of the rat SRIF (rSRIF) gene has
facilitated studies of the regulation of this gene. One
study has delineated regions of the 5'-flanking sequence of the SRIF gene which are involved in regulation of SRIF gene expression, while another has demonstrated the control of SRIF gene expression by cAMP
(13-15). A study of SRIF mRNA levels demonstrated
different developmental patterns of SRIF gene expression in whole brain and stomach of the rat (16), with
SRIF mRNA levels in whole brain reaching adult levels
at prenatal day 21 and remaining elevated at that level
throughout development. Previous immunocytochemical and RIA studies of the developing rat brain, however, have suggested that immunoreactive SRIF levels
change in different brain regions during development
(17-20).
In order to investigate this difference between the
SRIF mRNA levels and the SRIF peptide levels noted
above, as well as to elucidate further the role of SRIF
INTRODUCTION
Somatostatin (SRIF) is a polypeptide hormone which is
produced by multiple cell types including neuronal cells
0888-8809/87/0181 -0187$02.00/0
Molecular Endocrinology
Copyright © 1987 by The Endocrine Society
181
Vol. 1 No. 2
MOL ENDO-1987
182
in the brain and the control of SRIF gene expression,
we have studied the developmental expression of the
SRIF gene in various regions of the rat brain and in the
rat stomach which served as an example of SRIF gene
expression in a peripheral tissue.
base pairs (bp) in length at all stages of development
studied (Fig. 1 and data not shown). The size of the
SRIF mRNA was similar to that described by others
(16). Although quantitative differences in SRIF mRNA
levels were apparent, a hybridizing band was seen in
all tissues at all stages of development with the exception of the stomach, where SRIF mRNA was not seen
until postnatal day 3 (Fig. 1 and data not shown).
At postnatal day 22, SRIF mRNA is present in all
brain regions (Table 1). To further characterize SRIF
mRNA, poly (A)+ RNA was purified from total RNA
obtained from the whole brain of postnatal day 22 rats.
Hybridization of labeled rSRIF cDNA to a Northern blot
of poly (A)+ RNA and of the oligo d(T) cellulose column
flow through (poly (A)~ RNA) demonstrated a hybridizing band in the poly (A)+ lane only (Fig. 2). Similar results
were seen using poly (A)+ and poly (A)" RNA prepared
from prenatal day 21 brain (data not shown). This
RESULTS
Characterization of SRIF mRNA
RNA was isolated from multiple regions of the brain
and from the stomach during several stages of development between prenatal day 16 and postnatal day 82.
Hybridization of a labeled rSRIF cDNA probe to Northern blots of total RNA from olfactory bulb, cerebellum,
cortex, brainstem, hypothalamus, and stomach demonstrated a single hybridizing band approximately 670
Cortex
28S-
•
18S-
Brainstem
28S18S-
E16
E21
P1
P3
P8
P13
P22
P82
Fig. 1. Autoradiogram of Northern Blots of rSRIF mRNA from Different Brain Regions during Development
Total RNA was prepared from the indicated brain regions as described in Materials and Methods by pooling the brain parts from
eight to 12 male and female rats. Fifteen micrograms of total RNA from cortex (top) and brainstem (bottom) during several stages
of development were size separated by agarose gel electrophoresis, transferred to a nylon membrane, and hybridized to a 32Plabeled SRIF cDNA probe as described in Materials and Methods. The stages of development from prenatal day 16 (E16) to
postnatal day 82 (P82) are indicated at the bottom of each lane. The 28S and 18S markers correspond to the position of the
ribosomal RNA bands on the ethidium bromide stain of the gel.
SRIF Gene Expression in the Developing Brain
183
Table 1. SRIF mRNA Levels in Brain Regions and in Stomach during Development
Hypothalamus
Cortex
Cerebellum
Brainstem
Olfactory Bulb
Stomach
E16
E21
0.33 ± 0.03
0.37 ± 0.03
1.00 ±0.21
0.98 ± 0.26
0.39 ± 0.07
0.54 ± 0.08
1.10±0.17
2.50 ±1.37
0.06 ± 0.05
P1
P3
0.49 ± 0.02
1.50 ±0.47
0.94 ± 0.33
2.23 ± 0.59
0.04 ± 0.01
0.03 ±0.05 0.12 ±0.03
0.34 ± 0.04
0.95 ± 0.36
1.03 ±0.16
1.80 ±0.52
P8
P13
0.72 ±0.13
1.70 ±0.52
0.77 ±0.18
2.51 ± 0.76
0.04 ± 0.01
0.26 ± 0.09
0.99 ± 0.09
2.02 ± 0.71
0.69 ±0.11
1.91 ±0.64
0.09 ± 0.02
0.67 ± 0.22
P22
P82
0.86 ±0.15
1.00
2.11 ±0.47 2.49 ± 0.82
0.21 ± 0.05 0.04 ± 0.06
1.28 ± 0.47 0.60 ±0.12
0.11 ±0.01 0.30 ±0.12
2.77 ± 0.85 14.78 ± 7.35
SRIF mRNA levels were quantified from the autoradiograms of the slot blots in Fig. 3A and expressed in arbitrary densitometric
units. SRIF RNA levels were then normalized by using the amount of poly (A)+ RNA present in that sample as determined from the
autoradiogram of the hybridization with oligo d(T) in Fig. 3A. Each point represents the mean of three determinations ± SEM. For
all determinations the level of SRIF RNA in the hypothalamus at P82 was arbitrarily defined as 1.00 and the other values were
normalized accordingly. The stages of development for each region are represented by the day of development and the period of
development (E, embryonic day; P, postnatal day).
A
B
28S-
18S-
Fig. 2. Autoradiogram of a Northern Blot of rSRIF mRNA in
Poly (A)+ and Poly (A)" RNA from Rat Brain
Poly (A)+ and poly (A)~ RNA [i.e. oligo d(T) cellulose column
flow-through] were prepared from total RNA from the whole
brain of a postnatal day 22 rat. Fifteen micrograms of poly (A)"
(lane A) and 5.6 ng poly (A)+ RNA (lane B) were size separated
by agarose gel electrophoresis, transferred to a nylon membrane, and hybridized to a 32P-labeled probe as described in
Materials and Methods. The 28S and 18S markers correspond
to the position of the ribosomal RNA bands on the ethidium
bromide stain of the gel.
suggests that at postnatal day 22 and prenatal day 21,
and presumably, at all stages of development, SRIF
RNA is polyadenylated.
present was determined by hybridizing the slot blots
with a molar excess of poly d(T) (Fig. 3A). The resulting
autoradiograms were quantified by laser densitometry.
SRIF mRNA levels were normalized by using the total
poly (A)+ RNA levels, and when they were plotted as a
function of age, a unique pattern of SRIF gene expression was seen in each of the regions studied (Fig. 3B).
Similar patterns were seen when the SRIF mRNA levels
were normalized to the total amount of RNA (as determined by UV absorption) present on the blot (data not
shown). Interestingly, with the exception of the stomach
and olfactory bulb, significant levels of SRIF mRNA
were present in all regions during the early postnatal
period from day 3 to day 13 with a decline in levels in
the cerebellum and brainstem and an increase in levels
in the hypothalamus and cortex later in development at
postnatal days 22 and 82. In the stomach and olfactory
bulb, levels were low early in development with a
marked increase later in development.
In addition to the qualitative differences in the pattern
of SRIF gene expression in the various regions during
development, marked quantitative differences in SRIF
mRNA levels between regions were present as well
(Table 1). Early in development the highest levels of
SRIF mRNA were present in the brainstem and cerebellum. Later in development significant levels remained
in the brainstem, but levels were now high in the cortex,
hypothalamus, and stomach as well. Levels were low
in the olfactory bulb throughout development.
DISCUSSION
SRIF mRNA Quantification during Development
To further evaluate the apparent quantitative differences in SRIF mRNA levels during development, slot
blots of total RNA were prepared and hybridized with
the labeled rSRIF cDNA probe (Fig. 3A). SRIF mRNA
levels were determined by scanning the resulting autoradiogram with a laser densitometer and were expressed as arbitrary densitometric units (ADU). In order
to express the SRIF mRNA levels as a percentage of
the total poly (A)+ RNA present in each region at each
stage of development, the amount of poly (A)+ RNA
The developmental expression of the rSRIF gene in
several brain regions and in the stomach was studied
by determining SRIF mRNA levels. Consistent with
previous findings (16), a single species of rSRIF mRNA
approximately 670 bp in length was found in the brain
and stomach. A previous study of the developmental
pattern of SRIF mRNA accumulation in whole brain (16)
demonstrated an increase in SRIF mRNA levels to adult
levels at prenatal day 21 with the levels then remaining
relatively constant through adulthood. In the present
study, however, in which the developmental pattern of
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SRIF Gene Expression in the Developing Brain
SRIF gene expression was studied in individual brain
regions, a unique pattern of SRIF gene expression was
seen in each of the regions studied. The pattern of
SRIF accumulation found in the stomach in this study
was consistent with the previously reported findings
(16).
These quantitative differences in SRIF mRNA levels
may reflect quantitative differences in SRIF peptide
levels as well. Previous studies have provided RIA and
immunocytochemical findings which are consistent with
that idea (17-20). McGregor et al. (20) measured SRIF
levels in various brain regions during development by
RIA. They found significant levels of the peptide in the
cortex, cerebellum, brainstem, and hypothalamus during development, and the changes in the SRIF peptide
levels in each region during development were similar
to the developmental pattern of SRIF mRNA noted in
this study. Immunocytochemical studies (17-19) of
SRIF peptide levels in the lower brainstem, forebrain,
diencephalon, and cerebellum during rat development
also demonstrate patterns of SRIF accumulation similar
to those found in this study. Despite the general similarities between the developmental patterns of SRIF
mRNA accumulation and the appearance of SRIF peptide in the various brain regions, identical patterns were
not seen. For example, SRIF peptide levels in the
cortex, as measured by RIA, peaked at postnatal day
14 and decreased modestly at postnatal day 21 and in
adults (20), whereas in lower brainstem, SRIF levels as
measured by immunohistochemistry peaked on prenatal days 20 to 22 and decreased progressively through
adulthood (17). When these results are compared to
our findings illustrated in Fig. 3, the differences between
the mRNA and peptide levels are apparent. There exist
several possible explanations for these differences.
Measurement of SRIF levels by RIA will not distinguish
between SRIF produced locally and SRIF transported
to the region being assayed. Secondly, the SRIF mRNA
present in some regions may not be translated, which
would explain the discrepancy between mRNA and
peptide levels. Finally, the SRIF prohormone may be
processed differently in some brain regions during different periods of development resulting in the formation
of peptides which would not be measured by antibodies
directed against SRIF-14 and SRIF-28. Such alternative
185
peptides have been found in the brain (9), but their
prevalence, importance, and physiological significance
remain unknown.
The demonstration of SRIF mRNA in multiple brain
regions with varying patterns of expression in the different regions suggests the possibility of heterogeneous functions of SRIF in the brain. Interestingly, the
presence of SRIF mRNA in the brainstem and cerebellum before the development of synaptic transmission
(17, 19) and the presence of significant amounts of
SRIF mRNA in multiple brain regions during the early
postnatal period when rapid brain growth is occurring
in the rat (21) suggest that SRIF may play a role in brain
development.
Despite the identification of regions of the 5'-flanking
sequence of the rSRIF gene which are apparently involved in positive and negative regulation of SRIF gene
expression (13), and the demonstration of in vitro regulation of SRIF gene expression by cAMP (14, 15), the
factors which regulate SRIF gene expression in vivo
are still undefined. The marked differences in SRIF gene
expression during development in different brain regions may provide a model for further study of SRIF
gene expression in vivo.
In summary, SRIF mRNA is present in multiple brain
regions and the stomach during several stages of development. Marked quantitative differences of SRIF
mRNA levels are present during development in these
various regions. Further study of this phenomenon may
provide insights into the function of SRIF in the brain
as well as the regulation of SRIF gene expression.
MATERIALS AND METHODS
Preparation of Tissues
Pregnant, newborn, and early postnatal Sprague-Dawley male
and female rats obtained from Zivic-Miller Laboratories (Allison
Park, PA) and adult Sprague-Dawley male and female rats
obtained from Taconic Farms (Germantown, NY) were housed
under constant light and dark cycles and were provided free
access to laboratory chow and water. Fetuses were removed
from ether-anesthesized pregnant rats. All animals were killed
by decapitation. After immediate removal of whole brains,
individual brain parts were removed under a dissecting microscope and immediately frozen in liquid nitrogen. Stomachs
Fig. 3. A, Autoradiogram of Slot Blots of Total RNA Isolated from Different Brain Regions and the Stomach during Development
and Hybridized with rSRIF cDNA or Oligo d(T)18
RNA was prepared from eight to 12 male and female rats as described in the legend to Fig. 1. Total RNA (5 ng, left lane; 6 ^g.
center lane; 3 /*g> right lane) from different brain regions and from the stomach at the stage of development indicated (E, embryonic
day; P, postnatal day) was fixed to a nylon membrane as described in Materials and Methods. The left lane and center lane were
hybridized with a 32P-labeled rSRIF cDNA probe while the right lane was hybridized with a 32P-labeled poly d(T)18 probe to estimate
poly (A)+ RNA content. The autoradiograms were quantified by laser densitometry. RNA levels were expressed as arbitrary
densitometric units. B, Plots of SRIF mRNA levels in different brain regions and the stomach as a function of age. SRIF mRNA
levels were quantified as described above. The SRIF mRNA level in each region at each stage of development (expressed in ADU)
was divided by the poly (A)+ RNA concentration (expressed in ADU) of the same sample in order to express the SRIF mRNA level
as a function of the poly (A)+ RNA present. The normalized SRIF mRNA levels were plotted as a function of age and as a
percentage of the maximum SRIF mRNA level present in each region. Each point represents the mean ± SEM of three determinations
(i.e. three blots) of SRIF mRNA levels from a single total RNA preparation. As the left-hand two columns of slot blots in A show,
the patterns in different experiments seem similar.
MOL ENDO-1987
186
were removed, cleaned of their contents, and frozen in liquid
nitrogen. Tissue samples were stored at - 8 0 C.
RNA Isolation
RNA was prepared using a modification (Graham, D., personal
communication) of the method of Cathala et al. (22). The
individual brain parts or stomachs from eight to 12 male and
female rats were pooled, using all the animals in a litter for all
stages of development through P22, and five males and five
females for the studies on adult animals. The tissues were
thawed and homogenized with a Polytron homogenizer (Brinkmann Instruments, Westbury, NY) in 7 volumes (wt/vol) of 5
M guanidine thiocyanate (Fluka AG, Buchs, Switzerland), 10
mM EDTA, 50 mM Tris, pH 7.5, and 8% (vol/vol) |8-mercaptoethanol followed by extraction with an equal volume of 1:24
(vol/vol) isoamyl alcohol-chloroform (IAC). RNAs were precipitated by the addition of 5 volumes of 4 M lithium chloride (LiCI)
followed by incubation for 15-20 h at - 2 0 C. The precipitate
was pelleted by centrifugation and was washed by resuspension in 2 volumes of 3 M LiCI with the Polytron homogenizer.
After recentrifugation, the resulting pellet was resuspended
with the polytron homogenizer in 1.2 volumes of RNA suspension buffer [10 mM Tris, pH 7.5, 0.3% Triton X-100, 10 mM
ribonucleoside-vanadyl complex (New England Biolabs, Beverly, MA)] and was extracted twice with an equal volume of
isoamyl alcohol-chloroform. After the second extraction, EDTA
was added to a final concentration of 100 mM, and the solution
was incubated for 15 min at 22 C. One-tenth volume of 6 M
ammonium acetate and 2 volumes of ethanol were added and
the RNA was precipitated for 18 h at - 2 0 C. The precipitate
was collected by centrifugation and was washed three times
in 70% ethanol. The RNA was resuspended in H2O and was
quantified by reading the absorbance at 260 nm.
Preparation of Poly (A) + RNA
Poly (A)+ RNA was prepared with oligo d(T) cellulose (Bethesda
Research Laboratories, Gaithersburg, MD) according to the
manufacturer's instructions which were a modification of previously described techniques (23). One milliliter of oligo d(T)
cellulose was washed with 30 ml elution buffer [10 mM Tris,
pH 7.6, 1 mM EDTA, 0.2% sodium dodecyl sulfate (SDS)j
followed by a wash with 30 ml binding buffer (0.5 M NaCI, 10
mM Tris, pH 7.6,1 mM EDTA, and 0.5% SDS). Total RNA was
diluted with H2O to a concentration of 1 mg/ml, heat-denatured
at 70 C for 1 min, and chilled on ice. The sample was diluted
with an equal volume of 2x binding buffer and passed over
the column six times. The column was washed with 35 ml
binding buffer, and the bound RNA was eluted with 4 ml
elution buffer. One-tenth volume of 5 M NaCI was added to
the eluate. The samples were ethanol precipitated, collected
by centrifugation, and then reprecipitated with ethanol. The
final pellet was resuspended in H2O, quantified as above, and
stored at - 8 0 C.
RNA Gel Electrophoresis and Blotting
These were performed using a modification of methods described previously (24). Briefly, for Northern blots RNA was
denatured and loaded onto a 1.5% agarose gel made up in
17.5% formaldehyde and 1x MOPS (3-[A/-morpholino]propanesulfonic acid). Electrophoresis was in 1 x MOPS and 0.05
mg/ml ethidium bromide as described (24). After electrophoresis the gels were washed in 1 M ammonium acetate for 30
min. RNAs were transferred to a nylon membrane (Gene
Screen, New England Nuclear, Boston, MA) using 1 M ammonium acetate as the transfer buffer. After transfer the
membrane was rinsed briefly and air dried. RNA was fixed to
the membrane by baking in vacuo at 80 C for 2 h. The
membranes were washed in 1 x SSPE and stored at - 2 0 C.
RNA slot blots were prepared by heating RNAs diluted in
Vol. 1 No. 2
1 x SSPE to 65 C for 15 min and chilling immediately on ice.
RNA samples were spotted onto Gene Screen using the
Minifold II Slot-Blotter (Schleicher and Schuell, Keene, NH).
RNA was fixed to the membrane by air drying followed by
baking in vacuo for 2 h at 80 C. Membranes were rinsed in
1 x SSPE and stored at - 2 0 C.
Hybridizations
Hybridizations were performed using a cloned rSRIF cDNA
(kindly provided by Dr. J. Dixon); this probe included the entire
coding region as well as short portions of the 5'- and 3'untranslated regions (6). The probe was labeled with «-[32P]
dCTP using a modification of the previously described technique of random priming (25). One hundred nanograms of
rSRIF cDNA was denatured by boiling for 2 min and was
chilled on dry ice. The denatured probe was suspended in 50
mM glycine, pH 9.2, 50 ^M each of dGTP, dATP, and dTTP,
7.5 mg/ml calf thymus DNA oligodeoxynucleotide primers, and
60 ^Ci «-[32P]dCTP. The reaction was initiated by the addition
of 5 U of the large fragment of DNA polymerase I (International
Biotechnologies, Inc., New Haven, CT) and incubated for 5 h
at 22 C. Unincorporated label was separated from labeled
probe by purification with Elutip-d Columns (Schleicher and
Schuell) according to the manufacturer's instructions. Specific
activities ranged from 3-5 x 108 cpm//tg DNA.
Hybridizations were performed using a modification of previously described methods (24). Northern blots and slot blots
were prehybridized for 2 h at 50 C in a heat sealed bag in a
solution containing 50% formamide-dextran solution (prepared
as described) (24), 1 % SDS, 5x SSPE, 0.1% Denhardt's
solution, and 10 Mg/ml denatured, sheared salmon sperm DNA.
Labeled probe was prehybridized in an identical solution for 2
h at 50 C with blank Gene Screen (previously baked for 2 h at
80 C) in order to decrease background on the final blot (D.
Graham, personal communication). After the 2 h prehybridization, the solution with labeled probe was recovered and
heated to 80 C for 10 min. The solution then was added to
the prehybridization buffer of the Northern or slot blots, and
the blots were incubated for 15 h at 50 C. The final concentration of probe was approximately 1.0 x 106 cpm/ml hybridization buffer. After hybridization the blots were washed twice in
2x SSPE with 0.2% SDS at 37 C for 30 min followed by a 15min and a 5-min wash in 0.1 x SSPE at 60 C. The blot was
exposed to Kodak X-Omat AR film with two intensifying
screens at - 7 0 C for 1 to 8 days. The autoradiograms of the
slot blots were quantified by densitometric scanning with an
LKB 2202 Ultrascan Laser Densitometer coupled to an Apple
HE computer.
Quantification of RNA samples was done by hybridization
of the slot blots with octadecathymidylic acid [poly d(T)18 (New
England Biolabs, Beverly, MA)]. Poly d(T)18 was end labeled
with 7-[32P]ATP using polynucleotide kinase (Boehringer
Mannheim Biochemicals, Indianapolis, IN) (23). Labeled poly
d(T)18 was diluted with unlabeled poly d(T)18 to a final specific
activity of approximately 4.0 x 10" cpm/^g DNA. The slot
blots were hybridized in the presence of an approximately 8fold molar excess of poly d(T)18 (relative to the estimated
amount of poly (A)+ RNA present on the blots) after prehybridization of the slot blots with 5x SSPE, 5% Denhardt's
solution, and 1 % SDS for 2 h at 30 C and prehybridization of
the probe with blank baked Gene Screen under the same
conditions. The blots were hybridized with the poly d(T)18 for
15 h at 30 C and were washed with 5x SSPE and 0.75% SDS
for 30 min at 22 C and with 5x SSPE for 30 min at 22 C. The
blots were exposed to film for 4 to 24 h, and the autoradiograms were quantified as described above.
Acknowledgments
We would like to thank Dr. Jack Dixon of Purdue University
(Lafayette, IN) for kindly providing us with the rat SRIF cDNA.
We would also like to thank Dr. Charles Bevins for critical
SRIF Gene Expression in the Developing Brain
reading of the manuscript as well as Dr. Dale Graham for her
helpful comments and advice and Ms. Violet Katz for her
secretarial assistance.
Received June 23,1986.
Address correspondence and requests for reprints to: William Lowe, Jr., Diabetes Branch, National Institute of Diabetes,
Digestive and Kidney Diseases, National Institutes of Health,
Building 10, Room 8S-243, 9000 Rockville Pike, Bethesda,
Maryland 20892.
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