hb-gam inhibits proliferation and enhances differentiation of neural stem cells

www.elsevier.com/locate/ymcne

Mol. Cell. Neurosci. 26 (2004) 75–88

HB-GAM inhibits proliferation and enhances differentiation of

neural stem cells$

Anni Hienola,a,* Mari Pekkanen,b Erkki Raulo,a Paivi Vanttola,a and Heikki Rauvalaa,*

aNeuroscience Center, Department of Biosciences and Institute of Biotechnology, University of Helsinki, Helsinki 00014, FinlandbFinnzymes Oy, 02201 Espoo, Finland

Received 15 October 2003; revised 23 January 2004; accepted 30 January 2004

Available online 27 March 2004

Proliferation of neural stem cells in the embryonic cerebral cortex is

regulated by many growth factors and their receptors. Among the key

molecules stimulating stem cell proliferation are FGF-2 and the FGF

receptor-1. This ligand-receptor system is highly dependent on the

surrounding heparan sulfates. We have found that heparin-binding

growth-associated molecule (HB-GAM, also designated as pleiotro-

phin) regulates neural stem cell proliferation in vivo and in vitro.

Deficiency of HB-GAM results in a pronounced, up to 50% increase in

neuronal density in the adult mouse cerebral cortex. This phenotype

arises during cortical neurogenesis, when HB-GAM knockout embryos

display an enhanced proliferation rate as compared to wild-type

embryos. Further, our in vitro studies show that exogenously added

HB-GAM inhibits formation and growth of FGF-2, but not EGF,

stimulated neurospheres, restricts the number of nestin-positive neural

stem cells, and inhibits FGF receptor phosphorylation. We propose that

HB-GAM functions as an endogenous inhibitor of FGF-2 in stem cell

proliferation in the developing cortex.

D 2004 Elsevier Inc. All rights reserved.

Introduction

Early development of the mammalian cerebral cortex can be

roughly divided into two phases: the establishment of the founder

stem cell population in the ventricular epithelium by symmetrical

stem cell divisions, and the neurogenetic interval, when post-mitotic

migrating neurons are born by asymmetric stem cell divisions

(Rakic, 1995; Takahashi et al., 1994). Cell cycle length and the

number of symmetrical and asymmetrical divisions are strictly

regulated. The differentiation rate of born neural stem cells is also

heavily controlled and very minor changes in proliferation kinetics

1044-7431/$ - see front matter D 2004 Elsevier Inc. All rights reserved.

doi:10.1016/j.mcn.2004.01.018

$ Supplementary data associated with this article can be found, in the

online version, at doi:10.1016/j.mcn.2004.01.018.

* Corresponding authors. Neuroscience Center, Department of Bio-

sciences and Institute of Biotechnology, University of Helsinki, Viikinkaari

4, PO Box 56, Helsinki 00014, Finland. Fax: +358-9-19157620.

E-mail addresses: [email protected] (A. Hienola),

[email protected] (H. Rauvala).

Available online on ScienceDirect (www.sciencedirect.com.)

can result in astonishing changes in cell number and laminar thick-

ness (Caviness et al., 1995; Rakic, 1995; Takahashi et al., 1997).

The molecular mechanisms regulating the final number and

quality of appearing cortical cells and cell connections are very

complex and poorly understood. Ubiquitous growth factors like

FGFs are known to function in growth and differentiation of cortical

neurons and glia in a very general manner (for examples and

reviews, see Burrows et al., 1997; Junier, 2000; Tropepe et al.,

1999). The concept of stem cell ‘‘niche’’ has become important in

discussions about stem cell regulation. In developing cerebral

cortex, an important part of this niche may be the extracellular

matrix (ECM), which functions as a dynamic local storage for

growth factors and may regulate their binding to cell surfaces.

Heparin-binding growth-associated molecule (HB-GAM, pleio-

trophin, OSF-1) was initially isolated as a neurite outgrowth-

promoting factor from perinatal rat brain (Merenmies and Rauvala,

1990; Rauvala, 1989). It is a basement membrane-associated

protein in embryonic nonneural tissues (Mitsiadis et al., 1995)

and associates with growing axonal pathways in developing brain

(Kinnunen et al., 1999; Rauvala et al., 1994). HB-GAM expression

in the developing cerebral cortex starts at the onset of neurogenesis

(embryonic days 11–12 in mouse), when it is abundant in the

ventricular zone (VZ) of the cortical lobes and the medial gangli-

onic eminence. There it is localized in the radially orientated

processes of neuroepithelial cells (Kinnunen et al., 1998a,b; Rau-

vala et al., 1994; Silos-Santiago et al., 1996). The prominent

expression of HB-GAM in the germinal zones would be compatible

with a role in cortical development, but this possible function

remains unexplored.

HB-GAM consists of two thrombospondin type 1 (TSR)

domains flanked by lysine-rich N- and C-termini that are random

coils in solution (Kilpelainen et al., 2000). The TSR domains are

most likely responsible for binding to heparan sulfates and to the

cell surface, while the role of the unstructured lysine tails is

unknown (Kilpelainen et al., 2000). HB-GAM has a close relative,

midkine (MK), that was isolated and cloned as a retinoic acid-

induced differentiation factor (Matsubara et al., 1990; Nurcombe et

al., 1992). The in vivo functions of HB-GAM are still largely

unsolved, although it is known to affect adult hippocampal plastic-

ity in rodents (Amet et al., 2001; Lauri et al., 1996, 1998; Pavlov et

al., 2002).

A. Hienola et al. / Mol. Cell. Neurosci. 26 (2004) 75–8876

HB-GAM-induced effects on neurite outgrowth and synaptic

plasticity are mediated at least partially by the heparan sulfate

proteoglycan syndecan-3, also designated as N-syndecan (Carey et

al., 1992; Lauri et al., 1999; Raulo et al., 1994). This receptor

signals further through the cortactin–src-kinase pathway (Kinnu-

nen et al., 1996, 1998a,b), inducing cytoskeletal changes. HB-

GAM has a high affinity to heparan sulfate chains similar to those

which also bind FGF-2. In fact, HB-GAM and FGF-2 compete for

binding to syndecan-3 in vitro (Kinnunen et al., 1996; Raulo et al.,

1994). Both of them also bind to phosphacan, an abundant ECM

molecule in the brain and an extracellular splice variant of another

known receptor for HB-GAM, receptor type protein tyrosine

Fig. 1. Neuronal density is increased in the parietal cortex of adult HB-GAM kno

HB-GAM�/� mice populate all cortical layers. (B) However, most of the excess

increased in the cell population that forms the major group of neurons as regardin

and knockout cortices stained with hematoxylin and eosin. The photographs also s

(E) The relative difference in the density of glutamatergic neurons resembles the re

mice. (F) The number of GABAergic cells has not dramatically changed in the H

layers II– IV. Note that the samples in A, E, and F were photographed and measur

methods). In A, HB-GAM wild type n = 5, knockout n = 4. In E and F, HB-GA

calculated with single-factor ANOVA.

phosphatase h/~ (RPTPh/~ , Maeda et al., 1996, Milev et al.,

1998a,b). This suggests a possible interplay between FGF-2 and

HB-GAM also in vivo. The structure, biochemical properties, and

functional roles of HB-GAM have been recently reviewed (Rau-

vala and Peng, 1997; Rauvala et al., 2000).

The gene encoding HB-GAM has been knocked out and the

resulting homozygous knockout mice (HB-GAM�/�) show a

normal gross morphological phenotype (Amet et al., 2001; Pavlov

et al., 2002). We searched in the present study for morphological

anomalies in the HB-GAM knockout brain and studied the behavior

of HB-GAM knockout forebrain cells in vivo and in vitro. We found

out that although the gross brain morphology appears normal, the

ckout animals compared to the wild-type cortex. (A) The excess neurons in

cells are found in layers II– III and IV. (C) The neuron number is mainly

g the nuclear volume. (D) Coronal paraffin sections of HB-GAM wild-type

how the determination of columnar layers used in the morphometrical study.

lative difference in the total cell density between the wild-type and knockout

B-GAM knockout cortex; however, there is a slightly increased density in

ed with differently calibrated microscope imaging setups (see Experimental

M wild type n = 7, knockout n = 5. Error bars show SD; P values were

A. Hienola et al. / Mol. Cell. Neurosci. 26 (2004) 75–88 77

neuronal number is in fact increased in the adult cerebral cortex of

the HB-GAM knockout mice compared to their wild-type litter-

mates. This phenotype is due to enhanced proliferation of ventric-

ular zone neural stem cells in the early embryonic cortex and

possibly delayed maturation of neurons in the postnatal brain.

Our experiments in vitro show that both recombinant HB-GAM

and its truncated form containing only the TSR domains (di-TSR-

fragment) can inhibit proliferation and induce differentiation in

FGF-2-stimulated neural stem cells. Furthermore, while FGF-2

stimulation of neural stem cells increases the phosphorylation of

FGF receptor, exogenous HB-GAM and its di-TSR-fragment re-

duce this phosphorylation.

Fig. 2. In the perinatal brain, cell density differences are visible between HB-G

thickening of VZ and SVZ in E16 cortex and (B) increased neuronal density in

increased proliferation in E12.5 HB-GAM knockout cortex. (E) The bars show B

Error bars show SD, P values calculated with single-factor ANOVA. Scale bars:

zone; Hi, ventricular zone of hippocampal formation; GE, ganglionic eminence. Ph

taken with 63� objective.

Based on our findings, we postulate that inactivating HB-GAM

in vivo removes an important differentiation-regulating factor from

the developing brain, thus allowing enhanced proliferation of neural

stem cells. This restrictive behavior is likely to be tightly linked to

the proteoglycan environment in the surface of, or surrounding,

neural stem cells. We suggest that HB-GAM specifically inhibits

FGF-2’s function via FGF receptor both in vitro and in vivo.

In this paper, we refer to cells isolated from embryonic mouse

forebrain and cultured as neurospheres as neural stem cells (for

discussion on stem cell or progenitor cell definitions, see Seaberg

and van der Kooy, 2003). Under culture conditions described in this

work, these cells continue self-renewing proliferation and formation

AM knockout and wild-type animals. (A) Lack of HB-GAM results in

the SVZ of P3 pups. (C and D) Six hours of BrdU administration reveals

rdU+ cell densities in E12.5 cortex. HB-GAM�/�, n = 5; wild type, n = 5.

A, B, and D = 100 Am, C = 200 Am. LV, lateral ventricle; VZ, ventricular

otos A, B, and C were taken with 20� objective, A and C are collases. D is

Fig. 3. HB-GAM and its di-TSR-fragment inhibit forebrain cell proliferation and neurosphere formation. (A) HB-GAM contains two domains that are

homologous to the TSR repeat. The TSR domains are connected with a flexible linker and flanked by lysine-rich N- and C-terminal tails marked as p-Lys. The

di-TSR-fragment is the truncated form of HB-GAM, missing the lysine-rich tails. Midkine, used as a control in vitro, has the same domain arrangement and a

very similar secondary structure (for discussion, see Kilpelainen et al., 2000). (B) HB-GAM knockout cells derived from E12.5 embryos proliferate more than

wild-type cells when assayed with BrdU incorporation for 24 h. (C) Exogenous HB-GAM and its di-TSR-fragment cause a modest inhibition in proliferation of

E12.5 cortical cultures measured by 24 h BrdU incorporation in vitro. (D) The FGF-2-driven clone formation is totally blocked by HB-GAM and can be

reverted by adding heparin. The values below the figure are based on three experiments where clonal growth was followed for 5 days. The photographs show

the biggest clonal cell formations found among the observed clones. Scale bar = 50 Am; photos were taken with 20� objective. (E) Dose-dependent inhibition

by HB-GAM was demonstrated with cells derived from FGF-2- and EGF-stimulated cultures. Bars show relative change in clone formation compared to

cultures without HB-GAM. Error bars in A show SD (n = 3) and in E the variation of means (n = 2); P values in A are calculated with single-factor ANOVA.



of clonal neurospheres at least for six passages from isolation. The

cells, when plated on laminin, are nestin positive, respond to FGF-2

and EGF, and generate both neurons and glia.

Fig. 4. HB-GAM induces differentiation of neural stem cells. The

maintenance of neurospheres on non-coated glass was determined by

observing the differentiation of the spheres. (a) Shows a neurosphere with

undifferentiated morphology and (b) shows a sphere that has differentiated.

Based on morphological differences, the number of undifferentiated spheres

was counted after 3 days in culture under different conditions: without

FGF-2, with FGF-2, with FGF-2 and HB-GAM, and with FGF-2, HB-

GAM, and heparin. Scale bar = 50 Am; photos were taken with 20�objective. Error bars show SD (n = 3), except for the assay with heparin,

which gave in two independent experiments the percentage values 76.0 and

76.7; P values were calculated with single-factor ANOVA.

Results

Disruption of HB-GAM gene causes an increased neuronal density

in the cerebral cortex

In adult HB-GAM-deficient mice, the gross morphology of the

brain appeared normal as has been previously described (Amet et al.,

2001). When a more detailed stereological analysis of both parietal

and hind limb motor cortex was carried out, an average increase of

39% in neuronal density was found. The increase was detectable

throughout the whole cortical thickness but was most obvious in

layers II– IV (54% increase) and VI (31% increase, Figs. 1A and

1B). The neuronal identity of the counted cells was verified with

anti-GFAP and anti-NF immunostaining. We also calculated the size

distribution curve for the counted andmeasured nuclei of the cortical

cells (Fig. 1C). This showed that the excess cells in the knockout

mice belonged to the size-group that was the most populated also in

the wild-type mice, with nuclear volume ranging from 350 to 750

Am3. The neuronal number in this particular size-group was almost

doubled in the knockout mice in comparison with the wild type. No

increase in the number of the very large pyramidal neurons (>1000

Am3) populating the lamina V could be seen. This may be due to the

relative scarcity of these cells.

We further examined the distribution of different neuronal

subtypes in the cortical laminae. We used GABA and glutamate

receptor 2 immunopositivity for distinguishing between interneur-

ons and pyramidal neurons. We could see an increase in the density

of both of these neuronal types, but the increase in the density of

the GABAergic neurons was less prominent than the density of

glutamatergic neurons. Moreover, the density increase in GABA+

cells was only visible in the high laminae II– IV, whereas the

density of glutamatergic neurons was increased in all layers of the

knockout cortex in approximately the same proportions as the

measured total density (Fig. 1E). The size distribution based on

nuclear volume of the glutamatergic neurons was similar to that

observed in the whole cortical population, meaning that excess

neurons were once again found in the most populated size groups

and only some increase was found in the group of very large

pyramidal neurons populating lamina V (data not shown).

During this more careful histological examination of the adult

cortex, we could not find anything unusual in the structure or

morphology of either neurons or glial cells. Bielschowsky-silver-

staining and immunohistochemistry with anti-neurofilament (NF,

200, 145, and 68 kDa, from Chemicon) and glial fibrillary acidic

protein (GFAP, from Chemicon) antibodies revealed no abnormal-

ities in the distribution of neurites or axonal pathways in the cortex

(not shown). However, based on neurofilament expression, the

neuronal differentiation is delayed in HB-GAM�/� mice (see

Supplementary Fig. 1).

Enhanced cell proliferation in the germinal zones of

HB-GAM�/� embryos

Histology of embryonic (E16) and postnatal (P3) brain sug-

gested increased cell numbers in the cerebral cortex (Figs. 2A and

2B). Immunostaining with an M-phase cell cycle marker, phos-

phorylated histone 3, showed an increase in the density of mitotic

cells in E12 knockout embryos (0.55 F 0.06 � 106 1/mm3, n = 3)

in comparison with wild-type littermate samples (0.32 F 0.04 �106 1/mm3, n = 3). This type of assay, however, only shows the

number of mitotic cells at the time point of sample preparation and

is thus unreliable in estimation of the proliferation rate.

To study more carefully the in vivo stem cell proliferation, we

gave 5-bromo-2V-deoxy-uridine (BrdU) injections to timed-preg-

nant HB-GAM+/� females for 6 h at E12.5. The injection protocol

was based on a study by Takahashi et al., 1996, where the authors

determined the length of the stem cell cycle in the mouse embryonic

VZ at different embryonic ages. The BrdU incorporation time used

in our protocol should result in labeling of all VZ cells undergoing

cell divisions at embryonic day 12.5. A visible increase was found

in the BrdU+ cell density in the HB-GAM knockout ventricular

zone when compared to the wild type (Figs. 2C and 2D). The BrdU+

cell density was on the average 50% higher in the knockouts than in

the wild types (1.84� 106/mm3 F 0.22� 106 and 1.23� 106/mm3

F 0.17 � 106, respectively). The 6-h BrdU administration thus

labeled 60% of the total amount of cells in the knockout and only

38% in the wild-type VZ (Fig. 2E).

HB-GAM and its di-TSR-fragment inhibit proliferation of

embryonic mouse forebrain cells

We compared the amount of BrdU incorporation in the HB-

GAM knockout and wild-type cortical cultures and saw a clear


increase of 55% in the number of BrdU+ knockout cells (Fig. 3B).

This finding together with the phenotype of the adult HB-GAM

knockout mice raised the question of whether exogenously added

recombinant HB-GAM would induce proliferation arrest and

change the differentiation fates of neural stem cells in vitro. It

was also interesting to see if HB-GAM’s di-TSR-fragment (Fig.

3A) would act in a similar manner as HB-GAM in vitro. E12.5

forebrain cells from HB-GAM knockout embryos were cultured for

24 h in the presence or absence of recombinant HB-GAM, HB-

GAM’s di-TSR-fragment, or midkine, and the amount of cell

proliferation was measured. The proliferation inhibition caused

by recombinant HB-GAM or its di-TSR-fragment within this time

period was less pronounced (30%) than what was expected based

on the knockout phenotype and BrdU incorporation in vivo (Fig.

3C). On the other hand, midkine did not induce any changes at all

as compared to the baseline proliferation (Fig. 3C).

HB-GAM inhibits clone formation in FGF-2 but not in EGF

stimulated neural stem cells

From previous studies, we knew that HB-GAM can compete

with FGF-2 in heparin binding (see Introduction and Discussion)

and that FGF-2 is one of the key factors regulating stem cells in

early VZ. We isolated neural stem cells as neurospheres from

Fig. 5. HB-GAM and its di-TSR-fragment cause growth inhibition on neurospher

grow for 5 days, after which the colonies were photographed and fixed for immu

neurospheres form large discs of cells migrating away from the central neurospher

discs did not spread but stayed compact and sent only few radial processes. When

clearly diminished. Again, adding midkine to the neurosphere solution did not ha

mouse embryos to see if HB-GAM could induce a more pro-

nounced growth arrest in FGF-2-stimulated stem cells than it had

in primary forebrain cells. FGF-2 was used to induce neurosphere

formation and to maintain the stem cells during the assays. We

used EGF-stimulated neurospheres to control the specificity of the

effects of HB-GAM. The concentration of FGF-2 that induced

most efficient clonal growth in our experiments was determined to

be between 1 and 10 ng/ml in a titration assay (data not shown).

Based on this finding and the available literature, we chose the

concentration of FGF-2 to be 10 ng/ml and of EGF 20 ng/ml

(Hulspas et al., 1997).

First we derived neurospheres with FGF-2 as the only stimu-

lating growth factor. The cells were plated in clonal densities in the

presence of FGF-2 and additional HB-GAM or midkine. The FGF-

2-stimulated cells failed to grow any clones in the presence of HB-

GAM after 5 days of observation. With FGF-2 alone, the clonal

growth took place (Fig. 3D). The inhibitory effect of HB-GAM

could be reverted by adding heparin to the culture medium.

Midkine did not display a significant effect on the formation of

spheres in this assay.

Our next experiment was to demonstrate the dose dependency

of HB-GAM-induced proliferation arrest in FGF-2-stimulated stem

cells. We produced neurospheres using both FGF-2 and EGF as

stimulating growth factors in the culture medium. The cells were

e discs grown on laminin. Neurospheres were plated on laminin and let to

nostaining. In the presence of neurosphere medium containing FGF-2, the

e core. When the medium contained no growth factor or just HB-GAM, the

HB-GAM was added to the normal neurosphere medium, the disc size was

ve any effect on disc size or appearance. Scale bar is 100 Am.

Fig. 6. Reduction in the number of nestin-positive cells in neurosphere

cultures induced by HB-GAM and its di-TSR-fragment. FGF-2-stimulated

neurospheres grow large stem cell discs on laminin. The number of nestin-

positive cells in these discs is diminished in HB-GAM or TSR-di-domain

containing stem cell cultures in comparison with FGF-2-stimulated cultures.

This inhibition can be reverted by adding heparin. Using MK in the

medium does not change the number of nestin+ cells. Scale bars = 50 Am;

photos were taken with 63� objective. Error bars show SD (n = 4); P value

was calculated with single-factor ANOVA.


cultured in clonal densities with single growth factor and different

concentrations of HB-GAM. The clonal growth was followed for 7

days. HB-GAM had a clear dose-dependent inhibition on FGF-2-

stimulated clone formation, and although EGF-stimulated clone

formation was also slightly inhibited, there was no dose depen-

dency in the effect of HB-GAM on EGF-induced neurospheres

(Fig. 3E).

HB-GAM and its di-TSR-fragment induce differentiation in neural

stem cells

To study the differentiation of neurospheres, we cultured them on

object glasses with additional HB-GAM or its di-TSR-fragment.

Neurospheres plated on non-coated glass did not spread but kept

their round morphology in the presence of FGF-2. FGF-2-starved

spheres diminished in size and flattened loosing their round,

undifferentiated morphology within a few days. At 10-fold molar

concentration, HB-GAM and the di-TSR-fragment both inhibited

the stem cell supporting effect of FGF-2 and induced neurosphere

differentiation similar to the cultures, where no FGF-2 was present

(Fig. 4). After 3 days under these conditions, 75.2(F6.0)% of FGF-

2-stimulated neurospheres still possessed their undifferentiated

morphology, in FGF-2-starved and HB-GAM-containing cultures,

only 36.1(F11.1)% and 47.6(F3.4)% of the spheres, respectively,

were undifferentiated. When heparin was added to the HB-GAM-

containing cultures to reverse its effect, the number of undifferen-

tiated neurospheres stayed at the level of FGF-2-stimulated cultures

(76.7%).

On laminin-coated glasses the FGF-2-stimulated neurospheres

rapidly spread and produced a growing disc of cells which were

initially more than 90% nestin positive (data not shown). When

neurospheres were plated without FGF-2 or with HB-GAM or the

di-TSR-fragment, the stem cell discs were after 5 days clearly

smaller (Fig. 5) and possessed significantly less nestin-positive

cells (Fig. 6). This clearly demonstrated HB-GAM’s inhibitory

effect on neural stem cells and showed that HB-GAM participates

in the onset of differentiation in these cells. Remarkably, MK did

not have any effect on the disc size or the number of nestin+ cells

(Figs. 5 and 6). The effect of HB-GAM on the number of nestin+

cells was reversed by low concentrations of heparin (Fig. 6).

EGF-stimulated neurospheres in the same conditions did not

show any variations in the proportions of nestin+ cells (data not

shown).

To see the possible changes in the differentiation pattern of these

cells, we immunostained the fixed spheres with beta-tubulin and

GFAP antibodies (Chemicon). The number of GFAP+ cells did not

differ between FGF-2 and FGF-2–HB-GAM cultured cells (59.8F10.3% and 68.5F 6.0%, respectively, n = 4). On the other hand, the

relative number of h-tubulin+ cells was elevated in FGF-2–HB-

GAM cultures (with FGF-2, 27.9 F 4.8%; and with FGF-2–HB-

GAM, 47.3 F 7.0%, n = 4, P = 0.012).

HB-GAM reduces phosphorylation of FGF receptor in

FGF-2-stimulated neural stem cells

We examined the direct effect of HB-GAM on FGF receptor

activation in FGF-2-stimulated neural stem cells. After 15 min of

stimulation with FGF-2, about 200% increase in phospho-FGFR

receptor level was detected. If HB-GAM or the di-TSR-fragment

was present in the culture medium during stimulation, the phos-

phorylation was diminished to only 78% or 62% of the level of

FGF-2 stimulation, respectively (Fig. 7). The same band detected

with anti-phospho-FGFR was also phospho-tyrosine immunopos-

itive (Fig. 7).

HB-GAM does not affect apoptotic cell death in vivo or in vitro

Histological sections never revealed any cellular abnormalities

in the HB-GAM knockout cortex, besides the increased neuronal

density, and adding HB-GAM or the di-TSR-fragment in vitro on

neural stem cells did not induce cell death as observed by phase-

contrast microscopy. To rule out the possibility of diminished

apoptotic cell death in the cerebral cortex of the knockout mice,

Fig. 7. HB-GAM inhibits phosphorylation of the FGF receptor. Neural stem

cells plated on laminin were first deprived of growth factor and then

stimulated with FGF-2 for 15 min. In total cell lysates the a-phospho-FGFR

antibody detected an increased signal in FGF-2-stimulated cells as

compared to nonstimulated controls. HB-GAM and di-TSR-fragment

prevented the increase. a-phospho-tyrosine immunoblotting detected a

band of the same size (120 kDa) as a-phospho-FGFR and showed a similar

variation in the level of phosphorylation. a-FGFR1 immunoblotting was

used to verify the constant amount of FGFR1 in all samples. Error bars

show SD (n = 3); P values were calculated with paired t test for means.


we stained E12, E16, and P10 brain sections and embryonic

primary neurons in culture with anti-active caspase-3 antibody.

Activation of caspase-3 is considered to be an irreversible phase in

the complex protein activation cascade that leads to programmed

cell death (Grutter, 2000). Seven to 10 sample sections spanning the

whole cortical hemispheres from four animals per genotype were

averaged. At E12, 3.6 F 1.2 and 3.5 F 1.2 apoptotic figures per

section were detected in wild-type and knockout animals, respec-

tively. The corresponding figures at E16 were 3.4 F 0.3 and 3.4 F0.5, and at P10 13.1 F 2.9 and 12.0 F 2.1 in wild-type and

knockout animals. Similarly, in vitro apoptotic activity during HB-

GAM treatment stayed the same as in control cultures: 16.9F 1.3%

of HB-GAM-treated and 14.5F 5.7% of untreated cells were active

caspase-3 positive.

Discussion

In this study, we show that depleting HB-GAM in mice results in

increased neuronal density in the cerebral cortex. The increase in the

laminae II– IV can exceed 50%, which we think is a very significant

change. The magnitude of this phenotype indicates an important

regulatory role for HB-GAM in cortical development. We also show

that exogenous recombinant HB-GAM and its heparin-binding di-

TSR-fragment can inhibit neural stem cell proliferation and neuro-

sphere formation in vitro, and induce differentiation towards a

neuronal phenotype. We also show that this inhibition is rather

specific for FGF-2-responsive neural stem cells because a similar

inhibition was not seen in EGF-derived neural stem cells. In

addition, FGF-2-induced FGF receptor phosphorylation detected

in stem cell lysates is reduced by exogenous HB-GAM and its di-

TSR-fragment.

We suggest that the mechanism of inhibition is likely due to

competition between HB-GAM and FGF-2 in binding to proteo-

glycan co-receptor(s) required for FGF-2-induced proliferation. We

base this claim on several findings presented both in this paper and

in studies by others: first, the knockout phenotype described in the

present study is essentially a reverse situation of the phenotype

found in the FGF-2 knockout (Dono et al., 1998; Raballo et al.,

2000). In the FGF-2 knockout, a decreased neuronal density has

been found essentially in the same layers where we have found an

increased neuronal density in the present study.

Second, the present paper demonstrates that HB-GAM and its

heparin–heparan sulfate binding di-TSR-fragment are able to in-

hibit neural stem cell proliferation and suppress FGF-2-driven

neurosphere formation to the level seen during growth factor

deprivation. The effect on neural stem cell growth is clearly

dependent on heparan sulfate proteoglycans because the growth

arrest can be reversed with low concentrations of heparin. Further,

no such inhibition can be seen in the EGF-stimulated neural stem cell

cultures.

Third, HB-GAM and the di-TSR-fragment also reduce FGF

receptor phosphorylation in FGF-2-stimulated neural stem cells. In

addition, HB-GAM and its di-TSR-fragment reduce the number of

nestin-immunopositive cells in neural stem cell cultures, indicating

the start of differentiation of these cells. This differentiation is

directed towards the neuronal phenotype, indicated by h-tubulinexpression. Specificity of the effect is suggested by the unexpected

finding that the closest HB-GAM homologue midkine that binds to

heparin–heparan sulfates as strongly as HB-GAM does not inhibit

proliferation of the stem cells or diminish the number of nestin-

expressing cells. Interestingly, HB-GAM has been previously

linked to cell differentiation at a more general level in limb bud

development and chondrogenesis involving FGF-2 (Dreyfus et al.,

1998; Szabat and Rauvala, 1996).

Fourth, HB-GAM binds strongly to heparin and co-purifies with

FGF-2 in salt gradient elution from heparin-Sepharose (Rauvala,

1989). Naturally occurring heparan sulfates contain a wide variety

of carbohydrate epitopes binding growth factors and matrix mole-

cules; the HB-GAM-binding carbohydrate epitope overlaps with

that binding FGF-2 (Kinnunen et al., 1996). In addition, previous in

vitro results suggest competition in the binding of HB-GAM and

FGF-2 to heparin–heparan sulfate (Raulo et al., 1994; Szabat and

Rauvala, 1996). In particular, HB-GAM and FGF-2 display a

similar binding to brain-derived N-syndecan and are competitive

in direct binding assays (Raulo et al., 1994).

Finally, HB-GAM is expressed in the rodent brain up to 100-

fold molar excess as compared to FGF-2 (Rauvala, 1989). From

this viewpoint and considering high local expression of HB-GAM

in the germinal zones at the stage when FGF-2 is an essential

proliferation-enhancing factor (Rauvala et al., 1994), it seems

obvious that the expression level of HB-GAM in early develop-


ing brain should be high enough to brake FGF-driven prolifera-

tion. Our finding that the density of both GABAergic and

glutamatergic neurons is increased in the knockout mice supports

the idea about HB-GAM’s general inhibitory function in the

germinal areas of the early brain. These two distinct types of

cortical neurons have separate sources—glutamatergic pyramidal

neurons are born in the VZ underlying their future cortical

column, to where they will migrate radially, and GABAergic

interneurons arise from the ventral parts of the cortex, migrating

tangentially to their target areas. However, the HB-GAM muta-

tion has a more prominent effect on the glutamatergic population,

suggesting that in vivo HB-GAM has more impact on the VZ

stem cells during neurogenesis.

The proliferation phenotype that we have described in this

study could reflect either a shorter stem cell cycle or a decreased

number of cells escaping the stem cell cycle, or a combination of

these two. Our methods cannot, unfortunately, distinguish be-

tween these two alternatives. The number of cells in the M-phase

at E12 was clearly elevated in HB-GAM knockout embryos, and

labeling the whole proliferating cell population with BrdU

revealed an increase of similar magnitude. In both of these

assays, the results could be due to an increase in the total number

of proliferating cells or to a decrease in the cell cycle length,

which increases the probability of an M-phase cell to be captured

by the assay. A more sophisticated method involving prolonged

labeling of the proliferating populations with two separate mitotic

markers (Caviness et al., 1999) should be used to determine the

Fig. 8. Function of HB-GAM in neural stem cell proliferation arrest and in

proteoglycans regulates dynamically the amount of glycosaminoglycan (GAG)-b

growth factors, including FGF-2. FGF-2 binding to its receptor FGFR1 is regulat

proteoglycans like phosphacan and RPTPh/~ . The activity of FGFR1 keeps the ne

stem cells, it is able to displace FGF-2 from the proteoglycan co-receptor(s) and

binding to N-syndecan and RPTPh/~ . Cell signaling upon HB-GAM binding to the

src-kinase pathway (Kinnunen et al., 1998a,b; Maeda and Noda, 1998). ECM, ex

cell cycle length. Because FGF-2 is known to simultaneously

decrease the G1 duration in stem cells and increase the proportion

of proliferative divisions (Lukaszewicz et al., 2002) and HB-

GAM inhibits FGF-2, it seems reasonable that HB-GAM may

affect the same parameters as FGF-2.

Our observations speak for the importance of glycosamino-

glycan (GAG) epitopes in the regulation of neural stem cells. It

has been shown that several proteins associated with neural

growth bind brain-derived GAGs (Herndon et al., 1999). This

raises the questions of whether there are any differences in the

functional specificity between different GAG epitopes and what

the carrier proteins for these GAGs could be. Evidence exists for

the distinction between FGF-2 and FGF-1 (aFGF) binding hep-

aran sulfate epitopes in developing murine brain (Chipperfield et

al., 2002; Joseph et al., 1996; Nurcombe et al., 1993). Syndecan

family members and perlecan are known to interact with FGF-2

and modify its function in several tissue types (Nugent and Iozzo,

2000; Tumova et al., 2000; Whitelock et al., 1996). Also,

chondroitin sulfate proteoglycans like phosphacan have been

shown to enhance growth factor function (Milev et al., 1998b).

The extracellular matrix protein phosphacan and its transmem-

brane form RPTPh/~ (receptor-type protein tyrosine phosphatase

h/~) bind HB-GAM, too, and phosphacan can reverse the

inhibitory effect of HB-GAM in neural stem cells in vitro

(Hienola and Rauvala, unpublished results). Interestingly, low

concentrations of heparin inhibit HB-GAM binding to phospha-

can (Maeda et al., 1996), suggesting a similarity in HB-GAM

neuronal differentiation and migration. A population of transmembrane

inding molecules at the cell surface. Among these molecules are several

ed strongly at least by syndecans and possibly by some chondroitin sulfate

ural stem cells in a self-renewing cell cycle. If HB-GAM is present near the

can thus inhibit FGF-2 activity. HB-GAM also induces cell signaling via

transmembrane proteoglycans is mediated, at least in part, by the cortactin–

tracellular matrix.


binding carbohydrate epitope(s) found in heparin–heparan sulfate

and phosphacan. Mouse mutants have been created at least for

the HB-GAM receptors syndecan-3 (Kaksonen et al., 2002;

Reizes et al., 2001) and RPTPh/~ (Harroch et al., 2000), and

for perlecan (Arikawa-Hirasawa et al., 1999; Hart et al., 2001).

They offer opportunities to study more closely the specific roles

of these molecules in the germinal zones of the brain.

A wide number of proteins containing TSR domains are

expressed in the developing nervous system in addition to HB-

GAM and midkine, including F-spondin, semaphorins F and G,

and thrombospondins (for review, see Adams and Tucker, 2000).

For example, TSR domains closely resembling those of HB-

GAM are found in F-spondin (Klar et al., 1992; Tzarfaty-Majar et

al., 2001). Very recent studies have suggested that the TSR

sequence motif defines a similar h-structure in different proteins

of the family and that the TSR domains mediate interactions with

heparin–heparan sulfate and the cell surface (Kilpelainen et al.,

2000; Tan et al., 2002). The present results suggest that the TSR

domains of HB-GAM specifically restrict proliferation of neural

stem cells because midkine did not display this effect. Further

studies are however warranted for understanding whether other

TSR proteins play a role in stem cell regulation in neural or

nonneural tissues.

HB-GAM promotes neurite outgrowth and migration through

binding to N-syndecan (syndecan-3) and RPTPh/~ (Imai et al.,

1998; Kinnunen et al., 1996; Maeda and Noda, 1998). The

migration-promoting effect of HB-GAM in neuronal cells

depends on signaling through the cortactin–Src-kinase pathway

(Kinnunen et al., 1998a,b). These findings suggest that HB-GAM

may have a role in the radial migration of new neurons. The

current histological analysis of HB-GAM-deficient mice supports

this view. First, HB-GAM mRNA colocalizes with RC-2 immu-

noreactivity in embryonic cortical slices (Hienola and Rauvala,

unpublished results). Second, in the prenatal phases of cortical

histogenesis, we can detect most of the excess cells in HB-GAM

knockouts in the lower layers of the cortex, for example, the SVZ

at E16, but in the adult animals, the most dramatic density

differences can be seen in the relatively high layers, namely in

II– III and IV. In addition, about 10 days after birth, we can still

see in the cortices of HB-GAM knockouts radially oriented cell

stripes that are no longer detectable in the wild-type animals and

resemble late migrating, clonally related cells (Hienola and

Rauvala, unpublished results). It therefore appears that radial

migration is delayed in the HB-GAM knockout brain. In addition,

we find it interesting that the excess GABAergic neurons in the

HB-GAM knockout cortex are localized almost exclusively in the

higher laminae II– IV. The tangential migration of interneurons

ends in an event when the cells make a decision to migrate

radially to their final destination (Nadarajah et al., 2003). Why an

excess of these neurons should end up in the upper layers in HB-

GAM knockout remains to be solved. We hope to shed more

light on these issues in a separate study characterizing the

migration phenotype in these animals.

This study reveals an important endogenous inhibitor for the very

basic stem cell regulating growth factor, FGF-2. The regulation is

most likely due to competition between FGF-2 and HB-GAM in

binding the specific GAG epitopes at the cell surfaces and in the

extracellular matrix. Fig. 8 summarizes our current view, according

to which HB-GAM binding to cell surface proteoglycans links

proliferation arrest to the start of differentiation and migration of

developing neural cells.

Experimental methods

Cell culture reagents and proteins

All cell culture experiments were done in Dulbecco’s modified

Eagle’s medium (DMEM) with additional penicillin–streptomycin

and L-glutamine. Either bovine serum albumin (BSA, 10 mg/ml) or

fetal calf serum (FCS, 10%) was added to the medium. In neuro-

sphere medium B27 cell culture supplement (Gibco) was used

together with human recombinant FGF-2 (10 ng/ml or approxi-

mately 0.6 nM, Promega) or with FGF-2 and human recombinant

EGF (20 ng/ml or approximately 3 nM, Promega).

Except for FGF-2 and EGF, the other recombinant proteins

were self-produced. Baculovirus expression system was used for

recombinant HB-GAM (Raulo et al., 1992). Recombinant midkine

and HB-GAM’s di-TSR-fragment production in Escherichia coli

and their purification were carried out as described previously

(Kilpelainen et al., 2000). Low-molecular weight heparin (Sigma)

was used at low concentrations (100 ng/ml) to specifically block

HB-GAM binding to heparan sulfates in cell cultures.

Animals and organs

The HB-GAM male knockout mice in 129/SvEv background

were crossbred with 129/SvEv females and the inbred mutant

mouse line was established by sibling breeding. In all of our

studies, littermate wild-type controls were used. Genotyping and

sample preparation for histology of adult brain samples have been

described before (Amet et al., 2001).

Brain samples from P3 and P10 pups were collected by

decapitating the pups and dissecting the brains out on a petri dish

kept on ice. For histological stainings, the brains were cut in two

parts separating the cerebellum from the rostral part of the brain.

The samples were fixed in 4% PFA and 0.5% glutaraldehyde in PBS

overnight.

HB-GAM knockout embryos for histological staining and for

isolation of cortical cells were collected from timed-pregnant

females. The pregnant mice were killed with CO2 and by cervical

dislocation, and the uteri were dissected out and placed in sterile

PBS. Embryos were cut out and either the placenta or the leftover

torso of each embryo was used for genotyping.

Material for cortical and neurosphere cultures was collected

either from HB-GAM mutant or outbred NMRI mouse embryos

aged E12.5–E14.5. The frontal cortical lobes were separated and

transferred to cell culture medium with 10% FCS. The lobes were

dissociated and homogenized by passing the cell suspension

through 20 G needles attached to a sterile syringe several times.

After this, the FCS medium was changed and the cells were left to

recover for 30 min. Cells were collected and washed twice in BSA

medium. When mixed litters of HB-GAM knockout and wild-type

embryos were used, all embryos were handled separately.

Optical dissecting—selector method

Adult brain samples were sectioned as described before (Amet et

al., 2001). From each sectioned sample, a random series was

collected and stained. The sections were photographed with oil-

immersion (Olympus AX50, 60� objective). The random sample

consisted of about 40 sections/brain and the sample area covered

about 2 mm of the cortex in rostral–caudal direction. The sample

volume was located and chosen based on the size and shape of the


dentate gyrus in hippocampal formation. Photocollases of parietal

cortices were divided into nine counting layers (Fig. 1D). Neurons

were identified during counting from hematoxylin-eosin or Nissl-

stained sections according to their nuclear morphology. In the case

of E12.5 embryos, the whole embryo was sectioned in the coronal

direction and the stereological sample volume was chosen from the

rostral tip of the brain to the caudal edge of lateral ventricles. In the

case of embryonic brain samples, the random sample consisted of

20 sections.

The photographs were measured with an image processing

software ImagePro. The optical quantification was based on

selector-method (McMillan and Sorensen, 1992) that allows one

to ignore the sample thickness (Everall et al., 1997). In selector-

method, every morphologically identified neuronal nucleus (based

on size and presence of Nissl bodies) found inside the square-

shaped probe (approximately 500 Am2) or hitting its inclusion lines

was counted and its radius was measured. The relative area (Irel)

occupied by all measured objects on the counting area was then

calculated. The shape of the nuclei was assumed to be sphere-like

and the volume of the objects was calculated based on that

assumption. The average volume (Vmean) of all counted objects

in the counting field was then used with the Irel to calculate the

neuronal density (ND = Irel / Vmean) as a number per cubic

millimeter. The observers were not aware of the genotype of

photographed and measured samples at any point of the analysis.

The measured data were afterwards grouped according to geno-

types for statistical evaluation. Single-factor analysis of variance

(ANOVA) was used to determine the significance of difference

between HB-GAM wild-type and knockout samples.

Identification of the subtypes of cortical neurons

Cortical samples consisting of 5-Am-thick sections from HB-

GAM wild-type and knockouts were stained for GABA and

glutamate receptor 2 immunopositive cells to distinguish between

the populations of interneurons and glutamatergic, pyramidal

neurons, respectively. The mouse anti-GABA (1 Ag/ml, Chemicon)

and mouse anti-GluR2 (1 Ag/ml, Zymed) antibodies were diluted in

PBS with 0.3% Triton-X and 2% BSA and the sections were

incubated in the primary antibody solution overnight in +4jC. Thesections were then incubated with biotinylated anti-mouse IgG

(Zymed) overnight in +4jC. DAB colorimetry was used for

immunodetection. The sections were photographed with oil im-

mersion (Zeiss AxioPlan 2, 63� objective). The average density of

GABA+ and GluR2+ cells in the cortical laminae II– IV, V, and VI

was estimated as described above.

Proliferation in primary cortical cultures

Cortical cultures from HB-GAM knockout and wild-type em-

bryos were plated on polystyrene 96-well microplates, and cells

were plated at 150000/ml density, that is, 15000 per well. HB-

GAM, di-TSR-fragment, or MK was added on knockout cells in the

BSA-medium at 6-nM concentration. BSA medium alone was used

as a control for base level proliferation. Cell proliferation was

measuredwith BrdUCell Proliferation Kit (Boehringer-Mannheim).

Neurosphere assays

Neurospheres were derived from NMRI mouse embryos. Cor-

tical cells were collected at E14 as described above. After recovery

period, the cells were plated on non-coated petri dishes in neuro-

sphere medium. The next day the cells were collected, triturated,

and cultured at 150000/ml cell density in the neurosphere medium.

The grown neurospheres were used in assays after two or three

passages (about a week in culture).

Clonal neurosphere formation

We derived neurospheres from primary forebrain cultures by

using either FGF-2 only or both FGF-2 and EGF as growth factors

in the medium. Spheres derived from FGF-2-stimulated cultures

were used directly in the assays after three passages in culture. The

spheres were triturated and plated on nontreated 96-well plates at a

density of 2–10 cells/well. The clonal growth was followed for 5

days in the presence of FGF-2 (0.6 nM), HB-GAM (6 nM), FGF-2,

and HB-GAM, FGF-2, HB-GAM, and heparin (100 ng/ml), and

FGF-2 and midkine (6 nM), and the number of generated clones

per 40 plated cells was counted. Dose-dependent inhibition by HB-

GAM in clonal growth was studied in cultures derived from

cultures with combined growth factors. The cells were cultured

with both FGF-2 and EGF for three passages. After this the

cultures were split in half and subcultures were continued in the

presence of either FGF-2 or EGF only. The single-growth factor

cultures were continued for extra two passages, and the cells were

triturated and plated as described above. The molar concentration

of HB-GAM varied from 0- to 100-fold with respect to the growth

factor concentration. The minimum number of cells was 100 in

each condition, and the clone formation was followed for 7 days.

Maintenance and differentiation

The experiments were carried on object glasses with eight

separate wells (1 cm2/well, Costar). Forty to 80 neurospheres per

well were plated in neurosphere medium containing FGF-2. The

absolute number of spheres per well was counted after plating.

Spheres were cultured on non-coated object glasses for 3 days in

neurosphere medium without FGF-2, with FGF-2 (0.6 nM), with

FGF-2 and HB-GAM (6 nM), or di-TSR-fragment (6 nM). Heparin

was added to block HB-GAM activity in the solution (100 ng/ml).

Neurospheres remaining morphologically undifferentiated were

counted every day (Fig. 4).

A similar assay was carried on laminin-coated (10 Ag/ml) object

glasses in the presence of either FGF-2 or EGF. In this assay also the

effect of MK (6 nM) in growth factor-stimulated cells was studied.

After 5 days in culture the spreading neurospheres were photo-

graphed. The glasses were fixed with 4% PFA in PBS for 1 h and

stained with anti-nestin (DSHB, University of Iowa, US), anti-h-tubulin (Zymed), and anti-GFAP (Chemicon) antibodies according

to the recommendations from the manufacturers. FITC and TRITC-

conjugated secondary antibodies (Molecular Probes) were used for

detection with fluorescence microscopy.

BrdU labeling and immunohistochemistry in vivo

BrdU injections were given intraperitoneally to timed-pregnant

females 12 days after detection of vaginal plug (embryonic day

12.5). BrdU (50 Ag/g, Sigma) in PBS was injected three times in 2-

h intervals (cumulative dose of 150 Ag/g), and the embryos were

collected 6 h after the first injection. Four wild-type and four

knockout embryos from both age groups were fixed with 4% PFA

in PBS overnight and embedded in paraffin. Thin sections (4 Am)


were cut, hydrated, and incubated overnight with anti-BrdU ready-

to-use solution (Amersham, UK). Biotinylated secondary antibody

was detected with peroxidase–DAB reaction. Sections were photo-

graphed and BrdU-positive cells were counted with the selector-

method (see above).

Immunostaining for M-phase cells

We used antibodies against phosphorylated histone 3 (rabbit

IgG, Upstate Biotechnology, US) as an M-phase marker to reveal

the number of mitotic cells in E12 embryos at the moment of

preparation. Paraffin sections (5 Am thick) were incubated in 0.5

Ag/ml antibody in PBS with 0.3% Triton-X and 2% BSA

overnight. Biotinylated secondary IgG was used with peroxidase

and DAB for colorimetric immunodetection. The sections were

photographed (Zeiss Axioplan 2, 63� oil objective) and the

density of phosphohistone 3-positive cells was estimated as

described above.

Activation of FGF receptor in neural stem cells

Neural stem cells grown in FGF-2 neurosphere medium were

triturated and plated on laminin for 2 days. The cells were deprived

in growth factor-free medium for 24 h. Cells were stimulated with

FGF-2 (0.6 nM) for 15 min after which the cells were washed once

with cold PBS and lysed in cold RIPA buffer (1% nonidet P40,

0.5% Na-deoxycholate, and 0.1% SDS in PBS) containing protease

inhibitors PMSF (1 mM) and aprotinin (1 Ag/ml), and Na-o-

vanadate (1 mM) as a phosphatase inhibitor. HB-GAM (6 nM)

and di-TSR-fragment (6 nM) were used together with FGF-2 to

examine their effect on receptor activation. Cell lysates were

homogenized with a 20-G needle and a syringe, and the lysates

were boiled for 5 min with SDS containing gel-loading buffer. The

samples were loaded in polyacrylamide gel and transferred to

nitrocellulose membrane. The relative amount of FGF receptor

phosphorylation was determined by immunoblotting the samples

with anti-phospho-FGFR (Cell Signaling Technologies), and the

equality of sample loads was verified by stripping the membrane

and blotting it with anti-FGFR1 antibody (Cell Signaling Technol-

ogies). The blots were visualized with ECL (Amersham Pharma-

cia) on X-ray film, and the relative optical densities were measured

digitally from the scanned images (Quantity One, BioRad). Sam-

ples were also blotted with anti-phospho-tyrosine antibody (Sigma)

to verify changes in the phosphorylation level.

Caspase-3 immunostaining

Polyclonal anti-caspase-3 antibodies (Zymed) were diluted in

buffer with 0.3% Triton-X, applied on paraffin sections or on

methanol-fixed cultured cells on object glasses overnight, and

detected with biotinylated secondary antibodies and peroxidase–

DAB reaction.

Acknowledgments

The authors wish to thank Erja Huttu, Seija Lehto, and Eeva-

Liisa Saarikalle for their excellent technical assistance, and doctors

Sarka Tumova, Marjo Salminen, and Richard Margolis for their

valuable comments, critics, and suggestions. This work has been

supported by the Academy of Finland and the Technical Research

Centre of Finland (Programme of Molecular Neurobiology), and the

Sigrid Juselius Foundation.

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