 |
 Previous Article | Next Article 
The Journal of Neuroscience, March 15, 1998, 18(6):2118-2128
Insulin-Like Growth Factor-I Is a Differentiation Factor for
Postmitotic CNS Stem Cell-Derived Neuronal Precursors: Distinct Actions
from Those of Brain-Derived Neurotrophic Factor
Yvan
Arsenijevic and
Samuel
Weiss
Departments of Anatomy and Pharmacology and Therapeutics,
University of Calgary Faculty of Medicine, Calgary, Alberta, Canada
T2N4N1
 |
ABSTRACT |
Insulin-like growth factor-I (IGF-I) has been reported previously
to promote the proliferation, survival, and maturation of sympathetic
neuroblasts, the genesis of retinal neurons, and the survival of CNS
projection and motor neurons. Here we asked whether IGF-I could promote
the in vitro differentiation of postmitotic mammalian
CNS neuronal precursors derived from multipotent epidermal growth
factor (EGF)-responsive stem cells. In the absence of IGF-I, virtually
no neurons were present in cultured stem cell progeny, whereas IGF-I
increased neuron number by eight- to 40-fold. Brief exposures (2 hr) to
IGF-I were sufficient to allow for neuronal differentiation without
affecting proliferation or survival. IGF-I actions could be mimicked by
insulin and IGF-II at concentrations that correspond to the
pharmacology of the IGF-I receptor, the latter for which the mRNA was
detected in undifferentiated stem cell progeny. Although ineffectual
alone at low concentrations (10 nM) that would activate its
own receptor, insulin was able to potentiate the actions of IGF-I by
acting on mitotically active neural precursors. When neuronal precursor
differentiation by IGF-I was examined in relation to brain-derived
neurotrophic factor (BDNF), two important observations were made: (1)
BDNF could potentiate the differentiating actions of IGF-I plus
insulin, and (2) BDNF could act on a separate population of precursors
that did not require IGF-I plus insulin for differentiation. Taken
together, these results suggest that IGF-I and BDNF may act together or sequentially to promote neuronal precursor differentiation.
Key words:
neurogenesis; insulin; insulin-like growth factor-I; brain-derived neurotrophic factor; in vitro differentiation; GABAergic neurons
| |
INTRODUCTION |
Insulin-like growth factor-I
(IGF-I), insulin, and their respective receptors are present throughout
the CNS during embryogenesis, whereas at later stages of development
IGF-I and IGF-I receptor mRNAs are localized discretely to certain
neuronal populations (Petruzelli et al., 1986 ; Garofalo and Rosen,
1988; LeRoith et al., 1988; Bassas et al., 1989; Bondy et al., 1990,
1992; Bartlett et al., 1991; Bondy, 1991; Garcia-Segura et al., 1991;
Devaskar et al., 1993; Kar et al., 1993). The presence of IGF-I and its receptor during development suggests that this factor may play a role
in neurogenesis. This has been confirmed by reduced neuron numbers
in vivo when IGF-I was eliminated by knock-out or reduced by
antibody neutralization (Beck et al., 1995; Frade et al., 1996). Studies of the peripheral nervous system have shown that IGF-I is a
mitogen for sympathetic neuroblasts and a stimulator of sympathetic neurite outgrowth (Mill et al., 1985; DiCicco-Bloom and Black, 1988;
Caroni and Grandes, 1990) (for review, see Ishii, 1995; Zackenfels et
al., 1995). In studies of CNS cells in vitro, IGF-I has been
reported to increase neurogenesis from E5 proliferating chick retinal
neuroepithelial cells and to enhance survival of E10 rat
neuroepithelial cells and embryonic spinal cord motor neurons (Drago et
al., 1991; Hughes et al., 1993). In studies of postmitotic CNS
populations, IGF-I stimulated choline acetyltransferase activity and
increased enzyme immunoreactivity in septal and pontine neuronal
cultures and increased dopamine uptake in cultures of ventral
mesencephalon neurons (Knusel et al., 1990). In vivo, depending on the experimental condition used, IGF-I was found to rescue
29-94% of mouse and chick motor neurons after postnatal or embryonic
axotomy (Hughes et al., 1993; Neff et al., 1993; Li et al., 1994). In
summary, IGF-I appears to influence both early and late events in CNS
neurogenesis; however, it remains unclear whether IGF-I can influence
the differentiation of postmitotic neuronal precursors, e.g.,
acquisition of the neuronal phenotype independent of actions on
survival and maturation.
We have isolated a self-renewing stem cell from the embryonic and adult
mouse striatum that proliferates in response to epidermal growth factor
(EGF) and produces progenitor cells that can differentiate into
neurons, astrocytes, and oligodendrocytes (Reynolds and Weiss, 1992,
1996; Reynolds et al., 1992; Weiss et al., 1996). In vivo, the adult EGF-responsive cells participate in repopulation of the
subependyma and can be mobilized to generate new neurons in the
striatal parenchyma (Morshead et al., 1994; Craig et al., 1996).
Despite the extensive characterization of the in vitro properties of these stem cells, the steps by which new neurons are
produced are understood only partially. We have found that two types of
stem cell-derived neuronal precursors, a bipotent (neuron/astrocyte-producing) and an unipotent (neuron only), are stimulated to divide by basic fibroblast growth factor (bFGF; Vescovi
et al., 1993). Brain-derived neurotrophic factor (BDNF), on the other
hand, enhances the differentiation but not the survival of these
neuronal precursors (Ahmed et al., 1995). We suspect that other
specific factors also contribute to this process and have asked whether
IGF-I is one of these. Our findings suggest that IGF-I is indeed a
differentiation factor for stem cell-derived neuronal precursors, with
few or no actions on proliferation or survival. Moreover, our analyses
suggest that IGF-I actions are distinct from those of BDNF in regard to
the population/phenotype of precursors that they regulate.
| |
MATERIALS AND METHODS |
Primary culture and cell passaging. Striato-pallidum
complexes were removed from 14-d-old CD1 mouse embryos (Charles River, Wilmington, MA) in PBS buffer containing 0.6% of glucose plus 50 U/ml
penicillin and 50 mg/ml streptomycin, both from Life Technologies, Gaithersburg, MD). Tissue was dissociated mechanically with a fire-polished pipette in serum-free medium composed of a 1:1 mixture of
DMEM and F-12 nutrient (Life Technologies). Cells were grown in 40 ml
of growth medium in Corning T75 flasks at a concentration of 200,000 cells/ml. The growth medium contained DMEM and F-12 nutrient (1:1),
0.6% glucose, 2 mM glutamine, 3 mM sodium
bicarbonate, and 5 mM HEPES buffer [all from Sigma (St.
Louis, MO), except glutamine, which was obtained from Life
Technologies], 25 µg/ml insulin, 100 µg/ml transferrin, 20 nM progesterone, 60 µM putrescine, 30 nM selenium chloride, and 20 ng/ml EGF (Chiron, Emeryville, CA).
After 7 d in vitro (DIV), cells formed well developed
floating clusters (spheres). To passage spheres, we pelleted 7 DIV
spheres after centrifugation at 600 × g for 5 min,
resuspended them in fresh medium, and mechanically dissociated them
with a fire-polished Pasteur pipette. Single cells subsequently were
seeded into EGF-containing medium in a Corning T75 flask at a
concentration of 50,000 cells/ml. This procedure resulted in a second
generation of spheres.
Differentiation of EGF-generated stem cell progeny. After 7 DIV the second generation of spheres was centrifuged and dissociated in
EGF- and insulin-free medium. Cells were plated onto
poly-L-ornithine-coated coverslips in 24-well plates (2 cm2; Falcon, Oxnard, CA) at a density of 100,000 cells/ml, each well containing 1 ml of medium with 1% fetal bovine
serum (FBS). Human recombinant IGF-I, human recombinant IGF-II (both
from Chiron), insulin (Sigma), and human BDNF (PeproTech, Rocky Hill,
NJ) were added to the medium at the time of plating for an incubation
period of 7 DIV, except when indicated otherwise.
Antibodies. Primary antibodies for indirect
immunocytochemistry included a mouse monoclonal antibody to  -tubulin
isotype III (final concentration 1:1000; Sigma), rabbit antiserum to
glial fibrillary acidic protein (GFAP; 1:400; Biomedical Technologies, Stoughton, MA), and mouse monoclonal antibody to bromodeoxyuridine (1:5; Amersham, Arlington Heights, IL). The secondary antibodies (Jackson ImmunoResearch, West Grove, PA) used were as follows: rhodamine-conjugated affinity-purified goat antibody to mouse IgG
(1:200), fluorescein-conjugated affinity-purified goat antibody to
mouse IgG (1:100), and aminomethyl-coumarin (AMCA)-conjugated affinity-purified goat antibody to rabbit IgG (1:100).
Immunocytochemistry and cell counting. Indirect
immunochemistry using secondary antibodies conjugated to rhodamine,
fluorescein, or aminomethyl-coumarin (AMCA) was performed on cells
7 d after plating. Coverslips were fixed with 4% paraformaldehyde
for 20 min and washed three times successively with PBS (5 min). For double labeling of -tubulin and bromodeoxyuridine (BrdU), primary monoclonal antibody (anti--tubulin, 1:1000) was diluted in PBS containing 10% normal goat serum and 0.3% Triton X-100. Coverslips were incubated for 2 hr at 37°C and then were washed three times with
PBS for 5 min each. Rhodamine-conjugated secondary antibody was added
(1:200), and the coverslips were incubated for 30 min at 37°C. After
three 5 min PBS washes, coverslips were incubated with primary antibody
to BrdU (1:5) for 2 hr at 37°C. All primary antibodies used in this
study reached steady-state immunodetection after 2 hr at 37°C. The
coverslips were washed again three times and incubated for 30 min with
fluorescein-conjugated secondary antibody to mouse IgG (1:100).
Coverslips were washed twice with PBS, and then Hoechst nuclear stain
(1 mg/ml; Sigma) was added for 5 min at room temperature, followed by
two more 5 min PBS rinses. A rapid water wash preceded the mounting of
coverslips on glass slides with Fluorsave (Calbiochem, La Jolla, CA).
For double labeling of GFAP and -tubulin, the primary antibodies were incubated at the same time, with the rest of the procedure remaining the same. Fluorescence was detected and photographed with a
Nikon Optiphot photomicroscope.
Immunoreactive cells for -tubulin, GFAP, or fluorescent nuclei from
Hoechst labeling were counted over ten 20× fields. Homogeneous regions
for cell density representative of the coverslip were chosen.
Immunoreactive cells for -tubulin were counted only when the nucleus
was detectable on the 20× field investigated. After 7 DIV, the number
of -tubulin-immunoreactive neurons varied between culture
preparations  0-0.8% in control and 1.7-5.0% with 10 nM IGF-I. Thus in some of the experiments, to ensure
standardization with multiple comparisons, the number of neurons
present after 7 DIV in the presence of 10 nM IGF-I (15-50
-tubulin-immunoreactive neurons within 600-900 cells) serves as
100%, and the remaining data are presented as a percentage of 10 nM IGF-I. When n was <30, the nonparametric
Mann-Whitney U test was used at the two-tailed level to
distinguish the difference between experiments. Otherwise, unpaired
Student's t test was used. All results are expressed as a
mean ± SEM.
BrdU labeling and detection. To evaluate whether the
production of neurons or astrocytes was associated with proliferation, we added BrdU (1 µM; Sigma) at plating for 7 DIV. The
presence of BrdU did not modify neuronal number nor total cell number. Cells were double-labeled for -tubulin and BrdU, as described in
Immunocytochemistry and Cell Counting. In each field (~70 cells) virtually no -tubulin-immunoreactive cells were present in control conditions, whereas in the presence of 10 nM IGF-I, one to
four cells were -tubulin-positive. Thus, at least 90-120
-tubulin-immunoreactive cells per condition were counted in each
independent experiment (n = 3 to 4). From these data
the percentage of double-immunoreactive cells for -tubulin and BrdU
was calculated.
Reverse transcription and PCR. Total RNA was extracted from
EGF-generated spheres and from cells plated in the presence or absence
of 10 nM IGF-I for 7 DIV by single-phase phenol/guanidine isothiocyanate TRIzol reagent (BRL, Bethesda, MD). Cells were pelleted
at 500 × g and immediately frozen in liquid nitrogen. Then cells were lysed with TRIzol (200 µl/106
cells) on the day of reverse transcription. The organic and the aqueous
phases were separated by the addition of 0.2 vol of chloroform and
centrifuged at 3500 × g. Total RNA was precipitated
from the aqueous phase with the addition of an equal volume of
isopropanol at 20°C overnight. Afterward, the dry pellet was
dissolved in 4 M guanidium thiocyanate, followed by the
addition of 2 vol of 95% ethanol and precipitated at 20°C
overnight. After centrifugation, the pellet was resuspended in water
for direct use in the reverse transcriptase reactions. The 260:280
ratio for all samples was between 1.6 and 2. First-strand cDNA was made
with Superscript reverse transcriptase (BRL). Briefly, 1 µg of total
RNA and 0.5 µg oligo-dT were heated to 65°C and quickly chilled on
ice. The annealed mRNA-oligo-dT complexes were incubated at 42°C for
75-90 min with 200 U of reverse transcriptase in 20 µl total volume of (in mM) 0.5 dNTPs, 50 Tris, pH 8.3, 75 KCl, 3 MgCl2, and 10 DTT. The reaction was heat-inactivated
(65°C for 15 min) and diluted to 50 µl with water. The diluted
reaction mix was used directly in PCR reactions. Primers specific for
individual members of IGF receptor family were designed from
published sequence data as follows: (1) IGF-I receptor  -subunit,
accession number U00182 (Mus musculus), the upstream (5')
primer corresponds to position 129-148
(5'-TATCAGCAGCTGAAGCGCCT-3') and the downstream primer (3') is
complementary to position 590-571 (5'-GGTGGTCTTCTCACACATGG-3'), 464 bp
amplified product; (2) IGF-II receptor, accession number L19500
(Mus musculus), the upstream (5') primer corresponds to position 185-204 (5'-CTGCTTGCTGGCCTTACTGC-3') and the downstream primer (3') is complementary to position 566-547
(5'-CTTCAGGACCTTGCGCTGTG-3'), 382 bp amplified product; (3) insulin
receptor, accession number J05149 (Mus musculus), the
upstream (5') primer corresponds to position 3036-3055
(5'-TCCATCTTCTGTGTACGTGC-3') and the downstream primer (3') is
complementary to position 3782-3763 (5'-AGATAGCCTCCATCCATGAC-3'), 747 bp amplified product; (4) -actin, accession number X03672 (Mus musculus), the upstream (5') primer corresponds to
position 182-202 (5'-CGTGGGCCGCCCTAGGCACCA-3') and the downstream
primer (3') is complementary to position 424-404
(5'-TTGGCCTTAGGGTTCAGGGGG-3'), 243 bp amplified product. PCR
analyses were performed in a final volume of 50 µl containing 25 pmol
of each primer, 5 µl of the reverse transcription reaction, 2.5 U of
Taq DNA polymerase (0.5 µl), and (in mM) 10 Tris-HCl, pH 8.3, 50 KCl, 1.5 MgCl2, and 0.2 each of
the four dNTPs. After a denaturation step of 5 min at 97°C,
amplifications were started at 75°C (hot start) with Taq DNA polymerase for 30 cycles for insulin receptor and actin, and for 32 cycles for IGF-II receptor of 94°C for 45 sec, x°C/45
sec, and 72°C/1 min, x being the annealing temperature at
60°C for IGF-II receptors and actin primer and at 59°C for insulin
receptor primers. We used a two step PCR amplification for IGF-I
receptor, beginning with a hot start. Thirty cycles of 94°C for 45 sec and 68°C for 3 min were performed. Final products were analyzed
on a 1.5% agarose gel. PCR amplification products for all receptors were excised out of the agarose gel and isolated by spin columns (Qiagen, Hilden, Germany). The purified fragments were sequenced. No
genomic amplification was observed from any extracted RNA samples.
| |
RESULTS |
IGF-I induces the appearance of neurons in stem cell progeny via an
action at the IGF-I receptor
We have shown previously that EGF-responsive stem cell progeny
produce neurons, astrocytes, and oligodendrocytes when they are
differentiated on a poly-ornithine substrate in vitro
(Reynolds and Weiss, 1996). Here we asked whether specific activation
of IGF-I receptors could increase the number of differentiated neurons. All experiments were performed without insulin in the medium to avoid
its actions on insulin and/or IGF-I receptors. When dissociated cells
derived from EGF-responsive stem cell progeny were cultured on
poly-L-ornithine coverslips for 7 DIV in the absence of
insulin, virtually no or few cells immunoreactive for -tubulin (an
early neuronal marker) were present. However, neurons were generated in
a dose-dependent manner when IGF-I and, to a lesser extent, IGF-II and
insulin (Fig. 1A) were
added to the culture medium. The increase in neuronal number ranged
from eight- to 40-fold relative to control; the maximum number of
neurons generated varied from 1.7 to 5% of total cell number. The
half-maximal bioactivities (ED50) of IGF-I, IGF-II,
and insulin were 5.5 ± 1.9, 34.3 ± 11.4, and 262 ± 84 nM, respectively, corresponding to the pharmacology of the
IGF-I receptor (IGF-I > IGF-II > insulin; LeRoith et al., 1993). This suggests that the generation of neurons at physiological concentrations occurred via an IGF-I receptor. The percentage of
GFAP-immunoreactive cells (Fig. 1B) was not affected
significantly by any of the growth factors, and the total cell number
increased significantly only at supersaturating concentrations (1 µM) of IGF-I or IGF-II (Fig. 1C).
View larger version (14K):
[in this window]
[in a new window]
|
Figure 1.
Effects of IGFs on neuron numbers in cultures of
EGF-generated precursors. A, Increasing concentrations
of IGF-I, IGF-II, and insulin induced a dose-dependent increase in
neuron (-tubulin-immunoreactive) production, expressed as a
percentage of total cells derived from EGF-generated spheres. The rank
order of potency, IGF-I > IGF-II > insulin, is
characteristic of IGF-I receptor pharmacology (LeRoith et al., 1993)
(see Results for further discussion). Growth factors were added at
plating and were present for the entire culture period. Under the
identical experimental conditions the percentages of astrocytes
(GFAP-immunoreactive (B) and total cell number
(C) were not affected significantly, other than
total cell numbers at supersaturating (1 µM)
concentrations of IGF-I or IGF-II. Data represent the mean ± SEM
of four independent cultures; *p < 0.05.
|
|
To confirm that the generation of neurons by insulin and IGF-II
occurred via their actions at IGF-I receptors, we examined whether
IGF-I, IGF-II, and insulin had additive actions at the maximal
effective dose of each factor. Coupled with its potency, a lack of
additivity of either IGF-II or insulin with IGF-I would confirm that
the action is mediated by the IGF-I receptor. The addition of 1 µM IGF-II or 10 µM insulin to 100 nM IGF-I did not increase the production of neurons in
comparison to 100 nM IGF-I alone (data not shown). In the
next step of this initial characterization we performed reverse
transcription (RT) of total cellular RNA (from EGF-generated stem cell
progeny) and PCR for the IGF receptor family. The E14 whole brain
served as control tissue. RT-PCR found that IGF-I, insulin, and IGF-II
receptors were all expressed in stem cell progeny (Fig.
2). Taken together, these findings
suggest that, despite the presence of all three receptor subtypes, the principal receptor mediating the growth factor-induced increases in
neuronal number is the IGF-I receptor.
View larger version (72K):
[in this window]
[in a new window]
|
Figure 2.
Detection of IGF receptor subtypes in embryonic
brain and EGF-generated spheres. Reverse transcription and PCR of total
RNA extracted revealed that IGF-I, insulin, and IGF-II receptor mRNA are present in the E14 brain (Br) and in the
EGF-generated spheres of undifferentiated precursors
(S). First-strand cDNA was amplified by using
specific primers, as described in Materials and Methods. The ladder is
the 100 bp ladder (from BRL). Amplified products were run out on a
1.5% agarose/Tris-acetate-EDTA gel and visualized with ethidium
bromide. No genomic amplification was observed from any extracted RNA
samples.
|
|
Although their numbers were not changed, IGF-I did induce a change in
astrocyte morphology 10 nM IGF-I (or higher concentrations of insulin or IGF-II) induced a flattening and increased GFAP immunoreactivity (data not shown). These results confirmed previous results that used high concentrations of insulin (Toran-Allerand et
al., 1991). These findings raise the possibility that the action of
IGF-I on neuron production could be an indirect effect via the
maturation of astrocytes. To address this issue, we reexamined IGF-I
actions at a lower cell density. Thus, the actions of 10 and 100 nM IGF-I were examined on 50 EGF-generated
cells/mm2 (rather than 500 cells/mm2 in standard conditions). Under these
experimental conditions IGF-I remained a potent and effective enhancer
of neuronal production (Table 1). Again,
total cell number did not vary. However, the basal neuron number (in
the absence of IGF-I) was greater in low-density experiments. A
putative unidentified inhibitor for neuronal differentiation may be
effective only at higher density and attests to the presence of
multiple factors secreted by cells that could interact in neuronal differentiation. Nevertheless, taken together these results suggest that the numbers of neurons are increased by IGF-I activation of its
receptors on neuronal precursors.
IGF-I-induced increases in neuronal number are attributable to
differentiation and not to proliferation nor survival of neuronal
precursors
In control conditions virtually no (0-0.8%)
-tubulin-immunoreactive cells were observed in a field of ~70
cells, whereas in the presence of 10 nM IGF-I one to four
cells (1.7-5.0%) were -tubulin-positive. When 10 fields were
counted in three independent cultures, 10 nM IGF-I induced
a significant increase in neuron number (Fig.
3A). The increased neuron
number after IGF-I treatment could be attributable to one of several
possible actions: (1) the proliferation of neuronal precursors, (2) the
survival of neuronal precursors, (3) the survival of postmitotic
neurons, or (4) the differentiation of neuronal precursors into
neurons. These different possibilities were examined in the following
experiments.
View larger version (18K):
[in this window]
[in a new window]
|
Figure 3.
IGF-I increases neuron number without an apparent
survival action. A, EGF-generated precursors were
cultured in the absence or presence of 10 nM IGF-I for 7 DIV. The total number of neurons in 10 fields (total of 600-900 cells)
was determined. In subsequent experiments, to ensure standardization
with multiple comparisons, the number of neurons present after 7 DIV in
the presence of 10 nM IGF-I (15-50
-tubulin-immunoreactive neurons within 600-900 cells) serves as
100%. *p < 0.05 compared with control.
B, A 5 d delay (from plating) in exposure of
precursors to 10 nM IGF-I yielded neuron numbers that were
not significantly different from a 7 DIV exposure starting at plating.
C, Brief exposure to IGF-I was sufficient to induce
neuronal differentiation. IGF-I (10 nM) was added for 2 hr
(2 h) at plating or for 1 DIV, followed by extensive
washing and a total 7 DIV incubation, and then compared with control
(*significantly different, p < 0.05) or to a 7 DIV exposure (not significantly different). All data represent the mean ± SEM of three independent culture experiments.
|
|
Studies of PNS neuroblasts have demonstrated that IGF-I-induced
increases in neuronal numbers are attributable to a mitogenic action.
To determine whether the increased number of neurons caused by IGF-I
occurs via proliferation of precursors, we included the thymidine
analog BrdU (1 µM) in cultures of stem cell progeny for 7 DIV both in the absence or presence of 10 nM IGF-I. Because control studies indicated that BrdU-immunopositive neurons constituted 10-20% of the total -tubulin-positive neurons, additional
extensive counting was performed for all BrdU double-labeling
experiments. Thus, in each independent experiment (n = 3 and in each condition therein) at least 90-120
-tubulin-immunoreactive cells were counted as being either single-
or double-labeled. From this data the percentage of
double-immunoreactive cells for -tubulin and BrdU was calculated.
After 7 DIV in the absence of IGF-I, a small number of neurons
(18.6 ± 0.6%) incorporated BrdU. After 7 DIV with 10 nM IGF-I, a similarly small (11.3 ± 4.4%, not
significantly different) number of the neurons was BrdU-immunoreactive.
If the increased numbers of neurons caused by IGF-I were a result of
neuronal precursor proliferation, one would expect a significant
increase in the percentage of -tubulin-immunoreactive neurons that
incorporate BrdU, corresponding to the number of newly generated
neurons. However, this was not the case. Thus, it is apparent that the increased numbers of neurons cannot be attributable to proliferation; therefore, IGF-I does not have a mitogenic action on stem
cell-generated neuronal precursors.
The increasing number of neurons induced by IGF-I could be attributable
to an enhanced survival of neuronal precursors that then could
differentiate spontaneously. We define survival to be a dependence that
would require the continued presence of the factor throughout the
culture period. Withdrawal of the factor would result in death. Delay
in administering the factor should result in a dramatic and significant
attenuation, relative to continued presence. This was tested as
follows. We delayed the addition of IGF-I for 5 d and counted the
neuron number at 7 DIV. Under these conditions neuronal production was
slightly, but not significantly (p = 0.19; Fig.
3B), reduced relative to a 7 DIV administration. Then we
tested the possibility that IGF-I acts as a survival factor for
postmitotic neurons. This goal was achieved by short-term exposures of
IGF-I at plating. Then the culture was maintained until 7 DIV. If IGF-I
is a survival factor for neurons, we expected a significant reduction
in neuron number after 7 DIV. Exposures to IGF-I for as little as 2 hr
(p = 0.44) or 1 DIV (p = 0.19) essentially mimicked the action of IGF-I when it was present
throughout the culture period (Fig. 3C). It is particularly
noteworthy that a 2 hr exposure produced an equivalent number of
-tubulin-immunoreactive cells as a 7 DIV exposure. Thus, these
results suggest that the increased number of neurons produced by IGF-I
is not an action on the survival of postmitotic neuronal precursors or
neurons but rather an induction of differentiation.
IGF-I and insulin are synergized to produce neurons
Although the results of our study clearly point to an action of
IGF-I at its receptor for inducing neuronal differentiation, the
presence of receptors for both insulin and IGF-II (see Fig. 2) prompted
us to examine whether the activation of these receptors could modulate
the IGF-I response. Given our observation that short exposure (2 hr) to
IGF-I was a sufficient stimulus for differentiation, we performed these
and all subsequent experiments with an additional insulin-free prewash
(see Materials and Methods) during the dissociation procedure to ensure
that no activation occurred before plating. To examine the putative
interactions among ligands, we used 10 nM concentrations of
both IGF-I and insulin to allow for maximal stimulation of each
respective receptor without cross-reacting with the other (LeRoith
et al., 1993). When stem cell progeny were exposed to 10 nM
insulin alone for 7 DIV, a small number of neurons was generated (Fig.
4A). This result is in
accordance with those of Figure 1. IGF-I (10 nM) increased
neuron production by approximately eightfold. When insulin and IGF-I
were combined, the resultant number of neurons was 37%
(p = 0.02) greater than the additive effect of
the both factors alone, although total cell number did not change
(IGF-I = 608 ± 64 cells/10 fields; IGF-I plus insulin = 642 ± 44 cells/10 fields). This suggests that insulin and IGF-I
have synergistic actions on neuronal production. When IGF-II (10 nM) was coincubated with either insulin or IGF-I, no
synergistic actions were observed.
View larger version (13K):
[in this window]
[in a new window]
|
Figure 4.
IGF-I and insulin cooperate to produce enhanced
numbers of neurons. EGF-generated precursors were cultured with 1 µM BrdU in the absence or presence of 10 nM
insulin, 10 nM IGF-I, or the two combined, for 7 DIV.
A, With the numbers of -tubulin-immunoreactive neurons produced by 7 DIV of 10 nM IGF-I serving as 100%
(see legend to Fig. 3 and Materials and Methods for details), the
combined actions of 10 nM IGF-I plus 10 nM
insulin are found to be more than additive (*p < 0.05; n = 4 independent cultures).
B, In each condition, 100 -tubulin-immunoreactive
neurons were examined for incorporation of BrdU, and the percentage of
double-labeled cells was calculated. The combined actions of 10 nM IGF-I plus 10 nM insulin result in a twofold
increase in newly generated neurons (**p < 0.01 relative to control or insulin; p < 0.05 relative to IGF-I; n = 4 independent cultures). Taken
together, these data suggest that increased neuronal numbers caused by
combined insulin plus IGF-I is attributable to actions on mitotically
active neuronal precursors.
|
|
We next asked whether the neuronal production generated by the
coincubation of insulin and IGF-I was attributable to the proliferation of a neuronal precursor. In each condition 100 -tubulin-immunoreactive neurons were examined for the incorporation
of BrdU, and the percentage of double-labeled cells was calculated. If
the 37% increase in total neurons caused by the presence of insulin is
a result of proliferation, this would translate into ~27% of
additional (to IGF-I) BrdU/-tubulin double-labeled cells (indicating
that all additional neurons had undergone mitosis before
differentiation). We found that the combined actions of 10 nM IGF-I plus 10 nM insulin resulted in an
additional 25% in the population of BrdU/-tubulin double-labeled
cells (IGF-I = 27 ± 13%; IGF-I plus insulin = 52 ± 5%; Fig. 4B). These findings suggest that the
more than additive numbers of neurons observed when IGF-I and insulin
were coincubated likely are derived from a mitotically active precursor
population. Figure 5 demonstrates that,
despite the use of two monoclonal antibodies (against -tubulin and
BrdU), our incubation procedure did not generate nonspecific overlap,
as demonstrated by both coincident and noncoincident immunolabeling
(with steady-state immunofluorescent intensity) in the same field.
View larger version (68K):
[in this window]
[in a new window]
|
Figure 5.
Dual labeling for -tubulin and BrdU provides
evidence for newly generated neurons. EGF-generated spheres were
dissociated and plated for 7 DIV with 1 µM BrdU in the
presence of 10 nM IGF-I (A, C) or 10 nM IGF-I plus 10 nM insulin (B,
D). Cells were dual-labeled for -tubulin (A,
B) and BrdU (C, D) immunoreactivity.
Arrows illustrate -tubulin-immunoreactive neurons
that had incorporated BrdU. Despite the use of two monoclonal
antibodies, our incubation procedure did not generate nonspecific
overlap, as demonstrated by both coincident and noncoincident
immunolabeling (with steady-state immunofluorescent intensity) in the
same field (see Materials and Methods for details). Scale bar, 20 µm.
|
|
IGF-I and BDNF act on two populations of undifferentiated
neuronal precursors
We have demonstrated previously that BDNF is also a
differentiation factor for stem cell-derived neuronal precursors. Those experiments were performed with high (4 µM) insulin.
Interestingly, the actions of BDNF required >5 d to develop, in
comparison to IGF-I actions that required only short incubation periods
(see Fig. 3C). Thus, we sought to determine whether IGF-I
and BDNF actions could be distinguished. Dissociated EGF-generated
precursors were cultured in the absence of insulin or IGF-I and in the
presence of increasing concentrations of BDNF. BDNF induced a
dose-dependent (Fig.
6
View larger version (15K):
[in this window]
[in a new window]
|
Figure 6. Independent and cooperative actions of BDNF and
IGF-I plus insulin in neuronal differentiation. A,
EGF-generated precursors were cultured without IGF-I or insulin and
with increasing concentrations of BDNF for 7 DIV. A representative
experiment illustrates a dose-dependent increase in neuron numbers
(total cell number was unaffected; data not shown). B,
Cumulative data for saturating concentrations (50 ng/ml) of BDNF
illustrate a significant three- to fourfold increase in neurons
relative to control (**p < 0.01;
n = 8 independent cultures). C, BDNF
potentiates the actions of low concentrations of insulin and IGF-I. As
described in the legend to Figure 3 and Materials and Methods, the
number of neurons generated by 7 DIV incubation with 10 nM
IGF-I (15-50 -tubulin-immunoreactive neurons within 600-900 cells)
serves as 100%. The number of neurons generated by the combined
actions of 1 nM insulin plus 1 nM IGF-I plus 50 ng/ml BDNF was significantly greater than either insulin plus IGF-I or
BDNF alone (**p = 0.02). When the action of BDNF
was subtracted, the resultant insulin plus IGF-I response was 3.85-fold
greater than that generated in its absence
(p < 0.05). D, BDNF acts on
a distinct population of neuronal precursors. Effects of 10 nM IGF-I and/or insulin were tested with 50 ng/ml of BDNF.
An additive action of IGF-I and insulin with BDNF was observed.
*p = 0.03 in comparison to 10 nM IGF-I;
ap < 0.01 in comparison to BDNF or to
IGF-I coincubated with insulin (n = 5 independent
cultures).
|
|
A) increase in
neuronal numbers; total cell number was unaffected (data not shown). At
saturating concentrations (50 ng/ml), BDNF induced a significant three-
to fourfold increase in neuronal number (Fig. 6B). In
eight independent culture experiments we compared the maximal
efficacies of BDNF and IGF-I. Maximal efficacy of BDNF was
approximately one-third that of IGF-I.
These results suggested three possibilities. BDNF might act on the same
precursors as IGF-I (plus insulin), on a distinct population, or on
both. This was studied in two ways. First, we coincubated low
concentrations of insulin and IGF-I (both at 1 nM), which
were ineffectual alone (Fig. 6C), and this resulted in a
combined action that was ~20% of that produced by 10 nM
IGF-I alone. When 50 ng/ml of BDNF was added to 1 nM IGF-I
plus insulin, the resultant production was not significantly different
from 10 nM IGF-I. Furthermore, this combined response was
much more than additive. Total cell numbers were unaffected (data not
shown). If we subtract the BDNF action, the resultant stimulation by
IGF-I plus insulin is increased by 3.85-fold (p < 0.05), suggesting that BDNF can potentiate the actions of IGF-I plus
insulin. Yet these results cannot exclude an action of BDNF on a
different population of cells. To test this, we exposed stem cell
progeny to maximal concentrations of IGF-I plus insulin (10 nM each) or BDNF (50 ng/ml) or all three combined (Fig.
6D). When all three were coincubated, the resultant
neuron number was completely additive, but the total cell number was
not significantly different (IGF-I plus insulin = 642 ± 44 cells/10 fields; IGF-I plus insulin plus BDNF = 780 ± 121 cells/10 fields; n = 5). Taken together, these findings
suggest that there are at least two populations of neuronal precursors
that are differentially responsive to IGF-I plus insulin and BDNF.
We next asked whether these two populations could be distinguished by
neurotransmitter phenotype. In a previous study we found that all
neurons induced to differentiate in the presence of BDNF colocalized
GABA and substance P (Ahmed et al., 1995). This was confirmed in the
present study, whereby close to 100% of the BDNF-generated neurons
were GABA-immunoreactive (Fig.
7C). On the other hand, of
those neurons generated in the presence of IGF-I alone, 35% were
GABAergic, although the remainder were not. Attempts to identify the
neurotransmitter phenotype by testing antibodies that recognize tyrosine hydroxylase, calbindin, or parvalbumin were unsuccessful. Nevertheless, these data further support the contention that IGF-I and
BDNF may act on two distinct populations of stem cell-derived neuronal
precursors.
View larger version (46K):
[in this window]
[in a new window]
|
Figure 7.
BDNF induces differentiation of GABAergic neurons,
whereas IGF-I-generated neurons are principally non-GABAergic.
A, B, IGF-I generated a population of non-GABAergic
neurons. The arrow illustrates a cell that is
immunoreactive for -tubulin (A), but not for
GABA (B, arrowhead). C, Quantitative
analysis of the GABAergic neurons present after differentiation by
BDNF, IGF-I, or IGF-I plus insulin. The results reveal that BDNF
generated only GABAergic neurons, whereas IGF-I-generated and IGF-I
plus insulin-generated neurons were principally non-GABAergic. In all,
100-120 neurons were examined per condition and within each experiment
(n = 3); **p < 0.01.
|
|
| |
DISCUSSION |
The overall objective of this work was to ask whether IGF-I
influences the differentiation of EGF-responsive stem cell-generated neuronal precursors. Three conclusions may be drawn as a result of our
findings. First, IGF-I, acting via its cognate receptor, promotes the
differentiation (and not proliferation or survival) of postmitotic
neuronal precursors. Second, insulin, likely acting via its cognate
receptor, appears to recruit (in the presence of IGF-I)
a more primitive precursor, e.g., upstream and mitotically active.
Finally, the actions of IGF-I can be distinguished from those of BDNF,
in that BDNF can act alone to differentiate a separate population of
postmitotic neuronal precursors. Each of these conclusions will be
discussed in turn.
The first major finding in this study was that in absence of IGF
peptides virtually no neurons were present in our cultures of
dissociated EGF-generated stem cell progeny and that increasing concentrations of either IGF-I, insulin, or IGF-II could induce similar
increases in the number of differentiated neurons. Moreover, the
magnitude of the increased neuronal number was eight- to 40-fold. The
rank order of potency of the three growth factors (IGF-I > IGF-II > insulin) suggests action at a single receptor, the IGF-I receptor (LeRoith et al., 1993). That an IGF-I receptor should be
present on neuronal precursors is not surprising, given the evidence
for the receptor mRNA or binding sites on CNS neurons (Shemer et al.,
1987; Bondy, 1991) and for IGF-I-mediated neuronal signal transduction
(Heidenreich, 1993; Robinson et al., 1994). What is surprising and
novel, perhaps, is that the activation of this receptor results in a
single response, promoting the differentiation of a postmitotic
neuronal precursor. The fact that the complete differentiation response
can be induced in as little as 2 hr of exposure suggests that IGF-I
initiates a differentiation program. Such an induction was described
recently for platelet-derived growth factor (PDGF) action on primary
cortical neuronal precursors (Williams et al., 1997). PDGF similarly
was found to induce neuronal differentiation in as little as 2 hr of
exposure, a process that was dependent on mRNA synthesis. In
preliminary studies we recently found that a 2 hr stimulation by IGF-I
was able to induce the expression of the POU-III homeodomain containing
transcription factor Brn4 as well as the trkB
mRNAs (Y. Arsenijevic, T. Shimazaki, and S. Weiss, unpublished data).
Whether the expression of these mRNAs is required for neuronal
differentiation is currently under investigation. Moreover, the
continued differentiation in the absence of IGF-I (after the 2 hr
exposure), coupled with the ability to delay administration for up to
5 d (and still evoke an almost complete response), suggests that
IGF-I is not required as a long-term survival factor for neuronal
precursors. This is in contrast to the apparent requirement for IGF-I
as a survival factor for neuroepithelial cells as well as for cortical,
mesencephalic dopaminergic, and cerebellar neurons (Aizenman and de
Vellis, 1987; Drago et al., 1991; Beck et al., 1993; Torres-Aleman et
al., 1994). Of those studies, only that of Drago and colleagues (1991)
truly established a requirement for IGF-I for cell survival, whereas
the results of the latter studies could be accounted for easily by an
action of IGF-I on differentiation of postmitotic neuronal precursors. Of course, one could argue that in the absence of differentiation a
neuroblast/neuronal precursor ultimately might die. Either
interpretation could serve to explain the reduction in certain
populations of central neurons in IGF-I null mutation mice (Beck et
al., 1995).
The lack of BrdU incorporation in the majority of neurons detected in
the presence of IGF-I suggests that IGF-I is not a mitogen for these
precursors, as it clearly is for sympathetic neuroblasts (DiCicco-Bloom
and Black, 1988; Zackenfels et al., 1995). Moreover, the lack of BrdU
incorporation also implies that IGF-I likely exerts its actions on
undifferentiated neural precursors that already are committed to the
neuronal lineage. Generally, it is accepted that multipotential cells
are mainly mitotically active (for review, see Barbe, 1996) and that
phenotype change likely requires being close to S phase (McConnell,
1995). Consequently, the closer a cell is to its final fate, the less
sensitive it is to signals that can modify that fate (Levitt et al.,
1993). Had IGF-I acted as a commitment factor, one would have expected a larger proportion (relative to control) of the neurons to have incorporated BrdU. This was clearly not the case. This is in contrast to the laminin-dependent IGF-I-stimulated production of neurons from
avian E5 retinal neuroepithelial cells (Frade et al., 1996). All of the
neurons produced in the presence of IGF-I in that study were newly
generated, suggesting that IGF-I was acting as a commitment factor. In
our study the coincubation of insulin and IGF-I was synergistic and
could be attributable to the recruitment of uncommitted cells to the
neuronal lineage. In the presence of insulin plus IGF-I, the numbers of
BrdU-immunoreactive neurons increased to close to 50%, as compared
with 25% with IGF-I or under control conditions. Moreover,
BrdU-immunoreactive neurons could account entirely for the increased
numbers of neurons that were generated by coactivation. The present
results show clearly that insulin (in the presence of IGF-I) acts on a
more primitive, mitotically active precursor in the neuronal lineage,
but this study cannot discern whether insulin plus IGF-I is mitogenic
for a neuronal precursor or whether insulin recruits uncommitted
mitotically active cells to the neuronal lineage; these are, in turn,
differentiated by IGF-I. Clonal analysis, with both sequential and
cooperative application of insulin and IGF-I, will be required to
distinguish between these possibilities. Nevertheless, reports of
insulin regulation of neuroepithelial cell number (De la Rosa et al., 1994) and the recent identification of a NeuroD site on the insulin gene promoter (Lee et al., 1995; Naya et al., 1995) support a role for
insulin in programs leading to neuronal differentiation.
In a previous study we found that BDNF was a differentiation factor
(Ahmed et al., 1995) for the same CNS stem cell-derived neuronal
precursors studied here. In that study, which used a high insulin
concentration (4 µM; that would activate both insulin and
IGF receptors maximally), BDNF further increased the number of
differentiated neurons without any apparent proliferation or survival
actions. BDNF actions differed from those of IGF-I in that short
exposures, e.g., 48 hr, were not sufficient to induce the
differentiation response. In the present study we found that BDNF could
induce a small population of neuronal precursors to differentiate in
the absence of any IGF-I or insulin. This suggests that BDNF likely
acts on a separate population of neuronal precursors. Yet BDNF and
IGF-I both (1) use tyrosine kinase receptors (Kasuga et al., 1982;
Klein et al., 1989, 1991; Cheatham and Khan, 1995; LeRoith et al.,
1995), (2) activate p21ras as part of their signal transduction (Ng and
Shooter, 1993; Robinson et al., 1994), and (3) support the survival of
mesencephalic dopamine and motor neurons (Beck et al., 1993; Li et al.,
1994). Thus, it is reasonable to suggest that BDNF and IGF-I may
cooperate on the same populations of neuronal precursors. Indeed, our
results support both scenarios. First, we showed that when
low concentrations of IGF-I and insulin were coincubated
with maximal concentrations of BDNF, the resultant response was more
than additive. This suggested either that BDNF was potentiating the
IGF-I plus insulin response or vice versa. Thus, we used saturating
concentrations of IGF-I plus insulin, together with BDNF, and found the
resultant response completely additive. If the BDNF response was
amenable to potentiation, we still should have observed a more than
additive response. Taken together with the fact that BDNF actions can
occur in the absence of either insulin or IGF-I, we propose that BDNF
can (1) potentiate the actions of IGF-I plus insulin to the maximum
they would achieve via complete activation of their cognate receptors
and (2) act on a separate population of neuronal precursors that do not
require IGF-I plus insulin.
How might insulin, IGF-I, and BDNF operate in CNS neurogenesis in
vivo? Our in vitro investigations raise several
interesting possibilities. We have postulated two models that could
account for the apparently different populations that respond to IGF-I plus insulin (potentiated by BDNF) and to BDNF alone. These are illustrated as part of our working model of neurogenesis from EGF-responsive stem cells in Figure 8.
Our contentions for the roles for EGF and bFGF on mitotically active
multipotent and committed neuronal precursors are based on our previous
clonal analyses (Reynolds et al., 1992; Vescovi et al., 1993; Reynolds
and Weiss, 1996) and those of others (Gritti et al., 1996). The results
of this study suggest that insulin may act to recruit either
multipotent stem cells or bipotential neuronal precursors directly to
the postmitotic neuronal fate. Subsequently, two models may explain the
actions of IGF-I and BDNF on the differentiation of the postmitotic neuronal precursors. In the first model, the "lineage" model (in italic type), two phenotypically distinct populations generated by the
EGF-responsive precursor have differential requirements for IGF-I (plus
insulin) or for BDNF. In support of this model are the data whereby we
examined the neuronal phenotype of the IGF-I (IGF-I plus insulin) and
BDNF-responsive populations. Confirming the results of our previous
study (Ahmed et al., 1995), virtually all of the BDNF-responsive
neurons contained GABA, whereas significantly fewer of those that were
dependent on IGF-I (or IGF-I plus insulin) contained the amino acid
transmitter. The phenotype of the non-GABAergic populations has not
been determined. Of course, one could argue that perhaps this
population has yet to express its phenotype and thus would support the
second model (in bold type), the "birthdate" model. In this
scenario, neuronal precursors first would be dependent on IGF-I for
differentiation, followed by BDNF. BDNF previously has been termed a
late differentiation factor for cerebellar granule cells (Gao et al.,
1995). Several lines of evidence support this model. First, as
discussed above, the minimal time necessary for differentiation is 2 hr
for IGF-I and >48 hr for BDNF. Second, twice as many (~25%)
IGF-I-responsive precursors incorporated BrdU than did those that were
BDNF-responsive. This suggests that the BDNF-responsive neuronal
precursors are further downstream (further from S phase). Moreover, it
suggests that precursors develop BDNF responsiveness in the absence of
IGF-I and that IGF-I may enhance the onset of the response. In fact,
preliminary data from our lab have found that a 2 hr exposure of stem
cell progeny to IGF-I resulted in an immediate upregulation of the
catalytic domain-containing trkB transcript (Y. Arsenijevic,
T. Shimazaki and S. Weiss, unpublished observations). Taken together,
these findings support the contention that, although not required,
IGF-I and BDNF may act in a sequential manner as early and later
differentiation signals, with a feed-forward (and possible
feed-backward) regulation of receptor expression. Ongoing
experimentation examining birthdates and the sequential stimulation of
growth factor receptors should serve to reconcile these two models of
neuronal differentiation.
View larger version (19K):
[in this window]
[in a new window]
|
Figure 8.
A model for neurogenesis by multipotent stem cells
in vitro. Our working hypothesis is that multipotent
stem cells produce neurons in four steps during which neuron number and
differentiation are regulated. Both EGF and bFGF induce multipotent
stem cells to proliferate. Basic FGF can stimulate committed neuronal
precursors (e.g., neuron/astrocyte or neuron only), which are derived
from multipotent stem cells, to produce neurons. In either case,
insulin may act to recruit multipotential or bipotential precursors to a postmitotic neuronal fate. Subsequently, two possible models (lineage
model in italic type and birthdate model in bold type) may serve to
explain how IGF-I and BDNF act to induce the differentiation of
postmitotic neuronal precursors. See Discussion for details of the two
models.
|
|
In addition to new basic concepts regarding the actions of IGF-I and
BDNF on the differentiation of neuronal precursors, our results also
raise some interesting practical considerations. IGF-I and BDNF
recently have been demonstrated to have important neurotrophic actions
in both motor neuron and striatal neurons (Hughes et al., 1993; Neff et
al., 1993; Li et al., 1994; Ventimiglia et al., 1995; Nakao et al.,
1996). If the synergistic action among insulin, IGF-I, and BDNF
operates on postmitotic neurons in vivo, lower (and thus
likely less toxic) concentrations of these factors could be
administered in combination for therapeutic intervention in disorders
such as amyotrophic lateral sclerosis and Huntington's disease (Adem
et al., 1994; Dore et al., 1997).
| |
FOOTNOTES |
Received Nov. 11, 1997; revised Dec. 24, 1997; accepted Jan. 6, 1998.
This work was supported by the Medical Research Council of Canada
(MRC). Y.A. was the recipient of The Swiss Foundation for Medicine and
Biology Fellowship. S.W. is an Alberta Heritage Foundation for Medical
Research Scholar and an MRC Scientist. We thank Drs. Y. Sagot, D. van
der Kooy, and A. Represa for the critical reading of an earlier version
of this manuscript and Dr. Takuya Shimazaki for help with receptor
detection.
Correspondence should be addressed to Dr. Samuel Weiss, Departments of
Anatomy and Pharmacology and Therapeutics, University of Calgary
Faculty of Medicine, 3330 Hospital Drive NW, Calgary, Alberta, Canada
T2N4N1.
Dr. Arsenijevic's present address: Gene Therapy Center and Surgical
Research Division, Centre Hospitalier Universitaire Vaudois, Lausanne
University Medical School, Pavilion 3 and 4, 1011 Lausanne, Switzerland.
| |
REFERENCES |
-
Adem A,
Ekblom J,
Gillberg P-G
(1994)
Growth factor receptors in amyotrophic lateral sclerosis.
Mol Neurobiol
9:225-231[Web of Science][Medline].
-
Ahmed S,
Reynolds BA,
Weiss S
(1995)
BDNF enhances the differentiation but not the survival of CNS stem cell-derived neuronal precursors.
J Neurosci
15:5765-5778[Abstract].
-
Aizenman Y,
de Vellis J
(1987)
Brain neurons develop in a serum and glial free environment: effects of transferrin, insulin, insulin-like growth factor-I, and thyroid hormone on neuronal survival, growth, and differentiation.
Brain Res
406:32-42[Web of Science][Medline].
-
Barbe MF
(1996)
Tempting fate and commitment in the developing forebrain.
Neuron
16:1-4[Web of Science][Medline].
-
Bartlett WP,
Li X-S,
Williams M,
Benkovic S
(1991)
Localization of insulin-like growth factor-I mRNA in murine central nervous system during postnatal development.
Dev Biol
147:239-250[Web of Science][Medline].
-
Bassas L,
Girbau M,
Lesniak MA,
Roth J,
de Pablo F
(1989)
Development of receptors for insulin and insulin-like growth factor-I in head and brain of chick embryos: autoradiographic localization.
Endocrinology
125:2320-2326[Abstract/Free Full Text].
-
Beck KD,
Knüsel B,
Hefti F
(1993)
The nature of the trophic action of brain-derived neurotrophic factor, des(1-3)-insulin-like growth factor-I, and basic fibroblast growth factor on mesencephalic dopaminergic neurons developing in culture.
Neuroscience
52:855-866[Web of Science][Medline].
-
Beck K,
Powell-Braxton L,
Widmer H-R,
Valverde J,
Hefti F
(1995)
IGF-1 gene disruption results in reduced brain size, CNS hypomyelination, and loss of hippocampal granule and striatal parvalbumin-containing neurons.
Neuron
14:717-730[Web of Science][Medline].
-
Bondy CA
(1991)
Transient IGF-I gene expression during the maturation of functionally related central projection neurons.
J Neurosci
11:3442-3455[Abstract].
-
Bondy CA,
Werner H,
Roberts Jr CT,
LeRoith D
(1990)
Cellular pattern of insulin-like growth factor-I (IGF-I) and type I IGF receptor gene expression in early organogenesis: comparison with IGF-II gene expression.
Mol Endocrinol
4:1386-1398[Abstract/Free Full Text].
-
Bondy CA,
Werner H,
Roberts Jr CT,
LeRoith D
(1992)
Cellular pattern of type I insulin-like growth factor receptor gene expression during maturation of the rat brain: comparison with insulin-like growth factors I and II.
Neuroscience
4:909-923.
-
Caroni P,
Grandes P
(1990)
Nerve sprouting in innervated adult skeletal muscle induced by exposure to elevated levels of insulin-like growth factors.
J Cell Biol
110:1307-1317[Abstract/Free Full Text].
-
Cheatham B,
Kahn CR
(1995)
Insulin action and the insulin signaling network.
Endocr Rev
16:117-142[Abstract/Free Full Text].
-
Craig CG,
Tropepe V,
Morshead CM,
Reynolds BA,
Weiss S,
van der Kooy D
(1996)
In vivo growth factor expansion of endogenous subependymal neural precursor cell populations in the adult mouse brain.
J Neurosci
16:2649-2658[Abstract/Free Full Text].
-
de la Rosa EJ,
Bondy CA,
Hernandez-Sanchez C,
Wu X,
Zhou J,
Lopez-Carranza A,
Scavo LM,
de Pablo F
(1994)
Insulin and insulin-like growth factor system components gene expression in the chicken retina from early neurogenesis until late development and their effect on neuroepithelial cells.
Eur J Neurosci
6:1801-1810[Web of Science][Medline].
-
Devaskar SU,
Singh BS,
Carnaghi LR,
Rajakumar PA,
Giddings SJ
(1993)
Insulin II gene expression in rat central nervous system.
Regul Pept
48:55-63[Web of Science][Medline].
-
DiCicco-Bloom E,
Black I
(1988)
Insulin growth factors regulate the mitotic cycle in cultured rat sympathetic neuroblasts.
Proc Natl Acad Sci USA
85:4066-4070[Abstract/Free Full Text].
-
Dore S,
Kar S,
Quirion R
(1997)
Rediscovering an old friend, IGF-I: potential use in the treatment of neurodegenerative diseases.
Trends Neurosci
20:326-331[Web of Science][Medline].
-
Drago J,
Murphy M,
Carroll SM,
Harvey RP,
Bartlett PF
(1991)
Fibroblast growth factor-mediated proliferation of central nervous system precursors depends on endogenous production of insulin-like growth factor-I.
Proc Natl Acad Sci USA
88:2199-2203[Abstract/Free Full Text].
-
Frade JM,
Marti E,
Bovolenta P,
Rodriguez-Peña MA,
Perez-Garcia D,
Rohrer H,
Edgar D,
Rodriguez-Tébar A
(1996)
Insulin-like growth factor-I stimulates neurogenesis in chick retina by regulating expression of the 6 integrin subunit.
Development
122:2497-2506[Abstract].
-
Gao WQ,
Zheng JL,
Karihaloo M
(1995)
Neurotrophin-4/5 (NT-4/5) and brain-derived neurotrophic factor (BDNF) act at later stage of cerebellar granule cell differentiation.
J Neurosci
15:2656-2667[Abstract].
-
Garcia-Segura LM,
Perez J,
Pons S,
Rejas MT,
Torres-Aleman I
(1991)
Localization of insulin-like growth factor-I (IGF-I)-like immunoreactivity in the developing and adult rat brain.
Brain Res
560:167-174[Web of Science][Medline].
-
Garofalo RS,
Rosen OM
(1988)
Tissue localization of Drosophila melanogaster insulin receptor during development.
Mol Cell Biol
8:1638-1647[Abstract/Free Full Text].
-
Gritti A,
Parati EA,
Cova L,
Frolichsthal P,
Galli R,
Wanke E,
Faravelli L,
Morassutti DJ,
Roisen F,
Nickel DD,
Vescovi AL
(1996)
Multipotential stem cells from the adult mouse brain proliferate and self-renew in response to basic fibroblast growth factor.
J Neurosci
16:1091-1100[Abstract/Free Full Text].
-
Heidenreich KA
(1993)
Insulin and IGF-I receptor signaling in cultured neurons.
Ann NY Acad Sci
692:72-88[Web of Science][Medline].
-
Hughes RA,
Sendtner M,
Thoenen H
(1993)
Members of several gene families influence survival of rat motoneurons in vitro and in vivo.
J Neurosci Res
36:663-671[Web of Science][Medline].
-
Ishii DN
(1995)
Implication of insulin-like growth factors in the pathogenesis of diabetic neuropathy.
Brain Res Rev
20:47-67[Medline].
-
Kar S,
Chabot J-G,
Quirion R
(1993)
Quantitative autoradiographic localization of [125I]insulin-like growth factor-I, [125I]insulin-like growth factor-II, and [125I]insulin receptor binding sites in developing and adult rat brain.
J Comp Neurol
333:375-397[Web of Science][Medline].
-
Kasuga M,
Karlsson FA,
Kahn CR
(1982)
Insulin stimulates the phosphorylation of the 95,000 dalton subunit of its own receptor.
Science
215:185-187[Abstract/Free Full Text].
-
Klein R,
Parada LF,
Coulier F,
Barbacid M
(1989)
trkB, a novel tyrosine protein kinase receptor expressed during mouse neural development.
EMBO J
8:3701-3709[Web of Science][Medline].
-
Klein R,
Nanduri V,
Jing S,
Lamballe F,
Tapley P,
Bryant S,
Cordon-Cardo C,
Jones KR,
Reichardt LF,
Barbacid M
(1991)
The trkB tyrosine protein kinase is a receptor for brain-derived neurotrophic factor and neurotrophin-3.
Cell
66:395-403[Web of Science][Medline].
-
Knusel B,
Michel PP,
Schwaber JS,
Hefti F
(1990)
Selective and nonselective stimulation of central cholinergic and dopaminergic development in vitro by nerve growth factor, basic fibroblast growth factor, epidermal growth factor, insulin, and the insulin-like growth factor I and II.
J Neurosci
10:558-570[Abstract].
-
Lee JE,
Hollenberg SM,
Snider L,
Turner DL,
Lipnick N,
Weintraub H
(1995)
Conversion of Xenopus ectoderm into neurons by neuroD, a basic helix-loop-helix protein.
Science
268:836-844[Abstract/Free Full Text].
-
LeRoith D,
Lowe Jr WL,
Shemer J,
Raizada M,
Ota A
(1988)
Development of brain insulin receptors.
Int J Biochem
20:225-230[Web of Science][Medline].
-
LeRoith D,
Werner H,
Faria TN,
Kato H,
Adamo M,
Roberts Jr CT
(1993)
Insulin-like growth factor receptors.
Ann NY Acad Sci
692:23-32.
-
LeRoith D,
Werner H,
Beitner-Johnson D,
Roberts Jr CT
(1995)
Molecular and cellular aspects of the insulin-like growth factor-I receptor.
Endocr Rev
16:143-163[Abstract/Free Full Text].
-
Levitt P,
Ferri RT,
Barbe MF
(1993)
Progressive acquisition of cortical phenotypes as a mechanism for specifying the developing cerebral cortex.
Perspect Dev Neurol
1:65-74.
-
Li L,
Oppenheim RW,
Lei M,
Houenou LJ
(1994)
Neurotrophic agents prevent motoneuron death following sciatic nerve section in the neonatal mouse.
J Neurobiol
25:759-766[Web of Science][Medline].
-
McConnell SK
(1995)
Constructing the cerebral cortex: neurogenesis and fate determination.
Neuron
15:761-768[Web of Science][Medline].
-
Mill JF,
Chao MV,
Ishii DN
(1985)
Insulin, insulin-like growth factor-II, and nerve growth factor effects on tubulin mRNA levels and neurites formation.
Proc Natl Acad Sci USA
82:7126-7130[Abstract/Free Full Text].
-
Morshead CM,
Reynolds BA,
Craig CG,
McBurney MW,
Staines WA,
Morassutti D,
Weiss S,
van der Kooy D
(1994)
Neural stem cells in the adult mammalian forebrain: a relatively quiescent subpopulation of subependymal cells.
Neuron
13:1071-1082[Web of Science][Medline].
-
Nakao N,
Odin P,
Lindvall O,
Brundin P
(1996)
Differential trophic effects of basic fibroblast growth factor, insulin-like growth factor-I, and neurotrophin-3 on striatal neurons in culture.
Exp Neurol
138:144-157[Web of Science][Medline].
-
Naya FJ,
Stellrecht CMM,
Tsai M-J
(1995)
Tissue-specific regulation of the insulin gene by a novel basic helix-loop-helix transcription factor.
Genes Dev
9:1009-1019[Abstract/Free Full Text].
-
Neff NT,
Prevette D,
Houenou LJ,
Lewis ME,
Glicksman MA,
Yin W-W,
Oppenheim W
(1993)
Insulin-like growth factors: putative muscle-derived trophic agents that promote motoneuron survival.
J Neurobiol
24:1578-1588[Web of Science][Medline].
-
Ng NFL,
Shooter EM
(1993)
Activation of p21ras by nerve growth factor in embryonic sensory neurons and PC12 cells.
J Biol Chem
268:25329-25333[Abstract/Free Full Text].
-
Petruzelli L,
Herrera R,
Arenas-Garcia RA,
Fernandez R,
Birnbaum MJ,
Rosen OM
(1986)
Isolation of a Drosophila genomic sequence homologous to the kinase domain of the human insulin receptor and detection of the phosphorylated Drosophila receptor with an antipeptide antibody.
Proc Natl Acad Sci USA
83:4710-4714[Abstract/Free Full Text].
-
Reynolds BA,
Weiss S
(1992)
Generation of neurons and astrocytes from isolated cells of the adult mammalian central nervous system.
Science
255:1707-1710[Abstract/Free Full Text].
-
Reynolds BA,
Weiss S
(1996)
Clonal and population analyses demonstrate that an EGF-responsive mammalian embryonic CNS precursor is a stem cell.
Dev Biol
175:1-13[Web of Science][Medline].
-
Reynolds BA,
Tetzlaff W,
Weiss S
(1992)
A multipotent EGF-responsive striatal embryonic progenitor cell produces neurons and astrocytes.
J Neurosci
12:4565-4574[Abstract].
-
Robinson LJ,
Leitner W,
Draznin B,
Heidenreich KA
(1994)
Evidence that p21ras mediates the neurotrophic effects of insulin-like growth factor-I in chick forebrain neurons.
Endocrinology
135:2568-2573[Abstract].
-
Shemer J,
Raizada MK,
Masters BA,
Ota A,
LeRoith D
(1987)
Insulin-like growth factor-I receptors in neuronal and glial cells.
J Biol Chem
262:7693-7699[Abstract/Free Full Text].
-
Toran-Allerand CD,
Bentham W,
Miranda RC,
Anderson JP
(1991)
Insulin influences astroglial morphology and glial fibrillary acidic protein (GFAP) expression in organotypic cultures.
Brain Res
558:296-304[Web of Science][Medline].
-
Torres-Aleman I,
Pons S,
Arevalo MA
(1994)
The insulin-like growth factor-I system in the rat cerebellum: developmental regulation and role in neuronal survival and differentiation.
J Neurosci Res
39:117-126[Web of Science][Medline].
-
Ventimiglia R,
Mather PE,
Jones BE,
Lindsay RM
(1995)
The neurotrophins BDNF, NT-3, and NT-4/5 promote survival and morphological and biochemical differentiation of striatal neurons in vitro.
Eur J Neurosci
7:213-222[Web of Science][Medline].
-
Vescovi AL,
Reynolds BA,
Fraser DD,
Weiss S
(1993)
BFGF regulates the proliferative fate of unipotent (neuronal) and bipotent (neuronal/astroglial) EGF-generated CNS progenitor cells.
Neuron
11:951-966[Web of Science][Medline].
-
Weiss S,
Reynolds BA,
Vescovi AL,
Morshead C,
Craig CG,
van der Kooy D
(1996)
Is there a neural stem cell in the mammalian forebrain?
Trends Neurosci
19:387-393[Web of Science][Medline].
-
Williams BP,
Park JK,
Alberta JA,
Muhlebach SG,
Hwang GY,
Roberts TM,
Stiles CD
(1997)
A PDGF-regulated immediate early gene response initiates neuronal differentiation in ventricular zone progenitor cells.
Neuron
18:553-562[Web of Science][Medline].
-
Zackenfels K,
Oppenheim RW,
Rohrer H
(1995)
Evidence for an important role of IGF-I and IGF-II for the early development of chick sympathetic neurons.
Neuron
14:731-741[Web of Science][Medline].
Copyright © 1998 Society for Neuroscience 0270-6474/98/1862118-11$05.00/0
This article has been cited by other articles:
|
|
|
|
 
B. Lorenzen and L. L. Murray
Benefits of Physical Fitness Training in Healthy Aging and Neurogenic Patient Populations
Neurophysiology and Neurogenic Speech and Language Disorders,
October 1, 2008;
18(3):
99 - 106.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
U Shivraj Sohur, J. G Emsley, B. D Mitchell, and J. D Macklis
Adult neurogenesis and cellular brain repair with neural progenitors, precursors and stem cells
Phil Trans R Soc B,
September 29, 2006;
361(1473):
1477 - 1497.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
N. Kosaka, M. Kodama, H. Sasaki, Y. Yamamoto, F. Takeshita, Y. Takahama, H. Sakamoto, T. Kato, M. Terada, and T. Ochiya
FGF-4 regulates neural progenitor cell proliferation and neuronal differentiation
FASEB J,
July 1, 2006;
20(9):
1484 - 1485.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
E. Colombo, S. G. Giannelli, R. Galli, E. Tagliafico, C. Foroni, E. Tenedini, S. Ferrari, S. Ferrari, G. Corte, A. Vescovi, et al.
Embryonic Stem-Derived Versus Somatic Neural Stem Cells: A Comparative Analysis of Their Developmental Potential and Molecular Phenotype
Stem Cells,
April 1, 2006;
24(4):
825 - 834.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
B. Angenieux, D. F. Schorderet, and Y. Arsenijevic
Epidermal Growth Factor Is a Neuronal Differentiation Factor for Retinal Stem Cells In Vitro
Stem Cells,
March 1, 2006;
24(3):
696 - 706.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
D. N. Abrous, M. Koehl, and M. Le Moal
Adult Neurogenesis: From Precursors to Network and Physiology
Physiol Rev,
April 1, 2005;
85(2):
523 - 569.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
R. Glass, M. Synowitz, G. Kronenberg, J.-H. Walzlein, D. S. Markovic, L.-P. Wang, D. Gast, J. Kiwit, G. Kempermann, and H. Kettenmann
Glioblastoma-Induced Attraction of Endogenous Neural Precursor Cells Is Associated with Improved Survival
J. Neurosci.,
March 9, 2005;
25(10):
2637 - 2646.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
Y. Hirabayashi, Y. Itoh, H. Tabata, K. Nakajima, T. Akiyama, N. Masuyama, and Y. Gotoh
The Wnt/{beta}-catenin pathway directs neuronal differentiation of cortical neural precursor cells
Development,
June 15, 2004;
131(12):
2791 - 2801.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. Hsieh, J. B. Aimone, B. K. Kaspar, T. Kuwabara, K. Nakashima, and F. H. Gage
IGF-I instructs multipotent adult neural progenitor cells to become oligodendrocytes
J. Cell Biol.,
January 5, 2004;
164(1):
111 - 122.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
H. Shan, M. L. Messi, Z. Zheng, Z.-M. Wang, and O. Delbono
Preservation of motor neuron Ca2+ channel sensitivity to insulin-like growth factor-1 in brain motor cortex from senescent rat
J. Physiol.,
November 15, 2003;
553(1):
49 - 63.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
R. Ajo, L. Cacicedo, C. Navarro, and F. Sanchez-Franco
Growth Hormone Action on Proliferation and Differentiation of Cerebral Cortical Cells from Fetal Rat
Endocrinology,
March 1, 2003;
144(3):
1086 - 1097.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
Y. Arsenijevic, N. Taverney, C. Kostic, M. Tekaya, F. Riva, L. Zografos, D. Schorderet, and F. Munier
Non-neural Regions of the Adult Human Eye: A Potential Source of Neurons?
Invest. Ophthalmol. Vis. Sci.,
February 1, 2003;
44(2):
799 - 807.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
C. Vicario-Abejon, M. J. Yusta-Boyo, C. Fernandez-Moreno, and F. de Pablo
Locally Born Olfactory Bulb Stem Cells Proliferate in Response to Insulin-Related Factors and Require Endogenous Insulin-Like Growth Factor-I for Differentiation into Neurons and Glia
J. Neurosci.,
February 1, 2003;
23(3):
895 - 906.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. V.T. Lobo, F. J. M. Alonso, C. Redondo, M. A. Lopez-Toledano, E. Caso, A. S. Herranz, C. L. Paino, D. Reimers, and E. Bazan
Cellular Characterization of Epidermal Growth Factor-expanded Free-floating Neurospheres
J. Histochem. Cytochem.,
January 1, 2003;
51(1):
89 - 103.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
T. Shingo, S. T. Sorokan, T. Shimazaki, and S. Weiss
Erythropoietin Regulates the In Vitro and In Vivo Production of Neuronal Progenitors by Mammalian Forebrain Neural Stem Cells
J. Neurosci.,
December 15, 2001;
21(24):
9733 - 9743.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
Y. Arsenijevic, S. Weiss, B. Schneider, and P. Aebischer
Insulin-Like Growth Factor-I Is Necessary for Neural Stem Cell Proliferation and Demonstrates Distinct Actions of Epidermal Growth Factor and Fibroblast Growth Factor-2
J. Neurosci.,
September 15, 2001;
21(18):
7194 - 7202.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M.-C. Amoureux, B. A. Cunningham, G. M. Edelman, and K. L. Crossin
N-CAM Binding Inhibits the Proliferation of Hippocampal Progenitor Cells and Promotes Their Differentiation to a Neuronal Phenotype
J. Neurosci.,
May 15, 2000;
20(10):
3631 - 3640.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. A. I. Aberg, N. D. Aberg, H. Hedbacker, J. Oscarsson, and P. S. Eriksson
Peripheral Infusion of IGF-I Selectively Induces Neurogenesis in the Adult Rat Hippocampus
J. Neurosci.,
April 15, 2000;
20(8):
2896 - 2903.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. Morrione, G. Romano, M. Navarro, K. Reiss, B. Valentinis, M. Dews, E. Eves, M. R. Rosner, and R. Baserga
Insulin-like Growth Factor I Receptor Signaling in Differentiation of Neuronal H19-7 Cells
Cancer Res.,
April 1, 2000;
60(8):
2263 - 2272.
[Abstract]
[Full Text]
|
|
|
|
|
|
|
B Diaz, J Serna, F De Pablo, and E. de la Rosa
In vivo regulation of cell death by embryonic (pro)insulin and the insulin receptor during early retinal neurogenesis
Development,
January 4, 2000;
127(8):
1641 - 1649.
[Abstract]
[PDF]
|
|
|
|
|
|
|
D. A. Lim and A. Alvarez-Buylla
Interaction between astrocytes and adult subventricular zone precursors stimulates neurogenesis
PNAS,
June 22, 1999;
96(13):
7526 - 7531.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. M. Daadi and S. Weiss
Generation of Tyrosine Hydroxylase-Producing Neurons from Precursors of the Embryonic and Adult Forebrain
J. Neurosci.,
June 1, 1999;
19(11):
4484 - 4497.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
Top
Abstract
Introduction
