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Oligodendroglial Cells Express and Secrete Reelin.

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THE ANATOMICAL RECORD 294:759–763 (2011)
Oligodendroglial Cells Express and
Secrete Reelin
JUSTIN R. SIEBERT AND DONNA J. OSTERHOUT*
Department of Cell and Developmental Biology, State University of New York
Upstate Medical University, Syracuse, New York
ABSTRACT
Oligodendrocyte (OL) progenitor cells (OPCs) give rise to the myelinating cells of the central nervous system (CNS), the OL. To examine molecular changes involved in OPC differentiation, a microarray analysis was
performed at several time points during OPC maturation. The results
revealed significant expression levels of mRNA for reelin, one reelin receptor, very low density lipoprotein receptor (Vldlr), and the cytoplasmic adaptor molecule, disabled homolog 1 (Dab1). The expression of these proteins
in oligodendroglial (ODG) cells was confirmed by immunocytochemistry
and Western blot analysis. It was also discovered that both progenitors and
mature OLs secrete reelin. Although there is no known effect of reelin on
ODG cells, the data suggest that these cells may be a source of reelin in
C 2011 Wiley-Liss, Inc.
the CNS. Anat Rec, 294:759–763, 2011. V
Key words: oligodendrocyte progenitor cell; oligodendrocytes;
reelin; Dab1; VLDLR
Oligodendrocytes (OL) are the myelin-forming cells of
the central nervous system (CNS). These cells originate
from progenitors generated in specific regions of ventral
forebrain and migrate extensively throughout the CNS
during development (reviewed by Baumann and PhamDinh, 2001; Bradl and Lassmann, 2010). When an OL
progenitor cell (OPC) arrives at its appropriate location,
it begins to differentiate into a mature OL, extending
multiple processes, each of which contacts and
ensheathes an axonal segment. However, the molecular
cues that coordinate the migration and timing of myelin
formation in vivo are not well understood at the present
time.
Numerous in vitro studies have demonstrated that
OPC differentiation can be influenced by extrinsic and
intrinsic factors, including growth factors, extracellular
matrix molecules, and transcription factors (reviewed by
Pfeiffer et al., 1993; Jakovcevski et al., 2009; Li et al.,
2009). To further understanding of the molecular events
that regulate OPC differentiation, we conducted a
detailed microarray screening of OPCs in vitro using
cells at the progenitor, premyelinating, and mature
stages of differentiation. One unexpected finding was
the expression of the mRNA for reelin. Reelin is a large
glycoprotein that is secreted by Cajal–Retzius cells in
the marginal zone, and functions as a critical regulator
of neuronal migration (reviewed by D’Arcangelo, 2006).
The results of this study reveal that OPCs and OLs not
C 2011 WILEY-LISS, INC.
V
only synthesize and secrete reelin but also express a receptor and essential components of the intracellular signaling pathway for reelin. These data suggest that
oligodendroglial (ODG) cells are a source of reelin in the
CNS and that reelin may potentially modulate OL
behavior during development.
MATERIALS AND METHODS
All procedures involving animals were in compliance
with guidelines established by the institutional Committee for the Humane Use of Animals and Department of
Laboratory Animal Resources following recommendations from the Association for Assessment and Accreditation of Laboratory Animal Care.
Grant sponsor: New York State Department of Health
(NYSDOH), Spinal Cord Injury Research Board (SCIRB)
Mentored Scientist Grant, number C022046; Grant sponsor:
Craig H. Neilsen Foundation.
*Correspondence to: Donna J. Osterhout, Ph.D., Department
of Cell and Developmental Biology, SUNY Upstate Medical University, 750 East Adams Street, Syracuse, NY 13210. Fax: 315464-8584. E-mail: osterhod@upstate.edu
Received 4 November 2010; Accepted 26 January 2011
DOI 10.1002/ar.21370
Published online 23 March 2011 in Wiley Online Library
(wileyonlinelibrary.com).
760
SIEBERT AND OSTERHOUT
Primary Cell Culture
Primary mixed glial cell cultures were isolated from
neonatal (P2) Sprague-Dawley rats (Taconic Farms, Germantown, NY) as described previously (Osterhout et al.,
1997). In brief, the cortices of neonatal brains were disassociated, and the resulting cell suspension was plated
in poly-D-lysine polylysine coated tissue culture flasks.
When the monolayer of cells was confluent, OPCs were
isolated by shaking overnight at 37 C and further purified by differential plating.
Purified OPCs were plated onto culture dishes precoated with either laminin (Invitrogen, Carlsbad, CA) or
poly-D-lysine (Sigma, St. Louis, MO). Cells were cultured
for the first 24 hr in defined serum free media (adapted
from Osterhout et al., 1999, containing insulin, selenium, transferrin, and triiodothyronine) supplemented
with B104 neuroblastoma conditioned media (CM; B104CM, 30% v/v) to prevent OPC differentiation. For the
Western blot analysis of reelin levels, OPCs were maintained in defined serum free media containing 10 ng/mL
PDGF-AA (Peprotech, Rocky Hill, NJ) and 20 ng/mL
FGF2 (Peprotech, Rocky Hill, NJ) to prevent OPC differentiation. B104-CM was not included in the media for
these experiments as it contains reelin (data not shown),
and as such, we could not detect reelin secretion by
OPCs in the presence of B104-CM. To initiate OPC differentiation, the culture media was changed to the
defined serum free media without growth factors.
Microarray
RNA harvest. Six dishes of OPCs were harvested at
0, 4, or 6 days postplating for each substrate. Culture
dishes were placed on ice and rinsed three times in icecold 1x PBS. Cells were lysed by adding 300-lL RLT
lysis buffer (Qiagen, Valencia, CA) with 1% b-mercaptoethanol (Sigma Aldrich, St. Louis, MO). RNA samples
were snap frozen on dry ice and stored at 80 C until
further processing. RNA was purified and amplified
using the OvationTM Biotin RNA amplification and
Labeling System (NuGen, San Carlos, CA). An enriched
antisense cDNA probe was produced from the RNA
using the basic 30 Ribo-SPIATM (NuGen, San Carlos,
CA).
Microarray hybridization. Amplification and
labeling of cDNA probes was performed by the SUNY
Upstate Medical University Microarray Core Facility.
cDNA probes were hybridized to the Rat genome 230 2.0
gene array (Affymetrix, Santa Clara, CA). All hybridization, washing, labeling, and detecting steps were performed according to the manufacturers established
protocols.
Data analysis. Expression values were obtained
using RMA normalization of the .CEL files and data formatted for import into Microarray Experiment Viewer
4.2 (Saeed et al., 2003). Significant changes in gene
expression were detected using a two-way ANOVA to
compare the effects of plating substrate and differentiation time across the three time points.
Immunocytochemistry. Coverslips were fixed every
day over the time course of 1 week, using 4% parafor-
maldehyde. The fixed cultures were permeabilized in
0.25% Triton-X 100 in 1x PBS for 5 min and blocked in
an antibody blocking serum (3% FBS and 3% BSA in
HBSS) for 1 hr at room temperature. All primary antibodies were diluted in antibody blocking serum and left
on the cultures overnight at 4 C. Primary antibodies
included three for reelin (CR50, 1:200, a gift from Dr.
Huaiyu Hu; clone 142, 1:200, Millipore, Bellerica, MA,
and G10, 1:200, Abcam, Cambridge, MA), Dab1 polyclonal antibody (1:200; a gift from Dr. Brian Howell) and
very low density lipid receptor (VLDLR, 1:1000; gift
from Dr. Brian Howell). The coverslips were rinsed and
incubated with secondary antibodies, a goat-anti-mouse
or goat-anti-rabbit Alexafluor 488 or 555 (1:500, Invitrogen, Carlsbad, CA), for 1 hr at room temperature. Coverslips were rinsed and mounted on slides using Prolong
Gold mounting media with DAPI (Invitrogen, Carlsbad,
CA).
Western Blotting
CM and cell lysate collection. OPC CM was collected every 2 days and concentrated using the Amicon
Ultra-4 Centrifugal Filter Unit (Millipore, Bellerica,
MA). OPC cell harvest was done on ice. Cells were
washed three times in ice-cold 1x PBS and detached by
scraping. The resulting cell suspension was transferred
to a microfuge tube and pelleted by centrifugation. The
resulting cell pellet was resuspended in ice-cold lysis
buffer with protease inhibitors added fresh. All harvested CM and lysates were kept at –80 C.
Immunoprecipitation. Protein lysates were harvested in the same manner as for the Western blots.
Lysates were incubated overnight at 4 C with a polyclonal antibody to Dab1 (1:200, Biodesign, Saco, ME) and
precipitated out with Protein-A agarose beads (Santa
Cruz Biotech, Santa Cruz, CA).
Western blotting. Samples were run on a precast
gradient, range 3%–8%, Tris-Acetate Nupage gel (Invitrogen), followed by transfer to a PVDF membrane.
Membranes were blocked overnight in a solution of 5%
instant milk in TBS containing 0.1% Tween-20 and then
probed for reelin (G10, 1:200, Abcam, Cambridge, MA),
Dab1 (1:1000, from Dr. Howell), phosphorylated tyrosine
(4G10; 1 lg/mL), and VLDLR (1:1000; from Dr. Howell).
Primary antibodies were diluted in the blocking solution
and incubated overnight at 4 C. Membranes were
rinsed, and the bound antibodies were detected using an
ECF kit (GE Health Care) per the manufacturer protocol. Labeled membranes were imaged on a STORM 840.
RESULTS
ODG Cells Express the mRNA for
Reelin, Dab1, and VLDLR
To better understand the changes in gene expression
that occur as the OPC matures into an OL, a detailed
microarray analysis was performed on ODG cells at several stages of differentiation: the progenitor, premyelinating progenitors, and mature myelinating OL. Genes
that demonstrated a significant change in expression
were determined by a two-factor ANOVA analysis, (P 0.01) level based on 1,000 permutations, comparing the
ODG EXPRESS AND SECRETE REELIN
effect of the substrate, the OPCs were plated on against
the stage of differentiation. The results of this analysis
revealed two specific genes, which unexpectedly showed
a significant change in expression as OPCs underwent
differentiation.
The heat map in Fig. 1A summarizes the expression
profile for reelin and reelin-related genes. Reelin (Reln)
and disabled homolog 1 (Dab1) are both expressed at
high levels in the progenitors. However, at the premyelinating phase of differentiation, there is a significant
decrease in the expression levels of Reln (P < 0.0001)
and Dab1 (P < 0.001). As cell differentiation continues,
Reln and Dab1 maintain a constant but low level of
expression. The expression of Reln and Dab1 are
independent of the substrate, with similar expression
patterns on laminin and polylysine. Interestingly, the
gene for a reelin surface receptor, Vldlr (P ¼ 0.564) was
moderately expressed during all time points and did not
change over the course of differentiation.
In addition to the Reln, Dab1, and Vldlr genes, two
specific myelin genes with known expression patterns
were also identified: myelin-associated glycoprotein,
(Mag), and myelin basic protein, (Mbp). These genes
served as internal controls confirming the differentiation
of OPCs. The discovery that Reln and Dab1 genes were
present in ODG was unexpected and indicates that ODG
cells may express reelin and associated proteins.
ODG Cells Express Reelin Protein
Western blotting analysis of ODG whole cell lysates
demonstrated that reelin protein was expressed at all
stages, from the progenitor phase (lane 1; Fig. 1B) and
all subsequent days after the initiation of differentiation
(2, 3, 4, and 5 days; Fig. 1B). Reelin expression appears
to be maximal in progenitors and decreases as the cells
start to differentiate. A lower level of reelin expression
remains constant through the premyelinating progenitor
phase (3 and 4 days in vitro) and mature OLs (6 days
in vitro). Immunocytochemical studies showed the cellular localization of reelin, which is observed throughout
the cell body and proximal cell processes of progenitor
cells (day 1; Fig. 1C). However, as the cell differentiates
into the premyelinating stage, reelin localizes to the
perinuclear area and expression in the proximal processes is barely visible. In the mature OL, reelin is highly
perinuclear and completely absent in any of the cellular
processes (day 6; Fig. 1C).
Collectively, the western blotting and immunocytochemistry results confirm the genetic expression data,
revealing that ODG cells express reelin at every stage of
maturation and suggesting that ODG could be a source
of reelin in the CNS.
ODG Cells Express Components
of the Reelin Signaling Pathway
As with reelin, both Western blots and immunocytochemical data confirmed the protein expression of Dab1,
the reelin surface receptor, and VLDLR. VLDLR is present in all ODG cells, from progenitors to mature cells
(5 days postplating; Fig. 1B). VLDLR levels steadily
increase as OPCs differentiate into a mature OL, with
maximal expression being found at 5 days postplating
(4 days differentiation time). Within the cell, VLDLR is
761
observed at all stages of differentiation throughout the
cell body and processes (Fig. 1C). In mature OLs,
VLDLR expression can be seen not only in the cell body
and cytoplasmic processes but also in patchy areas of
the myelin membrane sheet proximal to the branching
points of processes (Fig. 1C).
The expression of the cytoplasmic adaptor protein
Dab1 appears to mirror the expression pattern of reelin.
Dab1 protein is present in both progenitors and differentiating cultures (Fig. 1B). Dab1 levels are highest in the
progenitor cells, and as OPCs differentiate, Dab1 levels
decrease. A low, but steady expression level of Dab1 is
maintained at all stages of differentiation. Dab1 appears
in the cytoplasmic processes and cell body in both the
progenitor and premyelinating progenitor phases (1 and
3 days postplating; Fig. 1C). In mature OLs, Dab1
remains in both the cell body and cytoplasmic processes
but appears to be excluded from the developing myelin
membrane sheet (Fig. 1C).
Collectively, these data show that ODG cells express
reelin, as well as key components of the reelin signaling
pathway, the receptor for reelin (VLDLR) and the cytoplasmic signaling molecule for reelin (Dab1). This suggests that ODG cells not only produce reelin but they
may also be responsive to the reelin in an autocrine
manner.
ODG Cells Secrete the Reelin Protein and
Display an Intracellular Dab1-Fyn Interaction
With evidence of reelin protein expression in the cell
lysates, it was next determined if ODG actually secrete
reelin. Conditioned OPC culture media (24 hr incubation) was analyzed by Western blotting to see if reelin
was detectable. When the CM is compared with the progenitor cell lysate (Lys), the amount of reelin found in
the media was dramatically higher than the protein levels found in the cell lysate (Fig. 1D). Culture medias
were conditioned by ODG cells at various time of differentiation (1, 3, 5, and 7 days) and probed for reelin,
which was detected at all time points (Fig. 1E). This
finding suggests that most of the reelin being produced
by these cells is being secreted.
Given that ODG express all three components of the
reelin signaling pathway, it might be expected that
reelin can bind to ODG and activate intracellular signaling pathways associated with reelin. Reelin–Dab1 interactions are critical for a cell to respond to reelin (Howell
et al., 1997). Dab1 can also interact with the Src family
kinase Fyn, which is a critical regulator of ODG differentiation (Osterhout et al., 1999). If these pathways are
involved, Dab1 would be phosphorylated in the presence
of reelin. To examine this, we immunoprecipitated Dab1
from the cell lysate and probed it for phosphorylated tyrosine, which would indicate protein activation. There
were consistent low levels of tyrosine phosphorylation at
all time points, but it is unclear if this is truly in
response to reelin in the culture media (data not shown).
We also probed for a Dab1–Fyn interaction by immunoprecipitating with Dab1 and probing for Fyn. We
observed Fyn–Dab1 associations at all time points along
a differentiation time course (Fig. 1F). Interestingly, the
interaction looks strongest in the ODG progenitors, a
stage before Fyn activation and the beginning of OPC
differentiation (Osterhout et al., 1999).
Fig. 1. ODG cells express and secrete reelin and reelin signaling
components. (A) Gene expression profiles for reelin (Reln), disabled
homolog 1 (Dab1), very low density lipoprotein receptor (Vldlr), and
two myelin genes, myelin-associated glycoprotein (Mag), myelin basic
protein (Mbp). This heat map illustrates the level of gene expression
on a colormetric scale. High levels of expression are indicated in red,
whereas low levels of gene expression are indicated in green. Gene
expression was examined at various time points during the differentiation of OPCs into mature OLs. Reln and Dab1 expression is high in
the progenitor cells (day 1) and downregulated after differentiation
begins, exhibiting a low level of expression by the premyelinating
phase of differentiation (day 4). (B) The expression of reelin and components of the reelin signaling pathways were confirmed by Western
blotting. The expression of these proteins in whole cell lysates of ODG
is observed at all stages of differentiation. The levels of protein
expression were dependent on the phase of differentiation, especially
for reelin and VLDLR expression. (C) A series of ODG cell cultures
were set up for immunofluorescent analysis to confirm the cellular
expression and localization of reelin, VLDLR and Dab1. As seen in the
top row of C, cultures were stained with stage specific markers
(A2B5, progenitor; O4, premyelinating progenitor cell; O1, mature mye-
linating OLs) to verify the presence of ODG cells in our cultures, and
the stage of differentiation. Sister cultures were stained for reelin,
Dab1, and VLDLR. In the progenitor phase, reelin is found throughout
the cell body and proximal processes (1-day post plating). However, in
the premyelinating progenitor phase, reelin begins to localize around
the nucleus (4 days postplating). In mature, fully differentiated Ols,
reelin is highly localized to the perinuclear region (7 days postplating).
Dab1 is expressed in the cell body and primary cytoplasmic processes of ODG cells. VLDLR, the reelin receptor is observed throughout
the cell body, processes, and in the myelin membrane sheet of mature
Ols. (D) The secretion of reelin was revealed by Western blot analysis
of OPC conditioned media and cell lysates. The levels of reelin
expressed in OPC lysates is much lower that the reelin found in the
conditioned culture media (CM). (E) Reelin is expressed by ODG at all
stages of differentiation, from progenitors (day 1) to fully mature Ols
(day 7). (F) Immunoprecipitation with Dab1 reveals the interaction of
the Dab1 protein with the tyrosine kinase Fyn. Fyn is detected at all
stages of differentiation, especially in the early progenitor cells.
Although the interaction of Fyn and Dab1 has been previously documented in neurons, this data shows the same relationship between
Fyn and Dab in OPCs. (DIV) ¼ Days in vitro, Scale bars ¼ 50 lm.
ODG EXPRESS AND SECRETE REELIN
These findings reveal that ODG cells, regardless of
their differentiation status, will secrete reelin into their
environment. ODG cells are also unique in that they
express all of the essential components of the reelin signaling pathway: reelin, a reelin receptor, and the cytoplasmic adaptor protein. Taken together, this suggests
that ODG cells may be a major source of reelin in the
CNS, and moreover, reelin may play a role in the biology
of ODG cells.
763
ACKNOWLEDGMENTS
DISCUSSION
The authors would like to thank Yijuan Lin for maintaining the primary OPC cultures. The authors are very
grateful to Drs. Huaiyu Hu (CR-50), and Brain Howell
(anti-Dab1, and VLDLR) in the Neuroscience and Physiology Department for the antibodies used this study, as well
as helpful discussions of our work. The authors gratefully
acknowledge Ms. Karen Gentile and the SUNY Upstate
Medical University Microarray Core Facility for processing all the RNA samples, and Dr. Frank Middleton for his
assistance with the microarray data analysis.
Reelin is a critical modulator of neuronal positioning
and migration during brain development (reviewed by
Rice and Curran, 2001; Tissir and Goffinet, 2003). It is
expressed by specific populations of migrating neurons,
whereas the reelin receptors are expressed by different
target cells. Reelin is not typically expressed by glial
cells; to date, it has only been observed in a small subset
of spinal cord astrocytes during development (Hochstim
et al., 2008). This study demonstrates that ODG cells
express reelin, Dab1, and VLDLR proteins and secrete
reelin into the surrounding environment. This finding is
unique, in that ODG express all three components
needed for reelin signaling, raising the possibility that
reelin secretion may have an autocrine effect. However,
any biological effects of reelin, whether through autocrine or paracrine signaling, on ODG cells has not been
previously investigated.
Our data also suggests that ODG may be a source of
reelin in the developing CNS. This is in agreement with
two studies that also provide indirect evidence that
ODGs may be a source of reelin. In this work, OPCs
were ablated in mice, which generated severe cerebellar
malformations. Subsequent analysis revealed decreased
reelin levels and a disorganization of the cellular architecture that included impaired interneuron cell migration (Mathis et al., 2003; Collin et al., 2007). However,
the possibility that cells in the ODG lineage are reelin
producers has not been examined before this work.
Studies of the intracellular signaling pathways activated by reelin have been only done in neurons, and
identified functional interactions between the cytoplasmic adaptor molecule Dab1, the serine-threonine kinases
P35 and Cdk5, and Src family kinase (Fyn), which may
yield a clue to the role reelin in the OL lineage (Howell
et al., 1997; Arnaud et al., 2003; Fatermi, 2005; Herz
and Chen, 2006). Of particular interest is the discovery
that reelin binding to its receptor activates the Src family kinase Fyn (Fatermi, 2005). Fyn activation is essential for the migration and differentiation of OPCs
(Osterhout et al., 1999; Miyamoto et al., 2008). This suggests reelin may play a role in OPC migration and differentiation. We show that both the reelin and Dab1
proteins are at maximal expression during the progenitor phase and are quickly down regulated during OPC
differentiation. This suggests that reelin may be more
important for migration, rather than differentiation.
Although the role of reelin in ODG biology is undetermined, studies are currently underway utilizing mutant
mouse models (reeler and scrambler) to understand how
reelin influences the behavior of ODG cells. The findings
from this study suggest that reelin may have a role in
ODG biology, and that ODG cells may be an important
source of reelin in the CNS.
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