1 Wellcome Trust/CR UK Gurdon Institute, University of Cambridge, Tennis Court
Road, Cambridge CB2 1QN, UK
2 Department of Biochemistry, University of Cambridge, Tennis Court Road,
Cambridge CB2 1QN, UK
3 Department of Biological Sciences, Gilbert Hall, Stanford University,
Stanford, CA 94305, USA
4 Department of Neuroscience, Albert Einstein College of Medicine of Yeshiva
University, Rose F. Kennedy Center, 1410 Pelham Parkway South, Room 903,
Bronx, NY 10461, USA
5 The Physiological Laboratory, University of Cambridge, Downing Site, Cambridge
CB2 3EG, UK
* Author for correspondence (e-mail: rick{at}gurdon.cam.ac.uk)
Accepted 22 June 2005
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SUMMARY |
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Key words: Neocortex, Protomap, Microarrays, Patterning, Mouse
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Introduction |
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The early formation of cortical areas, as assessed by gene expression,
occurs normally in the absence of thalamocortical innervation, suggesting that
arealisation is a process intrinsic to the neocortex
(Miyashita-Lin et al., 1999;
Nakagawa et al., 1999
). Two
homeobox transcription factors, Pax6 and Emx2, are expressed in opposing
rostrocaudal gradients in the developing neocortex
(Bishop et al., 2000
;
Mallamaci et al., 2000
). Mice
carrying mutations in either Pax6 or Emx2 have reductions in
the relative sizes of the cortical areas in which those genes are normally
highly expressed, and expansions of parts of the neocortex in which they are
expressed at low levels (Bishop et al.,
2000
; Mallamaci et al.,
2000
; Muzio et al.,
2002
). A similar finding has been made in the case of another
trancription factor, COUP-TFI (Nr2f1 Mouse Genome Informatics), which
is expressed in a high caudal to low rostral gradient
(Liu et al., 2000
;
Zhou et al., 2001
). Recent
data indicate that altering the absolute levels of Emx2 in the developing
neocortex is sufficient to alter rostrocaudal patterning, such that caudal
areas are expanded at the expense of rostral areas
(Hamasaki et al., 2004
). These
opposing gradients of transcription factor expression are reminiscent of
transcription factor expression in progenitor cells in the spinal cord and the
retina (Jessell, 2000
;
Koshiba-Takeuchi et al.,
2000
). In the developing spinal cord, combinatorial expression of
a set of transcription factors divides progenitor cells into several discrete
domains along the dorsoventral axis of the neural tube, with the progenitor
cells in each domain giving rise to a particular class of neuron
(Briscoe et al., 2000
).
Areal identity is conferred, at least in part, by extracellular signals.
Heterotopic transplantation of presumptive cortical areas early in development
results in the transplanted cortices assuming the identity of their
destination (Cohen-Tannoudji et al.,
1994). However, mouse neocortical tissue becomes intrinsically
specified with respect to spatial identity by embryonic day 13, two days after
the initiation of neurogenesis, at which point it becomes refractory to
transplantation (Gitton et al.,
1999
). Similar findings have been reported in the developing rat
cortex (Gaillard et al.,
2003
). It has been proposed that there are three potential
signalling centres around the margins of the neocortex: the anterior neural
ridge, the cortical hem and the cortical-subcortical boundary region
(Grove and Fukuchi-Shimogori,
2003
; O'Leary and Nakagawa,
2002
; Ragsdale and Grove,
2001
). Several BMP family members are produced at the cortical
hem, dorsal midline and caudal cortex
(Furuta et al., 1997
), and
there is evidence for a role for these proteins in mediating mediolateral
patterning of the neocortex (Monuki et
al., 2001
), although studies of Bmp receptor mutants suggest that
the primary role of Bmp signalling is local patterning around the dorsal
midline (Hebert et al., 2003a
;
Hebert et al., 2002
).
Fgf8 appears to be a primary regulator of rostral areal identity in the
neocortex. Expressed initially in the anterior neural ridge and then by
progenitor cells in the rostral pole of each cerebral hemisphere
(Crossley and Martin, 1995),
Fgf8 is necessary for patterning the rostral neocortex and is capable of
repressing caudal and inducing rostral neocortical identities
(Fukuchi-Shimogori and Grove,
2001
). These findings have been confirmed in an analysis of
hypomorphic Fgf8 mutants, in which there is both a relative reduction
in the domains of progenitor cells expressing rostral markers and a
proportional increase in the domains of progenitor cells expressing caudal
markers, as well as a loss of the most rostral cortical structure, the
olfactory bulbs (Garel et al.,
2003
). Fgf8 has been proposed to regulate area formation by
repressing progenitor cell expression of the transcription factor Emx2, which
is expressed in a high caudal to low rostral gradient
(Fukuchi-Shimogori and Grove,
2003
).
Therefore, a current model for cortical area formation proposes that
signalling centres co-ordinately determine the spatial or areal identity of a
neocortical progenitor cell, which is read out as spatially specific gene
expression (O'Leary and Nakagawa,
2002). By analogy with spinal cord development
(Jessell, 2000
), neocortical
progenitor cells then produce area-specific neurons based on their positional
identity. However, little is known of the components or the nature of the
proposed progenitor cell protomap. We report the results of a genomics-based
strategy for characterising the nature and composition of the protomap, in
which we identified known and novel rostrocaudal gradients of neocortical
progenitor cell gene expression.
Given the role of Fgf8 signalling in rostrocaudal patterning in the
neocortex, we proposed that protomap components would alter their expression
in response to changes in Fgf signalling. Therefore, we studied the
transcriptional consequences of forebrain-specific loss of Fgf receptor 1
(Fgfr1) function during the period of Fgf8-regulated patterning in the
neocortex, as the abnormalities in cortical development in these animals are
very similar to those occurring in Fgf8 hypomorphic mutants
(Garel et al., 2003;
Hebert et al., 2003b
). In
Fgfr1 mutant mice, a subset of the proposed protomap genes change in
expression in a manner consistent with their positive and negative regulation
by Fgf8-mediated signalling. One gene, Mest (also known as
Peg1), was identified by those analyses as a strong neocortical
patterning candidate gene, as in addition to its rostral expression and Fgf
regulation in vivo, we found that Mest expression is directly induced
by Fgf8 signalling in neocortical explant cultures. Anatomical and genomic
analysis of mice mutant for Mest/Peg1 identified significant
alterations in patterned gene expression in the developing neocortex.
Therefore, we propose that the early neocortical protomap is composed, at
least in part, of gradients, rather than domains, of gene expression along the
rostrocaudal axis that are regulated by an Fgf signalling system.
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Materials and methods |
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Microarray data analysis
Hybridised microarrays were scanned on a GenePix 4000B microarray scanner
(Axon Instruments) and the resulting images were analysed with GenePix 5 array
analysis software. Image data were archived and analysed in the Acuity system
(Axon Instruments). Individual arrays were lowess normalised by individual
printing block to correct for both intensity and position-dependent variations
in expression measurements. Data from each hybridisation were filtered to
remove array features that were not detectable significantly above local
background. To identify reproducible rostrocaudal differences in gene
expression, all hybridisations were carried out as dye-swapped technical
replicates. For statistical analysis, data from these technical replicates
were inverted and the average value of each pair of hybridisations was used in
subsequent analyses. The significance analysis of microarrays (SAM) algorithm
was used to identify significant rostracaudal differences in expression, using
a false discovery rate (FDR) cutoff of 5%
(Tusher et al., 2001).
Statistical analysis of expression data from the rostral/middle/caudal screen
and the Fgfr1 and Mest mutant cortices was carried out using
the t-test algorithm implemented in Acuity. Data from each analysis
were stored in Acuity and subsequently analysed by hierarchical and k-means
cluster analysis. Gene ontology annotation was assigned to gene sets using the
online FatiGO tool
(http://www.fatigo.org/)
(Al-Shahrour et al., 2004
). The
complete microarray datasets were deposited in the NCBI Gene Expression
Omnibus (GEO) data repository
(http://www.ncbi.nlm.nih.gov/projects/geo/index.cgi),
with accession numbers: GSE2854-GSE2858.
Analysis of gene expression in mutant mice
The dorsal telencephalon was dissected from subcortical structures from
single embryos of a litter of E12.5 Fgfr1 conditional mutant mice
(Hebert et al., 2003b), RNA
was extracted, and cDNA synthesised and amplified using the SMART system. To
control for differences in gene expression due to the presence of the modified
Fgfr1 allele and Foxg1 heterozygosity, gene expression was
compared among single embryos of two key genotypes,
Foxg1+/Cre;Fgfr1+/fl and
Foxg1+/Cre;Fgfr1fl/fl; that is, heterozygous
and homozygous Fgfr1 mutants. A total of nine different pair-wise
hybridisations was carried out, four of which were dye-reversed relative to
the other five. For the Peg1/Mest analysis, cortices were dissected from null
and wild-type littermate embryos at E12.5, and cDNA synthesised and amplified
from tissues from single embryos using the SMART system. As Mest is a
paternally imprinted gene, gene expression was compared between functional
null and wild-type tissues.
In situ hybridisation
Non-isotopic in situ hybridisation to whole-mount embryos and parasagittal
embryo sections was carried out as described
(Brent et al., 2003). Probes
were prepared from cDNA clones for the genes studied, selected from a Brain
Molecular Anatomy Project clone set (kind gift of Dr Bento Soares, University
of Iowa). All clones were sequenced and compared to GenBank sequences using
BLAST to confirm clone identity. For genes not represented in that set, PCR
primers were designed to amplify approximately 1 kb in the 3' region of
the corresponding RefSeq cDNA, the amplicons were cloned into TA cloning
vectors (Invitrogen) and the inserts sequenced. Whole-mount in situ
hybridisation was carried out as described using the same probes as were used
for sections (Bao and Cepko,
1997
). Fluorescent in situ hybridisation was carried out using
Tyramide Signal Amplification (TSA; Perkin Elmer) according to the
manufacturer's instructions with minor modifications. Cy3- and Cy5-labelled
hybridisation probes were visualised using the Axon 4000B microarray
scanner.
Neocortical explant culture and real-time PCR
Explants of the middle third of the E11.5 neocortex were dissected free of
surrounding tissues and placed on polycarbonate filters floating on serum-free
medium consisting of DMEM/F-12 and the supplements B-27 and N2 (Invitrogen),
to which poly-L-glutamine and heparin were added. At least two explants were
cultured on each filter. Recombinant mouse Fgf8b (R&D Systems) was added
to a concentration of 10 ng/ml. Explants were harvested 4 hours after Fgf8
addition, RNA extracted (Trizol, Invitrogen) and cDNA synthesised by oligo-dT
priming. Real-time PCR was carried out on a Roche LightCycler according to the
manufacturer's instructions. Expression of each gene was calculated relative
to that of the mRNA for the abundant ribosomal protein rpS17 in the same
sample and in three independent experiments.
Bioinformatics
Mest homologues were identified by BLAST comparison of the mouse and human
Mest protein sequences to the predicted proteins encoded by the other genomes
shown (see text and figures), using Ensembl and the public Caenorhabditis
elegans and Drosophila melanogaster genome databases (FlyBase
and WormBase). The best-scoring hits were compared by BLAST back against the
mouse genome to confirm the true homologue of Mest in each organism. Multiple
sequence cluster analysis was carried out by ClustalW on the EBI server.
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Results |
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To identify genes with spatial differences in expression in the developing
neocortex, we used oligonucleotide microarrays representing over 22,000 of the
genes expressed from the mouse genome. We analysed differences in rostrocaudal
neocortical gene expression prior to the arrival of the thalamocortical
afferents to the cortex, and at the onset and in the early phases of
neurogenesis (Molnar et al.,
1998; Takahashi et al.,
1999
). In order to systematically characterise rostrocaudal
differences in gene expression, we performed a detailed comparison of rostral
and caudal pools of E11 and E13 neocortex, applying the strategy illustrated
in Fig. 1. Two different
strains of mice, one inbred (C57Bl/6), the other outbred (MF1), were used to
control for strain-dependent variations in patterns of gene expression
(Pavlidis and Noble, 2001
;
Sandberg et al., 2000
). For
each strain at each timepoint, three sets of pooled rostral and caudal tissue
were collected from single litters. Gene expression screens were carried out
within single litters to match embryos by developmental age as closely as
possible. Individual pools contained material from the left and right cortices
of at least two embryos, to correct for variation among dissections. Given the
limited amount of RNA available when using such a screening strategy, the
corresponding cDNA was amplified using the SMART system to generate enough
material for synthesising array probes
(Matz et al., 1999
), as has
been used successfully for our previous studies
(Livesey et al., 2000
;
Livesey et al., 2004
).
To assess the efficacy of the screening strategy, we investigated the expression of a set of known differentially expressed genes in our dataset: Pax6, Emx2, Lhx2, COUP-TFI and Sfrp1. All five genes are represented on the array, and four were reliably detected by the arrays, with all four (Pax6, Lhx2, Sfrp1 and COUP-TFI) demonstrating the predicted expression patterns (Fig. 1B). Emx2 was detected in a subset of four arrays, and was detected as caudally expressed in all four (data not shown). Thus, the array strategy accurately identified the majority of known protomap components. Statistical analysis of the array data, both within and across timepoints, identified marked rostrocaudal differences in gene expression at each age, including temporally stable differences in gene expression between the rostral and caudal neocortex (Fig. 2).
Given the three rostrocaudal domains of gene expression observed in the
adult neocortex within neurons (Bishop et
al., 2000; Garel et al.,
2003
; Nakagawa et al.,
1999
), one possibility is that the progenitor cells that generate
those neurons are themselves organised into the three rostrocaudal domains
reflected in their gene expression. In order to look for genes differentially
expressed between the rostral, middle and caudal neocortex, we carried out a
further expression screen at E13.5, dissecting the neocortex into thirds along
the rostrocaudal axis (Fig. 2),
and collecting three pools of rostral, middle and caudal tissue. Each pool
contained tissue from four different embryos. Gene expression was compared
between each pair of groups (rostral versus middle; middle versus caudal;
rostral versus caudal) in a set of 18 independent hybridisations. Although we
found many genes to be differentially expressed in rank order between the
rostral, middle and caudal thirds, consistent with graded expression along the
rostrocaudal axis, we did not find any genes with significant peaks or troughs
of expression in the middle of the neocortex.
Differential expression between the rostral and caudal thirds of the neocortex could occur for a number of reasons, including as a result of genes with spatial differences in the expression in progenitor cells or neurons, the developmental gradient across the neocortex and the differing ratio of neurons to progenitor cells along the rostrocaudal axis (related to the developmental gradient). An analysis of temporal changes in gene expression in the neocortex over the period studied here demonstrated some overlap between spatially and temporally expressed transcripts, as had been expected (data not shown).
Genes expressed differentially between the rostral and caudal neocortex are expressed in gradients across the neocortex
A secondary in situ hybridisation screen was carried out to confirm
differential expression along the rostrocaudal axis, to assign expression to
cell types (progenitor cells, newly born neurons, differentiated neurons) and
to assess the nature of the rostrocaudal expression pattern (graded expression
or discrete domain of expression). Thirty-eight genes were selected for this
secondary expression screen according to two criteria: the rank q-value of
differential expression in the combined E11.5/E13.5 dataset, as calculated by
SAM (q-values approximate to false discovery rates), and functional
annotation. In the case of functional annotation, a thorough bioinformatics
annotation of all of the differentially expressed genes was carried out (data
not shown). For putative protomap components, by analogy with other systems,
transcription factors and signalling pathway components were prioritised for
the secondary screen.
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Many transcription factors and signalling proteins are differentially expressed along the rostrocaudal axis during early neocortical development
The set of differentially expressed genes appears to be functionally
diverse, as it includes genes encoding transcription factors, cell surface
proteins and signalling molecules, as well as many unannotated genes. At each
timepoint, over half of the differentially expressed transcripts encode
unannotated genes that encode mRNAs for which there are considerable expressed
sequence tag (EST) or full-length cDNA data, underlining the importance of
previously unstudied genes in cortical development. To analyse the functional
breakdown of the known genes in more detail, we assigned Gene Ontology (GO)
annotation to all genes for which it is currently available (data not shown).
Notably, genes involved in transcriptional regulation and neurogenesis are
highly represented.
Particularly noteworthy is the considerable number of differentially expressed transcription factors. A large set of transcription factors are expressed in high caudal to low rostral gradients, with Lhx2, Emx2, COUP-TFI, COUP-TFII, Hey1, Tcf4 and Fez-like all showing this expression pattern. Conversely, there are several transcription factors expressed in a high rostral to low caudal gradient, including Klf3, Pax6, Irx1, Etv5 (ERM) and Etv1 (ERF/ER81). All of the rostral and caudal transcription factors studied are expressed in neocortical progenitor cells in gradients that extend the entire length of the neocortex.
In addition to transcription factors, there is a marked enrichment of
extracellular signalling molecules and key components of their intracellular
signalling pathways in both rostral and caudal neocortex. Caudal neocortex
shows high expression of two bone morphogenetic proteins, Bmp2 and Bmp4, as
well as Tgfß3 and a key transcriptional regulator in the Bmp pathway,
Smad5. Furthermore, there is high caudal expression of both Lef1 and Tcf4, key
factors in the Wnt pathway that also integrate signals from different
pathways. In rostral neocortex, Fgf15 and the Wnt antagonist Sfrp1 are highly
expressed. Notably, sprouty 2 and Spred1, negative-feedback regulators of Fgf
signalling, are also highly expressed at the rostral pole of the neocortex.
The presence of Fgf15, Sprouty2 and Spred1, along with the Ets transcription
factors Etv5 and Etv1, is indicative of the active Fgf signalling taking place
around the rostral midline of the neocortex at this time in development
(Fukuchi-Shimogori and Grove,
2001; Fukuchi-Shimogori and
Grove, 2003
; Storm et al.,
2003
).
The expression of putative protomap components is regulated by Fgf signalling in the developing neocortex in vivo
Fgf8 signalling has a central role in controlling neocortical pattern
formation, as it is capable of both inducing rostral and repressing caudal
neocortical identities (Fukuchi-Shimogori
and Grove, 2001;
Fukuchi-Shimogori and Grove,
2003
; Grove and
Fukuchi-Shimogori, 2003
;
Ragsdale and Grove, 2001
). In
agreement with this, the expression gradients of caudally expressed genes are
shifted rostrally in the neocortex of Fgf8 hypomorphic mutants
(Garel et al., 2003
). Genes
encoding protomap components that are regulated by Fgf signalling should
change their expression pattern in predictable ways upon altering cortical Fgf
signalling, and this would serve to confirm their involvement in neocortical
patterning. To investigate this, we took an unbiased approach to identify gene
expression changes in the neocortex in response to altered Fgf signalling.
Fgfr1 mutant mice have a very similar phenotype to that of the
Fgf8 hypomorphic mutant, most notably the loss of the olfactory bulbs
(Garel et al., 2003
;
Hebert et al., 2003b
).
Therefore, we carried out a global analysis of changes in gene expression in
the dorsal telencephalon of forebrain-specific Fgfr1 mutant mice.
Gene expression was compared between the dorsal telencephalons of single E12.5
embryos of two key genotypes: Fgfr1 heterozygous and homozygous
mutants that were both heterozygous for the Foxg1 transcription factor, as Cre
recombinase expression is driven off the endogenous Foxg1 locus in
these animals (Hebert et al.,
2003b
; Hebert and McConnell,
2000
). Comparing Foxg1 heterozygous null tissues
controlled for possible changes in cortical gene expression due to
Foxg1 heterozygosity. Comparisons were carried out at E12.5, as this
developmental stage is early in the neurogenetic period, precedes the arrival
of incoming thalamocortical axons and falls between the two stages at which we
carried out the spatial expression screen.
Statistically significant differences in expression between the two
genotypes were identified, including both up- and downregulated transcripts
(Fig. 4). Notably, among the
genes whose expressions were altered in the homozygous null mutants were three
Ets-domain transcription factors, Etv1, Etv5 and ELF2A2, found rostrally
expressed in the array screen (Fig.
4). Two of these, Etv1 and Etv5, have been shown to be regulated
by Fgf8 in vivo (Fukuchi-Shimogori and
Grove, 2003). In addition, several other rostrally expressed
candidate protomap genes, including Mest/Peg1 and Tweety, were downregulated
in the Fgfr1 mutant cortex. Conversely, caudally expressed proposed
patterning genes, most notably the transcription factors COUP-TF1 and Tcf4,
were upregulated in the Fgfr1 mutant cortex
(Fig. 4).
A striking additional finding in the Fgfr1 E12.5 mutant cortex was
the significant upregulation of expression of a set of neurogenic genes,
including many normally expressed in differentiating neurons
(Fig. 4). These genes include
several transcription factor genes, such as Neurod1
(Schwab et al., 1998),
Myt1-like (S. Rahman and F.J.L., unpublished) and Tbr2
(Bulfone et al., 1999
), that
are all expressed in newly born, differentiating neurons. The coherent
upregulation of this set of genes suggests that, in addition to the changes in
spatial gene expression, there is an increase in neurogenesis and neural
differentiation in the E12.5 Fgfr1 mutant cortex.
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Expression of a set of six genes, including known (COUP-TF1, Emx2,
Pax6) and novel potential protomap components (Mest, Klf3,
Hey1), was studied following brief (4 hour) Fgf8 treatment of neocortical
explants in defined media in vitro. Real-time PCR analysis of the expression
levels of the three rostrally expressed genes, Etv5/ERM, Mest and Pax6, found
an Fgf8-induced upregulation of expression of all of these genes within 4
hours. Conversely, of the three caudally expressed genes examined,
COUP-TF1, Hey1 and Emx2, only COUP-TF1 expression
was significantly repressed in neocortical progenitor cells within four hours
of Fgf8 exposure. At this developmental stage, the overwhelming majority of
neocortical cells are progenitor cells with a cell cycle length of
approximately 10 hours (Takahashi et al.,
1996). Together with the short time period over which these
changes occur, we conclude that Fgf8 directly regulates the expression of
Mest within neocortical progenitor cells.
In vivo loss of function of Mest results in abnormal neocortical development, including changes in cortical patterning
To validate the approach taken to identify protomap components, we analysed
neocortical development in mice mutant for one of the strongest candidate
neocortical patterning genes, Mest/Peg1. Mest/Peg1
mRNA is expressed in a rostrocaudal gradient in neocortical progenitor cells
at E11, a gradient that has substantially retracted rostrally by E13 to leave
a relatively small region of the extreme rostral neocortex in which progenitor
cells express Mest. The rostral expression of this gene is dependent
on Fgf8 signalling, as it is significantly downregulated in Fgfr1
mutant cortices in vivo, and is upregulated in neocortical progenitor cells by
Fgf8 treatment in vitro. Notably, adult Mest mutant mice have a
behavioural phenotype in which Mest mutant females fail to nurture
newborns (Lefebvre et al.,
1998). Furthermore, there is an unexplained increase in perinatal
mortality in Mest/Peg1 mutant newborns
(Lefebvre et al., 1998
).
A cellular function for Mest is not suggested by homology searches. Mest encodes a protein composed almost completely of a hydrolase-like domain, with an amino-terminal hydrophobic region that is predicted to act as either a signal peptide or a transmembrane domain (Fig. 6). Such a composition indicates that Mest is an enzyme, but it does not indicate a subcellular localisation or any possible substrates, as this hydrolase fold is very common in prokaryotic and eukaryotic proteins. However, although the cellular function of Mest/Peg1 is unknown, this protein is highly conserved throughout its length in vertebrates (Fig. 6). Notably, an orthologous protein cannot be found in the Drosophila or C. elegans genomes (Fig. 6), suggesting that this particular protein is vertebrate specific.
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|
To characterise the nature of the changes in expression in the Mest mutant, we carried out an analysis of the intersection between genes identified as showing spatial expression at E13 and those genes showing changes in expression in the Mest mutant cortex (Fig. 7). Of the 118 rostrally expressed genes that show altered expression in the Mest mutant cortex, 97 are downregulated and 21 are upregulated (Fig. 7). However, the 21 upregulated genes contain many candidate rostral patterning genes, including the Ets genes Etv1 and Etv5, Klf3 and Fgf15, as well as other rostrally expressed genes such ephrin B2 and ephrin A5. Section in situ hybridisation for the rostrally expressed gene Klf3 confirmed its elevated expression in the rostral region of the Mest mutant cortex relative to that of a wild-type littermate (Fig. 7).
Conversely, of the 84 caudally expressed genes that show altered expression in the Mest mutant cortex, 16 are downregulated and 68 upregulated. However, as for the rostral genes, the smaller set of downregulated genes includes the candidate patterning genes Hey1, Tcf4 and Emx2 (Fig. 7). Two conclusions can be drawn from this analysis: that there appear to be two distinct neocortical phenotypes in Mest mutant embryos, one of which is an alteration in progenitor cell patterning; and that the patterning phenotype consists of an upregulation of rostrally expressed genes accompanied by a downregulation of caudally expressed genes.
Array analysis indicates that Mest may act as a negative regulator of Fgf-regulated neocortical patterning
The genomics screen for protomap components and the subsequent functional
experiments on the Fgf regulation of neocortical gene expression indicate that
the neocortex is patterned by Fgf-regulated gradients of gene expression.
Furthermore, analysis of neocortical gene expression in
Mest/Peg1 mutant E12.5 embryos suggested that Mest/Peg1 is a
potential negative regulator of the Fgf8-controlled cortical patterning
pathway. To test this formally, we carried out a combined analysis of the gene
expression data from both the Fgfr1 and Mest mutant cortices
(Fig. 8).
Statistical testing (t-test) for significant changes in gene expression common to both genotypes found no such changes above those expected by chance (data not shown). However, testing for significant (P<0.05) differences in gene expression changes between the genotypes identified 703 such genes (Fig. 8). A hierarchical cluster of those genes shows that there are over 300 genes upregulated in the Mest mutant that are downregulated in the Fgfr1 mutant, and conversely over 300 genes that show the opposite behaviour (Fig. 8).
The genes showing opposite behaviours in the two mutant genotypes include several candidate rostral and caudal patterning genes (Fig. 8). Notably, the rostral patterning genes show a marked reversal in their expression change in the two mutants, such that genes in this category were upregulated in the Mest mutant and downregulated in the Fgfr1 mutant cortex. These genes include several readouts of Fgf signalling, including Ets genes and sprouty 2, suggesting that there is increased Fgf signalling in the Mest mutant cortex.
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Discussion |
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Gene expression gradients and compartments in the developing neocortex
At least two alternative systems for conferring spatial identities on
neural progenitor cells have been described in other systems. Dorsoventral
gradients of expression of transcription factors have been found in retinal
progenitor cells (Barbieri et al.,
1999; Koshiba-Takeuchi et al.,
2000
; Ohsaki et al.,
1999
; Schulte et al.,
1999
), which are required for the graded expression of the ephrins
and eph receptors in ganglion cell neurons. This in turn is the basis for the
retinotopic mapping of spatial information from retinal ganglion cells onto
the tectum (McLaughlin et al.,
2003
). Within the developing spinal cord, progenitor populations
are defined by the combinatorial expression of several transcription factors
along the dorsoventral axis of the neural tube, with each population giving
rise to defined classes of neurons (Briscoe
et al., 2000
; Jessell,
2000
).
It has been proposed that a similar system of transcriptionally encoded
positional information is likely to operate in neocortical progenitor cells
(Grove and Fukuchi-Shimogori,
2003). Opposing gradients of expression of Pax6 and Emx2 have been
found in the neocortex, and loss of function of either gene results in shifts
in the relative sizes of neocortical areas
(Bishop et al., 2000
;
Mallamaci et al., 2000
).
Alterations in the absolute levels of Emx2 result in shifts in neocortical
areas, leading to the recently proposed cooperative-concentration model for
neocortical patterning, in which it is proposed the spatial identity of a
neocortical progenitor cell is encoded by the absolute levels of expression of
the patterning transcription factors
(Hamasaki et al., 2004
).
Consistent with this, we found little evidence for discrete domains of gene
expression across the field of neocortical progenitor cells, as described in
the developing spinal cord (Briscoe et al.,
2000). The initial genomics screens and the in situ hybridisation
studies together demonstrate that the characteristic patterns of gene
expression across the field of neocortical progenitor cells are gradient
based. We did not find any clear boundaries of gene expression within the
neocortex at these stages. This is in contrast with the sharp gene expression
boundaries between the developing hippocampus, a cortical structure, and
subcortical structures and the neocortex. This is illustrated by the
expression of the transcription factors Fez-like and COUP-TFII: at embryonic
day E13.5, both of these genes have sharp caudal boundaries of expression,
whereas they are expressed in gradients across the neocortex.
Given the comprehensive, unbiased array design used for the screens reported here, the accuracy of which were confirmed by in situ hybridisation data for the majority of the top protomap candidate genes, we conclude that few genes are expressed in discrete domains in the field of neocortical progenitor cells in the first half of the neuronogenetic interval. It is possible that a small number of genes that encode spatial identity are expressed in domains and were not detected by this screen. However, the screens reported here identified almost all of the known genes that show differential expression along the rostrocaudal axis, and the arrays represent over 22,000 genes expressed from the mouse genome, suggesting that gradients are the dominant pattern of gene expression along the rostrocaudal axis of the neocortex at the stages examined.
Fgf8 signalling and the formation of the neocortical protomap
A central tenet of our strategy for identifying protomap components is that
regionally expressed genes have roles in setting up or encoding the protomap,
and that a subset of these genes will be regulated directly or indirectly by
Fgf8 signalling. Several of the genes identified in our screen have been shown
to have roles in neocortical area formation, most notably Pax6, Emx2
and COUP-TFI (Bishop et al.,
2000; Mallamaci et al.,
2000
; Zhou et al.,
2001
). By expression profiling of the neocortices of
Fgfr1 mutant mice, we found that many genes showing patterned gene
expression in the neocortex (some of which encode proposed protomap
components) are regulated by Fgf signalling in vivo. However, we also found a
marked change in the expression of neurogenic and neural differentiation
genes, suggesting that there are alterations in progenitor cell proliferation
or neurogenesis, or both, in this mutant. Such a phenotype is similar to that
observed in the neocortex of Fgf2 mutant mice, in which neocortical
progenitors generate too many neurons at the expense of the progenitor cell
pool early in neocortical neurogenesis
(Korada et al., 2002
),
resulting in a reduction in total numbers of glutamatergic neurons in the
mature frontal cortex. Similarly, overexpression of a dominant-negative Fgfr1
in the early developing cortex results in an overall reduction of pyramidal
neurons in the adult frontal cortex (Shin
et al., 2004
).
Those findings raised the possibility that Fgf signalling may indirectly regulate cortical patterning by controlling the relative growth of the frontal cortex. We therefore tested whether Fgf8 could directly regulate neocortical progenitor cell expression of known and proposed neocortical patterning genes, finding that Fgf8 could induce or repress expression of a subset of such genes by as much as twofold within 4 hours. Together with the in vivo analysis of Fgf-regulated gene expression, we conclude that Fgf8 can directly regulate the graded expression of protomap components.
Mest/Peg1, an Fgf8-regulated gene, is a part of the rostral neocortex patterning system
To validate the strategy taken to identify protomap components, we carried
out a functional in vivo analysis of a leading candidate for a gene involved
in protomap formation, Mest/Peg1. This gene is rostrally
expressed normally, is induced almost twofold by Fgf8 treatment for 4 hours in
vitro and is downregulated in the Fgfr1 mutant. Mest loss of function
results in striking changes in neocortical patterning during development: a
set of rostrally expressed genes, downregulated in the Fgfr1 mutant,
is upregulated, whereas caudally expressed genes are downregulated.
Of the rostrally expressed genes upregulated in the Mest mutant, several are well-characterised targets genes for Fgf signalling, including the Ets transcription factor genes Etv1 and Etv5 and the Fgf-induced negative regulator sprouty 2. By contrast, all of these genes are downregulated in the Fgfr1 mutant neocortex. These opposite phenotypes are consistent with Mest being part of a pathway that negatively regulates Fgf-mediated rostral patterning in the neocortex. However, the cellular function of Mest is not clear from the nature of the Mest protein. The predicted signal peptide/transmembrane domain indicates that Mest may act in an intracellular compartment or may act extracellularly. Future experiments will clarify the cellular localisation and possible function of Mest as a negative regulator of Fgf signalling.
Rostrocaudal gradients and the nature of the neocortical protomap
The gradient-based system identified here is consistent with previous gene
expression analyses in primates (Donoghue
and Rakic, 1999a; Donoghue and
Rakic, 1999b
). In situ hybridisation studies of a set of genes
identified the characteristic spatial expression pattern across the field of
neocortical progenitor cells as being gradient based, although there was
strong evidence for domains of gene expression in developing neurons
(Donoghue and Rakic, 1999a
;
Sestan et al., 2001
), as also
seen in rodents (Miyashita-Lin et al.,
1999
; Nakagawa et al.,
1999
), corresponding to anatomical and functional neocortical
areas.
Further support for a gradient-based system for positional information
comes from in vivo manipulation of Fgf8 levels in the developing neocortex,
which alters the position of neocortical areas along the rostrocaudal axis
(Fukuchi-Shimogori and Grove,
2001). Detailed analyses of the morphology of one major component
of the somatosensory cortex, the whisker barrels, revealed that this
manipulation does not simply shift areas, but also alters their relative
proportions. In the case of the whisker barrels, the individual whisker
barrels are expanded or compressed with the reduction or augmentation of the
endogenous Fgf8 source (Fukuchi-Shimogori
and Grove, 2001
), leading to the suggestion that progenitor cell
spatial identity may be gradient based
(Grove and Fukuchi-Shimogori,
2003
).
Patterning of developing structures by gradients of extracellular factors
and morphogens is a common theme in many developmental systems, including the
neural tube and developing limb (Echelard
et al., 1993; Pearse and
Tabin, 1998
; Placzek et al.,
1991
; Riddle et al.,
1993
; Roelink et al.,
1994
). However, a classical question in developmental biology is
how such graded signals generate cells with discrete positional identities, as
reflected in the spatial expression of Hox genes in the developing limb
(Johnson et al., 1994
). This
is distinct from the findings on neocortical progenitor cell spatial identity,
which suggest that these cells are not organised into discrete spatial
populations based on graded extracellular signals. Rather, it appears that
each progenitor has a unique spatial identity, based on the gradient of gene
expression across the field of neocortical progenitor cells. Recent data have
clearly demonstrated that altering the nature of the gradient of expression of
the protomap gene Emx2, rather than removing its expression
completely, leads to shifts in both the position and morphology of neocortical
areas (Hamasaki et al., 2004
).
In this case, it appears that the absolute level of Emx2 expression
at any point along the gradient is a key regulator of neocortical pattern
formation (Hamasaki et al.,
2004
).
In conclusion, from the combined genomic and genetic analysis presented here, we propose that the protomap is composed of transcription factors with gradients of expression across the field of neocortical progenitor cells. Furthermore, we conclude that a key element in the nature of the protomap is gradient-based positional information within neocortical progenitor cells, the encoding of which is controlled via an Fgf8-regulated pathway of which Mest is a potential negative regulator.
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ACKNOWLEDGMENTS |
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