Development, cell lineage and differentiationP7-1The vital rule of cerebrospinal fluid on corticaldevelopmentF. Mashayekhi* and J. A. Miyan�

*The University of Guilan, Rasht, Iran; �UMIST, Manchester, UK

Brain development is a very complicated process in which neuronesare born in the germinal layer of the neural tube and then migrateaway to form the different parts of the brain including the cerebralcortex. In hydrocephalus the cerebrospinal fluid (CSF) pathway isblocked and fluid accumulates in the ventricles. Fluid accumulationresults in a rise in intracranial pressure. CSF is actively producedwithin the brain and does not switch off when a blockage occurs.Our recent work demonstrates that fluid blockage results inimmediate effects on cell division in the germinal layer resultingin fewer neurones being produced. We have also shown that thefluid which accumulates in the ventricles prevents cell division ofneural precursors grown outside the brain in culture. Fluidaccumulation does not immediately raise pressure nor lead to braindamage but affected individuals do have less cortical thickness/massthan normal. We investigated this using the substance BrDu thatallows us to identify neurones born at a particular time duringdevelopment. This demonstrates that the number of cells beinggenerated by the germinal layer is much less than normal and thatfewer cells arrive in the cortex. Accumulated CSF contains signalswhich prevent normal cell division in affected brains. We tested thisby taking cells from normal and abnormal brains and growing themin artificial medium. Released from the effects of the accumulatedfluid, cells from affected brains divide faster than those from normalbrains. Fluid from normal brains has no effect on the division ofneural precursors but fluid from affected brains prevents celldivision. These data show that the flow of CSF through and over thebrain is important to the normal development of the brain and,particularly, the cerebral cortex.Keywords: cell lineage and differentiation, development, stem cell biology.

P7-2Direct visualization of the presence of protein translationfactors in the dendritic spinesI. S. Moon

Department of Anatomy, College of Medicine, Dongguk University,

Gyeongju, Korea

Synapse-specific local dendritic protein synthesis may play an important role

in the regulation of synaptic plasticity. Previously, my laboratory identified

eukaryotic translation elongation factor-1A (eEF1A) and HSP70 in the rat

forebrain postsynaptic density, a cytoskeletal specialization that regulate

postsynaptic signaling. Immunoblot and immunocytochemistry revealed that

various translation initiation factors are also present at the postsynaptic sites.

Recently, we have further verified punctate distribution of these factors in

dendritic spines by direct visualization of GFP-fusion proteins expressed in

the primary rat cortical or hippocampal neurons. In another line of research

using a microarray technology, we have found that mRNAs for various eEFs

and eIFs, among many others, are present in subsynaptic sites. Our data

indicate that dendritic mRNAs may be translated by factors provided by

synapses, and that there may be a translation acceleration mechanism at or

near synapses.

Keywords: development, gene expression, learning and memory, plasticity,

signal transduction.

P7-3Expression analysis of the novel basic helix-loop-helixtranscription factor, Olig3L. Ding,* H. Takebayashi,* O. Chisaka� and K. Ikenaka*

*National Institute for Physiological Sciences, Okazaki; �GraduateSchool of Biostudies, Kyoto University, Kyoto, Japan

Olig family is a novel subfamily of basic helix-loop-helix transcriptional

factors recently identified. Olig1 and Olig2 were first reported to promote

oligodendrocyte differentiation, and later Olig2 was reported to be involved

in motoneuron specification as well. Olig3 was isolated as a third member of

Olig family and shown to be expressed in dorsal ventricular zone of the

embryonic neural tube at early stage. Moreover, Olig3 is expressed in ventral

migrating progenitors of specific interneurons at later stage. Therefore, we

speculated that Olig3 might have some role(s) in cell fate specification and

control of migration in developing central nervous system. In order to

understand the role of Olig3 during development, we decided to generate

Olig3 knockout mice. We generated lacZ knock-in mice, and first examined

the lacZ expression pattern in heterozygous Olig3þ=� embryos by X-gal

staining. As expected, lacZ was expressed strongly and continuously in

dorsal neural tube from the upper rhombic lip caudally to the tail at E10.5.

Moreover, lacZ expression was also detected in thalamus, and non-neural

tissue. We will show detailed expression analysis and discuss possible role of

Olig3 during development.

Keywords: bHLH, knockout mice, lacZ, olig3, transcription factor.

P7-4Stage- and site-specific DNA demethylation in neuralstem cells generated from embryonic stem cellsK. Shimozaki, M. Namihira, K. Nakashima and T. Taga

Department of Cell Fate Modulation, Institute of Molecular

Embryology and Genetics, Kumamoto University, Kumamoto,

Japan

The epigenetic modification of chromosomal DNA plays multiple important

roles in the regulation of cell-specific gene expression during embryogenesis.

We have shown that the activation of a transcription factor STAT3 is crucial

for astrocyte differentiation, and that CpG methylation of STAT3-binding site

in glial fibrillary acidic protein (GFAP) promoter diminishes at the stage

when progenitor cells become responsive to the STAT3 activation signal.

Methylation of this cytosine abolishes the accessibility of STAT3 and inhibits

transcriptional activation. Here we report that the methylation status of the

STAT3-binding site in the GFAP promoter is regulated in a developmental

stage-specific manner, by using an in vitro ES cell differentiation system.

This CpG dinucleotide in the STAT3-binding site in the GFAP promoter was

highly methylated in ES cells, but was demethylated in cells responsive to

the STAT3 activation signal to express GFAP. However, this CpG

demethylation was not detected in non-neural cells in contrast to those in a

neural lineage. We further show the cell fate-specific CpG demethylation in

mouse fetal tissues. A CpG dinucleotide within the STAT3-binding element

in the GFAP promoter was demethylated in mouse E14.5 neuroepithelial

cells, yet cells from E14.5 non-neural tissues kept the high methylation

status. Therefore, lineage specification in the brain appears to be regulated by

DNA methylation, and a neural gene-specific demethylation is programmed

only when pluripotent cells are committed to a neural lineage.

Keywords: cell lineage, cytokines, development, differentiation.

30 � 2004 International Society for Neurochemistry, Journal of Neurochemistry, 88 (Suppl. 1)

P7-5DNA methylation-mediated regulation of astrocytedifferentiation from neural precursor cells in mouse fetalbrainM. Namihira, K. Nakashima, T. Takizawa and T. Taga

Department of Cell Fate Modulation, Institute of Molecular

Embryology and Genetics, Kumamoto University, Kumamoto,

Japan

Astrocyte differentiation, which normally occurs at a late stage in brain

development, is largely dependent on the activation of a transcription factor,

signal transducer and activator of transcription 3 (STAT3). We here show that

astrocytes, as judged by expression of glial fibrillary acidic protein (GFAP),

do not emerge from neuroepithelial cells on embryonic day (E) 11.5 even

when STAT3 is activated, in contrast to E14.5 neuroepithelial cells. We

further demonstrate that a CpG dinucleotide within a STAT3 binding element

in the GFAP promoter is highly methylated in E11.5 neuroepithelial cells, but

is demethylated in cells responsive to the STAT3 activation signal to express

GFAP. This CpG methylation of STAT3 binding site leads to inaccessibility

of STAT3 and results in transcriptional repression of the GFAP gene.

Moreover, neither downregulation of maintenance DNA methyltransferase

(Dnmt1) nor genome-wide demethylation of DNA is observed when cells

become responsive to a STAT3-activating signal, suggesting that the

demethylation occurs at a specific site (or sites). In addition to the GFAP

gene promoter, a particular CpG site in a promoter region of the gene for

S100b, another maker for astrocytes, is also demethylated in accordance withbrain development, which coincide with developmental stage-dependent

expression of gene. Furthermore, the proximal region including its demeth-

ylated CpG site is important for transcriptional activation of S100b. It is thusconceivable that methylation of cell type-specific gene promoters is a critical

determinant in regulating lineage specification in the developing brain.

Keywords: astrocytes, cell lineage and differentiation, cytokines, develop-

ment, gene expression, regulation, stem cell biology.

P7-6Effect of oxidoreductase fragment on lipid compositionand aggregation of CHO cellsV. P. Ivanova, Z. V. Kovalyova, E. P. Shukolyukova,

S. A. Zabelinskii and A. I. Krivchenko

Institute of Evolutionary Physiology and Biochemistry, RAS,

St Petersburg, Russia

Many widespread diseases are accompanied by dissemination with the following

fibroplasia in affected tissue. Development and acceleration of these processes in

organism are related to the formation of biological factors stimulating the reactions

of focal and cell–cell adhesion. The latter ones may be accelerated by multiple

peptide fragments releasing from different human and/or bacterial proteins during

their limited proteolysis occurring in inflammatory infiltrations. The aim of the

present work was to study the effect of oxidoreductase fragment from Mycobac-

terium tuberculosis on the aggregation of Chinese hamster ovary (CHO) cells. To

estimate aggregating activity of the peptide, CHO cells (106/ml) were incubated

with or without the peptide for 30 min at 37�C and then 60 min at 4�C. Thepercentage of aggregates consisting of two and more cells was counted by phase-

contrast microscopy. Lipids were isolated by Folch procedure. Content of

phospholipids was evaluated by the amount of lipid-bound inorganic phosphorus.

Fatty acid methyl esters were separated by gas-liquid chromatography. The peptide

has been found to stimulate the CHO cell aggregation and to change the fatty acid

composition of cell membrane phospholipids. The data obtained indicate that the

aggregate-stimulating effect of the investigated peptide may be connected with its

influence on the composition and properties of lipid domains in cell membranes,

which affect the functional activity of receptors participating in cell aggregation.

Keywords: Chinese hamster ovary cells, fatty acids, phospholipids, regulatory

peptides.

P7-7Co-clustering of mitochondria and synaptic vesiclesduring presynaptic differentiation at the neuromuscularjunctionC. W. Lee and H. B. Peng

Biology Department, HK University of Science and Technology,

Clear Water Bay, HKSAR, China

During vertebrate neuromuscular junction (NMJ) development, the path-finding

axonal growth cone of a motor neuron differentiates into a specialized presynaptic

terminal. The clustering of synaptic vesicles (SVs) is a hallmark of this

transformation. Ultra-structural studies have demonstrated that presynaptic termi-

nals contain mitochondria in addition to SVs, but how mitochondria are targeted to

these sites is unknown. In this study, we investigated the localization and dynamics

of mitochondria relative to SVs in cultured Xenopus spinal neurons using the

mitochondrial marker MitoTracker. Mitochondria were present at varicosities and

growth cones of the neurons, and where neurites contacted muscle cells in nerve-

muscle co-cultures. As shown previously, SVs were also enriched at these

locations. Next, by treating neuronal cultures with growth factor-coated beads,

which can locally induce presynaptic differentiation, we found that mitochondrial

and SV clustering occurred in spatially and temporally similar manner. Time-lapse

imaging demonstrated that mitochondria were actively transported to and stably

clustered at the bead-neurite contacts, much like the SVs. Lastly, to gain insights

into the signaling involved in mitochondrial clustering, we treated bead-neuron

cultures with the ser/thr phosphatase inhibitor okadaic acid, which disperses SV

clusters. Intriguingly, in contrast to the effect on SVs, okadaic acid addition did not

significantly influence either the formation or maintenance of mitochondrial

clusters. Our results suggest that mitochondria are co-clustered with SVs during

presynaptic differentiation at the NMJ, but that the underlying mechanisms that

mediate the targeting and localization of these organelles are different.

Keywords: axonal growth and transport, development, cell lineage and differen-

tiation, signal transduction.

P7-8Differential expression of glutamate decarboxylaseisoforms in the horizontal cells of human and rat retinasS. X. Li,* S. Y. Shu,� X. M. Bao,� K. F. So,* D. Tay,* W. F. Kau,*

Y. M. Wu,� W. K. Yang� and H. K. Yip*

*Department of Anatomy, The University of Hong Kong, Hong

Kong SAR; �Department of Neurobiology, Zhujiang Hospital,

Guangzhou, China

L-glutamate decarboxylase (GAD) isoforms, GAD65 and GAD67, catalyze the

synthesis of c-aminobutyric acid (GABA). Previous studies demonstrated that

GAD65 and GAD67 appear only in the horizontal cells of monkey, and rodents

and cats, respectively; whereas both GAD65 and GAD67 can be found in the

rabbit horizontal cells. In the present study, we used immunohistochemistry and

Western blot to examine the expression of GAD isoforms in the retinas of human

fetuses and also in the developing and adult rat retinas. We found that both GAD

isoforms existed in the amacrine cells and the inner plexiform layer of both human

and rat retinas. GAD67 was found in the more early stage and more in the cell

bodies. Two GAD isoforms were detected in a different stratification pattern of the

inner plexiform layer. However, only GAD65 was observed transiently in the

horizontal cells, at 22 WG of human fetus; while both isoforms were found in rats.

GAD67 was first seen at birth and GAD65 at postnatal 14 days and through adult

in rats. Previous studies provided evidences that GABAc receptor is expressed in

the bipolar cells and cone photoreceptors. Thus GABA released by horizontal cells

was hypothesized to act as a forward feedback to bipolar cells and a backward

feedback to cone photoreceptors. In summary, GAD isoforms catalyzing GABA

synthesis in the horizontal cells are different among different species and only

GAD65 plays a role in the horizontal cells of human fetuses.

Acknowledgements: Support: The University of Hong Kong, Institute of

Molecular Technology for Drug Discovery and Synthesis of HKU, and Research

Grant Council of Hong Kong.

Keywords: development, glutamate decarboxylase, human, rat, retina.

� 2004 International Society for Neurochemistry, Journal of Neurochemistry, 88 (Suppl. 1) 31

P7-9Gender differences of the pontine nuclei between maleand female Sprague–Dawley rats as studied by 3-Dcomputer graphic systemK. Khankasikam, R. Tipyasang and N. Kotchabhakdi

Institute of Science and Technology for Research and Development,

Mahidol University, Salaya, Nakornpathom, Thailand

Many brain areas show dimorphic gender differences. The pontine

nuclei in the brainstem is an important relay system for the cerebro–

cerebellar interactions. Pontine nuclei receives direct projection

from specific cerebral cortical areas and project their axons as

mossy fiber afferents to specific areas of the cerebellar hemisphere.

The complex organization of the cerebro-ponto-cerebellar projec-

tion system has been studied by 3-D computer graphic and

morphometric system (Micro 3D). Ten male and female Sprague–

Dawley rats weighting between 250 and 300 gm were used in the

experiment. The rats perfused with 4% paraformaldehyde fixative in

PBS. The brainstem and the cerebellum were removed and stored in

sucrose solution. Serial 50-micron frozen sections were cut on

freezing microtome and stained with Cresyl-violet.The brainstem

sections were recorded by imaging system and the boundary of

pontine nuclei in all sections were systematically identified and

digitized as lines. The presence and location of various sizes of

neurons in the pontine nuclei were marked and digitized as dots and

symbols. Reconstruction of the pontine nuclei was performed with

computer graphic and morphometric system. The volume of pontine

nuclei was measured and the number of neurons was counted. The

differences in volume and neuronal distribution between adult male

and female rats were shown in 3-D atlas from each animal. Data

were analyzed for mean, standard deviation, and statistical differ-

ences between male and female. The data showed that there were

significant differences in volume and neuronal distribution between

male and female rats in different areas of the pontine nuclei.

Keywords: cerebro-ponto-cerebellar projection, dender differences,

morphometry, pontine nuclei, three-dimensional neuroanatomy.

P7-10Expression of telomerase activity and telomeric catalyticsubunit in goldfish (Carassius auratus) retinaW. M. Lau, S. X. Li, G. S. W. Tsao, A. O. L. Wong and H. K. Yip

The University of Hong Kong, HKSAR, China

Telomerase is an enzyme stabilizes eukaryotic chromosomes by

adding telomeric repeats (TTAGGG) to chromosomal ends. The

enzymatic activity is commonly found in proliferating cells and

organs with regenerating potential, but not postmitotic cells. Unlike

mammals, the retina of goldfish grows continuously through

neurogenesis. The aim of this study is to determine whether

telomerase catalytic subunit (TERT) and telomerase activity can be

detected in goldfish (Carassius auratus) retina. Eyes of adult

goldfish were dissected, enucleated and fixed for immunohisto-

chemistry while retinas were dissected for telomeric repeat ampli-

fication protocol (TRAP) assay. TRAP assay showed that

telomerase activity can be found in goldfish retina. Immunoreac-

tivity of TERT was shown strongly in inner nuclear layer and outer

nuclear layer. The results show that telomerase is present in goldfish

retina. As telomerase is correlated with prevention of apoptosis and

cell cycle progression in cells, it is possible that telomerase is

involved in continuous generation of neural retina in goldfish.

Unlike mammals, there are groups of neural progenitor cells in inner

nuclear layer of goldfish retina. These stem cells are the source of

neurons added during growth and after injury. They may be the

source of telomerase activity in goldfish retina. Further studies,

which confirm the correlation between telomerase and teleost retina

neurogenesis, are required to understand the role of telomerase in

teleost retina development.

Keywords: goldfish, retina, telomerase, telomeric repeat amplifica-

tion protocol assay.

32 � 2004 International Society for Neurochemistry, Journal of Neurochemistry, 88 (Suppl. 1)