BACKGROUND
down syndrome  results from the presence in three copies of human chromosome  <dig>  the smallest human autosome containing about  <dig> known protein-coding genes  <cit> .
the mechanisms by which this aneuploidy produces the complex and variable phenotype observed in ds patients are still under discussion.
the use of large scale gene expression methods such as microarrays were expected to shed light on which genes  contribute to the ds phenotype as well as to the phenotypic variability.
for the genes on chromosome  <dig>  all studies have confirmed a general increase of transcription following the chromosomal imbalance, or "primary gene dosage effect".
rna samples prepared from cells or tissues of ds patients or mouse models showed a global over-expression of the three-copy genes  <cit> .
however, even if the mean over-expression was generally reported to be close to the expected value of  <dig> , recent studies in ds cell lines have reported that about 70% of the three-copy genes were significantly below the  <dig>  ratio.
in these particular cell lines at least, a large proportion of the chromosome  <dig> transcripts were compensated for the primary gene dosage effect  <cit> .
as for non-chromosome  <dig> genes, results are less consistent.
the aneuploidy of an entire chromosome could affect the expression of either a limited number genes or a large number in a more random and extensive way  <cit> .
this question is still debated and more comprehensive studies assessing the variability among samples, tissues and development stages are needed.
we thus designed a large scale gene expression study in which we could measure the effects of trisomy  <dig> on a large number of samples in a tissue that is affected in ds where we could quantify the defect during postnatal development in order to correlate gene expression changes with the phenotype observed.
the ts1cje mouse model of ds is a segmental trisomy of mouse chromosome  <dig> with many genes orthologous to human chromosome  <dig> present in three copies .
this mouse model has the advantage of being available as large colonies of mice on b6c3snf1/orl mixed genetic background and rapidly screened  <cit> .
we focused our study on cerebellum since adult ts1cje mice show a reduction in cerebellar volume that parallel the observations in ds patients and in another mouse model of ds   <cit> .
the reduced size of the cerebellum and the reduced cerebellar granule cell number in ts65dn adults originate around birth because of a defect in granule cell precursor proliferation  <cit> .
in the present study four early postnatal time points that are crucial for cerebellar development were investigated which could provide a read-out of genes involved in cerebellar hypoplasia in ds.
these four time points correspond to birth  and postnatal days  <dig> ,  <dig>  and  <dig> .
during this time period granule cells proliferate and migrate from the external to the internal granule cell layer and purkinje cells start differentiating and growing their highly dense dendritic tree.
we quantified the proliferation of granule cell precursors on fixed cerebellum slices of ts1cje and euploid mice at p <dig>  p <dig> and p <dig> using immunohistochemistry and histology.
a significant 30% decrease of their mitotic index was observed at p <dig> but not at p <dig> and p <dig>  in agreement with the results obtained in ts65dn mice  <cit> .
finally and in order to find gene expression variations in granule cell precursor rich cerebellar regions, external granule cell layers of newborn ts1cje and euploid mice were dissected and analyzed on microarrays.
RESULTS
the cerebellar volume in ts1cje adult mice is reduced
using magnetic resonance imaging  we measured the volumes of whole brain and cerebellum of six male adult ts1cje and nine euploid littermates with a very high-resolution of  <dig> μm .
total brain volumes were not different between trisomic and euploid mice .
however we found a significant 16% decrease in the cerebellum volume of ts1cje mice as compared to euploids .
average volumes are given in cubic mm; cb = cerebellum; brain volume includes cb.
normalized cb refers to the cb volume divided by the total brain volume.
granule cell proliferation is decreased in newborn ts1cje mice
granule cell proliferation was measured in the cerebellum of ts1cje and euploid mice at p <dig>  p <dig> and p <dig> at a time when granule cell precursors proliferate in the external granule cell layer.
cryostat sections of cerebellar cortex were immunostained using an antibody against ki <dig> , a specific cellular proliferation marker  <cit> .
mitotic index was calculated as the number of ki67-positive cells divided by the total cell number determined by cresyl violet counterstaining.
we observed a significant  <dig> % reduction of the mitotic index in the external granule cell layer of ts1cje mice at p <dig>  but not at p <dig> and p <dig> .
this result is similar to the 22% decrease found in the cerebellum of ts65dn mice at birth but not at p <dig> using hematoxylin and cresyl violet stainings  <cit> .
altogether these data suggest a similar early event occurring around birth that affects granule cell proliferation in the cerebellum of ts1cje and ts65dn mice.
we did not observe any change in the number of pyknotic nuclei between ts1cje and euploid mice .
gene expression changes in the cerebellum of ts1cje and wild type mice between p <dig> and p10
in order to find genes or pathways involved in the cerebellar hypoplasia, we analyzed gene expression in the cerebellum of ts1cje and euploid mice at p <dig>  p <dig>  p <dig> and p <dig> using pangenomic two-color microarrays containing  <dig>  <dig> probes representing approximately  <dig>  <dig> mouse genes.
the main objective was to detect differentially expressed genes between ts1cje and euploid samples.
thus we hybridized ts1cje versus euploid samples at each postnatal developmental stage .
in order to estimate the impact of postnatal development on gene expression while still evaluating the difference between ts1cje and euploid samples with a minimum of experiments, we performed an additional series of comparisons between ts1cje and euploid samples at two successive stages  .
fifty-six samples from individual cerebella were hybridized on  <dig> microarrays.
to detect genes differentially expressed during cerebellar postnatal development and/or in ts1cje mice, we performed an analysis of variance  with two factors: the postnatal developmental stage  and the genotype .
in addition we compared the expression of  <dig> previously selected genes in ts1cje and euploid samples at p <dig> using quantitative real-time pcr .
our model considers the effects of  <dig> factors: the development  and the trisomy effect .
μ represent the mean gene expression.
effect of trisomy on gene expression at p <dig>  p <dig>  p <dig> and p10
in ts1cje mice at p <dig>  p <dig>  p <dig> and p <dig>  between  <dig> and  <dig> genes were found to be significantly differentially expressed .
the differentially expressed genes represented between  <dig> % to  <dig> % of the expressed genes present on the microarray.
these results indicate that trisomy does not lead to a significant global change in gene expression during early cerebellar postnatal development.
number of differentially expressed genes in ts1cje mice at p <dig>  p <dig>  p <dig> and p <dig>  and during normal cerebelelar development p0–p <dig>  p0–p <dig> and p0–p <dig>  obtained by anova.
we then listed genes that were dysregulated in ts1cje cerebella at all four postnatal development stages studied and found only  <dig>  with  <dig> up-regulated genes .
among them,  <dig> are triplicated genes.
one of them, son or 'son cell proliferation protein', had been previously reported as a candidate gene for ds cerebellar phenotype  <cit> .
all ratios have a p-value <  <dig>  .
in bold are three-copy genes.
effect of development  on gene expression
between p <dig> and p <dig>  p <dig> and p <dig> and p <dig> and p <dig>  the following number of genes were differentially expressed, respectively,  <dig>   <dig> and  <dig>  representing about  <dig>  to  <dig> % of expressed genes .
these results demonstrate that postnatal development impacts on the expression of a greater number of genes than trisomy does, as already seen in previous analysis  <cit> .
combined effects of trisomy and postnatal development
a high proportion of genes whose expression is regulated during cerebellar postnatal development were also differentially expressed in ts1cje mice at least at one time point .
among these,  <dig> are triplicated genes: girk <dig>  olig <dig> and dscam .
girk <dig> is the only triplicated gene differentially expressed during cerebellar postnatal development and constantly over-expressed in ts1cje mice between p <dig> and p <dig>  girk <dig>  also know as kcnj <dig>  is a voltage-gated potassium channel mainly expressed in granule cells of the cerebellum  <cit> .
a point mutation of girk <dig> in weaver mice  leads to granule cell degeneration  <cit> .
olig <dig> is a transcription factor involved in neuron fate commitment  <cit>  and dscam is a cell-surface receptor implicated in cell adhesion  <cit> .
expression ratio between trisomic and euploid mice  and betwen two time points .
.
ns: not significant.
effect of trisomy on the expression of genes involved in neuron proliferation
in order to identify specific pathways involved in cerebellar postnatal development and disrupted in ts1cje mice, we performed qpcr experiments on a selection of genes using the cerebellum of  <dig> ts1cje mice and  <dig> euploid mice from three litters at birth.
the list contained  <dig> genes belonging to pathways involved in cell proliferation and differentiation of the cerebellum or directly in the ds cerebellar phenotype  <cit> : the notch signalling pathway  <cit> , numb  <cit> , gli <dig>  gli <dig> and ptch <dig> involved in the shh signaling pathway  <cit> , nfat <dig> and nfat <dig>  <cit>  and rcan <dig>  <cit>  regulating ca2+ signalling and son and hmgn <dig>  two triplicated genes that have been proposed to be candidate genes for the cerebellar phenotype  <cit> .
expression ratios between ts1cje and euploid cerebellum are shown in figure  <dig>  the six triplicated genes tested were significantly over-expressed with a mean ratio greater than  <dig> .
surprisingly, of the  <dig> euploid genes tested,  <dig> were found to be slightly but significantly over-expressed: shh  and its receptor ptch <dig> .
major primary effect of trisomy exists in the external granule cell layer
in order to find gene expression variations in specific regions of the cerebellum where proliferation is impaired in ts1cje mice, external granule cell layers of  <dig> newborn ts1cje and  <dig> euploid mice from three litters were dissected and analyzed individually on microarrays.
we used illumina pangenomic microarrays containing more than  <dig>  <dig> transcripts corresponding to about  <dig>  <dig> genes.
gene expression data were analyzed using t-test .
of the  <dig>  <dig> expressed genes only 4%  were found to be significantly differentially expressed, with ratios between  <dig>  and  <dig> .
this low percentage was similar to those observed in the entire cerebellum at p <dig>  p <dig>  p <dig> and p <dig>  as shown on figure 3a, the distribution of expression ratios for euploid genes was approximately  <dig>   while the distribution of the three-copy genes was shifted towards higher values corresponding to the gene dosage effect .
figure  <dig> shows the mean expression ratios for each individual chromosome.
only the part of mmu <dig> present in three copies in ts1cje  gave a mean expression ratio that was significantly different from  <dig>  .
no difference was observed in the expression ratio variances, particularly between the mmu16b and all other chromosomes.
the distributions of p-values were also very different for the euploid and the three-copy genes.
for the euploid genes the p-value distribution was uniform indicating that very few genes were differentially expressed.
however for the three-copy genes, all p-values were below  <dig>  with a higher proportion under  <dig> .
all three-copy genes expressed in the external granule cell layer at p <dig> were found to be significantly over-expressed  .
in order to reduce the number of false positives that occur during multiple testing, we applied the false discovery rate  controlling procedure of benjamini and hochberg on the t-test p-values  <cit> .
as reported in table  <dig>  we found only  <dig> significantly dysregulated genes .
of these  <dig> genes,  <dig> are three-copy genes, representing more than 70% of the dysregulated genes.
the other nine are located on eight other chromosomes and are related to ion transport, signal transduction, cell proliferation or dna processes.
altogether these results show a major primary gene dosage effect on the three-copy genes.
this effect was also clearly detected using principal component analysis .
pca is a technique used to represent multidimensional data sets along principal component axes that reffect the degree of variance in the data.
ts1cje and euploid samples were grouped separately only when considering the three-copy genes .
<dig> genes were found significantly differentially expressed by t-test  followed by false discovery rate controlling procedure of benjamini and hochberg .
among them,  <dig> are three-copy genes, indicated in bold.
twelve three-copy genes were found significantly differentially expressed by student t-test  but failed the false discovery rate controlling procedure of benjamini and hochberg .
a partial monosomy of mmu <dig> is present in ts1cje mice
among the  <dig> genes whose expression was down-regulated in the external granule cell layer of ts1cje mice,  <dig> were located on the telomeric part of mmu12: cdca7l and sp <dig> .
their expression ratios  were  <dig>  and  <dig>  respectively.
since ts1cje mice are the result of the translocation of the distal part of mmu <dig> from sod <dig> to znf <dig> to the telomeric part of mmu <dig>  we suspected a deletion of these two down-regulated genes.
to confirm this chromosomal rearrangement, a comparative genomic hybridization using high resolution  agilent genomic microarrays was performed.
a 50% reduction of the genomic content corresponding to the chromosomal segment on mmu <dig> located between positions  <dig>  <dig>  <dig> and  <dig>  <dig>  <dig> was found .
the deleted part represented about  <dig> mb and contained  <dig> genes: dnahc <dig>  sp <dig>  sp <dig>  abcb <dig> and itgb <dig>  all of them are related to either neuronal proliferation or maturation, embryogenesis or peripheral and central nervous system development.
the additional translocated mmu <dig> segment started at position  <dig>  <dig>  <dig>  just proximal to the sod <dig> gene  and spanned to position  <dig>  <dig>  <dig> at the telomeric end of mmu <dig>  this genomic interval represented about  <dig> mb .
discussion
ds is caused by an aneuploidy corresponding to the trisomy or partial trisomy of chromosome  <dig>  the publication of the dna sequence of the human genome revealed the presence of about  <dig> genes on chromosome  <dig>  although this number is low as compared to other human autosomes, it is still too large to develop transgenic mouse models for all of them.
moreover transgenic mouse models for single genes are not adequate to address fundamental questions on gene interactions.
thus in order to find the genes that contribute to the ds phenotype and to the phenotypic variability, gene expression profiling has been applied to mouse models with segmental duplications of dna segments orthologous to human chromosome  <dig>  the studies published so far were conducted in different mouse models, on different tissues and at various developmental stages.
in addition the technology and the statistical tools were different thereby reducing the possibility of grouping the analysis and making insights into the genotype-phenotype status of ds.
we thus designed a gene expression study in which we could measure the effects of trisomy  <dig> in the ts1cje mouse model, on a large number of samples, in a tissue that is affected in ds where we could quantify the effetcs during postnatal development in order to correlate gene expression changes to the phenotype observed.
ds affects many aspects of brain development and represents the most common cause of inherited human mental retardation.
motor deficits are among the most frequently occurring features of ds.
individuals with ds exhibit hypotonia and motor dysfunction that could be related to cerebellar dysfunction  <cit> .
ds patients present a consistent reduction of the total brain volume with a disproportionally greater reduction in the cerebellum  <cit> .
one of the major findings in ts65dn and ts1cje mouse models of ds carrying segmental duplications, is the significant reduction in cerebellar volume paralleling that observed in ds patients  <cit> .
this cerebellar hypoplasia likely results from a decrease in the density of granule cells, the most abundant neuronal cell population.
moreover, behavioural studies have shown motor dysfunctions in ts65dn that are more severely affected in tasks involving a high degree of balance and coordination  <cit> .
this hypoplasia occurs during cerebellar postnatal development in the first weeks after birth and does not result from a neurodegenerative process  <cit> .
more recently, a reduction of about 20% in the number of granule cell precursors has been reported at birth  and  <dig> days after birth  in the cerebellar external granule cell layer of ts65dn mice  <cit> .
this reduction is much more significant at p <dig>  than at p <dig> .
in the present study we have shown a 17% significant decrease of volume in the cerebellum of adult ts1cje mice and a  <dig> % significant decrease of granule cell precursor proliferation only at birth and not at p <dig> or p <dig>  we analyzed global gene expression during postnatal development of the cerebellum in ts1cje mice at the time when major developmental changes occur: at birth  at p <dig>  p <dig> and p <dig>  during this period granule cells proliferate, migrate from the external to the internal layer of the cerebellum, and differentiate.
our previous study performed at p <dig>  p <dig> and p <dig> showed a clear over expression of triplicated genes in the cerebellum of ts1cje mice and a small effect on the expression of euploid genes  <cit> .
however since the proliferative external granule cell layer disappears by p <dig>  <cit> , this first study was not designed to detect gene expression changes between p <dig> and p <dig>  to increase the statistical power of gene expression analysis, we used a large number of samples:  <dig> cerebella from ts1cje and euploid mice bred on the mixed  genetic background.
we performed an anova with a type i error risk of 5% and we found between  <dig> % to  <dig> % of genes regulated at each time point including some three-copy genes.
then we applied a false discovery rate  with a  <dig>  threshold  on the gene list and found no significant gene at all time points, not even three-copy genes.
by increasing the fdr threshold up to  <dig>  we found only  <dig> significant genes among which  <dig> are three-copy genes .
these results suggest a limited secondary effect of trisomy on euploid genes.
most three-copy genes were not found significantly differentially expressed suggesting that compensatory mechanisms take place during postnatal development.
only  <dig> three-copy genes were constantly over-expressed in the cerebellum of ts1cje mice at p <dig>  p <dig>  p <dig> and p <dig>  using qpcr, we confirmed the increased expression of six three-copy genes at p <dig>  four of these genes  were also found over-expressed in the study of mao et al.
conducted on ds foetal tissues, which included three cerebella  <cit> .
we then performed a second transcriptome analysis using dissected cerebellar regions enriched in granule cell precursors from  <dig> ts1cje and nine euploid mice at p <dig>  at the time when we observed a significant decrease of cell proliferation.
about 4% of all genes were significantly differentially expressed in ts1cje samples as compared to euploid samples.
this value was close the ones obtained in the entire cerebellum at p <dig>  p <dig>  p <dig> or p <dig>  all the expressed three-copy genes  were significantly over-expressed in ts1cje with 12genes showing expression ratio significantly lower than  <dig>  .
after multi-test correction using the fdr procedure,  <dig> genes were still significantly modulated in ts1cje mice.
among them  <dig> are three-copy genes, indicating a major primary effect of trisomy on the three-copy genes.
from the euploid genes,  <dig> map to a chromosomal deletion  and are under-expressed.
the other seven are not well characterized and could be new gene candidates for cerebellar ds phenotypes and cerebellum postnatal development.
by using an enriched and more homogeneous cell population , we hoped to clarify the secondary effects of trisomy.
surprisingly, not only did we observed a main primary gene dosage effect  but we also identified for the first time a deletion in the ts1cje mice .
alternatively it is possible that the secondary effect of trisomy is variable among cells or since we demonstrated a proliferation defect in the granule cell precursors, it is plausible that we failed to identify cell cycle-regulated genes because cells from tissue are not synchronized.
in addition to the three-copy genes, we were able to detect a gene dosage effect on two genes mapping to mouse chromosome  <dig> , close to the translocation breakpoint of the trisomic segment.
a  <dig> mb segment of mmu <dig> was found to be deleted in ts1cje mice.
this segment contains  <dig> genes: dnahc <dig>  sp <dig>  sp <dig>  abcb <dig> and itgb <dig>  sp <dig> is a member of a family of zinc finger transcription factors and is highly expressed in the developing hippocampus and cerebellum, in particular in granule cells  <cit> .
knock-down of sp <dig> leads to an increased number of highly branched dendrites during maturation of granule cells in the cerebellar cortex  <cit> .
a reduced cell proliferation in hippocampus but not cerebellum has also been reported in sp <dig> null mutant newborns  <cit> .
thus, sp <dig>  the only gene found expressed in the external granule cell layer is more likely to affect neuron maturation than proliferation.
however it is unlikely that the cerebellum phenotype observed in ts1cje mice results from the mmu <dig> deletion since a very similar phenotype is present in the ts65dn mouse model that do not show any rearrangement on mmu <dig>  we found a decrease in cdca7l gene expression, a transcription factor involved in cell apoptosis  <cit>  and medulloblastoma transformation  <cit> .
since cdca7l is located approximately  <dig> kb proximal to the translocation breakpoint on mmu <dig>  we concluded that the gene dosage effect extends to regions that are close to the chromosomal rearrangement.
we then looked for genes that were regulated during normal postnatal development of the cerebellum and that were also differentially expressed between ts1cje and euploid cerebellum at one of the postnatal stages tested.
among the  <dig> genes found, only three were triplicated: olig <dig>  dscam and girk <dig>  olig <dig> encodes a basic helix-loop-helix transcription factor that is expressed in both the developing and mature vertebrate central nervous system.
olig <dig> has critical function during the formation of motor neurons and oligodendrocytes of the ventral neural tube .
dscam encodes an axon guidance molecule that is expressed by neurons in the central nervous system during development and throughout adult life.
its expression in the developing cerebellum is stronger in purkinje neurons.
a dscam mutation in mice leads to a subtle defect in the caudal folium of the cerebellum  <cit> .
girk <dig> is the only gene over-expressed in ts1cje across postnatal development of the cerebellum.
it is also over-expressed in the external granule cell layer of ts1cje newborns.
girk <dig> is a member of the g protein-gated inwardly rectifying potassium channel family that regulates cellular excitability and neurotransmission.
it is mainly expressed in the cerebellum granule cells  <cit> .
a missense mutation in girk <dig> leads to abnormalities of the cell cycle and apoptosis in the external granule cell layer of the cerebellum in the weaver mice  <cit> .
the girk <dig> mutated gene encodes channels that exhibit loss of potassium selectivity.
in contrast, girk <dig> knock-out mice are morphologically indistinguishable from wild-type mice, suggesting that the weaver phenotype is likely due to abnormal girk <dig> function  <cit> .
ion channels can regulate cell cycle and implication of transmembrane potassium fluxes via inward rectifier channels in the regulation of cell cycle has been proposed  <cit> .
moreover it has been hypothesized that over-expression of the girk <dig> subunit in trisomic mice will likely produce a hyperpolarization of the membrane  <cit> .
depolarization enhances calcium entry via voltage-sensitive ca2+ channels and activates calmodulin kinase and calcineurin phophatase.
the activation of calcineurin induces many genes encoding extracellular and intracellular signalling molecules involved in granule cell development.
<cit> .
thus, hyperpolarization of the membrane may prevent or reduce calcium entry in cells and decrease cell proliferation.
to further identify gene candidates for the ds cerebellar phenotype, we selected gene candidates involved in genetic regulation of the proliferation of the granule cell precursors  <cit>  and looked at their expression in the cerebellum of ts1cje and euploid mice at birth.
sonic hedgehog  is secreted by purkinje cells and regulates proliferation of the granule cell progenitors  <cit> .
the mitotic response of cultured granule cell precursors to shh was shown to be reduced in ts65dn mice as compared to euploids  <cit> .
systemic treatment of newborn ts65dn mice with an agonist of the sonic hedgehog pathway increased the mitotic index of trisomic granule cell precursors and even restored the granule cell population in one week.
however, to date, genes involved in the shh pathway have not been assigned to human chromosome  <dig> nor have they been shown to be differentially expressed in trisomic mice or in people with ds.
thus the positive effect of treatment with agonists of the shh pathway could result from an over-stimulation of shh receptors that are present on granule cell precursors.
it remains to be shown whether this positive effect will normalize the cerebellar volume and improve the cerebellar-related behavioural deficits observed in ts65dn mice  <cit> .
other genes have been reported to regulate the proliferation of granule cell progenitors.
some interact with the shh pathway, such as numb which is a suppressor of shh signaling  <cit> .
numb also regulates notch <dig> pathway  <cit> .
notch signalling is another crucial development-regulated pathway that appears to maintain cells in an undifferentiated state in the early mammalian central nervous system  <cit> .
in addition, we tested other genes related to granule cell proliferation or neural proliferation including igf-i  <cit> , bfgf  <cit> , pax <dig>  <cit> , olig <dig> and olig <dig>  <cit> .
we were not able to show any consistent difference of expression for any of these genes except a slight but significant over-expression of shh.
functional profiling of the differentially expressed genes from microarray experiments didn't reveal any enrichment in gene ontology categories related to cell proliferation.
two genes over-expressed in the external granule cell layer of ts1cje mice at birth, s100a <dig> and dlk <dig>  encode calcium ion binding proteins.
s100a <dig> belongs to the s <dig> family proteins that play an important role in cell growth, differentiation, and motility through calcium-dependent signalling pathways  <cit> .
s100a <dig> encodes the calcyclin protein whose expression is associated with neuronal differentiation  <cit> .
this gene is thus a candidate for the cerebellar hypoplasia.
dlk <dig>  is a transmembrane and secreted protein from the epidermal growth factor-like homeotic family.
dlk <dig> expression is increased in gliomas and over-expression in transfected cells promotes cell proliferation by inducing the expression of cyclin d <dig>  cdk <dig>  and e2f <dig>  <cit> .
however, we could not detect any change in expression of any of these genes suggesting that either dlk <dig> has no effect on granule cell proliferation or that the effect is not visible because of lack of cell synchronization in tissues.
recently, the generation of a collection of haploid yeast strains that each bear an extra copy of one or more of almost all of the yeast chromosomes has been published  <cit> .
their characterization revealed that aneuploid strains share a number of phenotypes, including defects in cell cycle progression, that are independent of the identity of the individual extra chromosomes.
it was thus proposed that disruption of cell homeostasy in ds could be due to the additional chromosomal material rather than the gene content  <cit> .
however aneuploidy is a condition frequently found in tumor cells which often display high rate of proliferation, suggesting that yeast and mammalian cells can respond differently to aneuploidy.
moreover in ds, reduced proliferation rates have not been observed in all proliferative cells across development and in adulthood.
we show here a reduced proliferation of granule cell precursors only at birth.
several studies have reported evidence of neurogenesis impairment in the developing neocortex, in the dentate gyrus of ds foetuses and in mouse models  <cit> .
in the dentate gyrus, cell proliferation is decreased in aged but not young adult ts65dn mice  <cit>  suggesting a neurodegenerative related process occurring in trisomic mice.
another study has reported a lower cell proliferation due to cell cycle alteration in the dentate gyrus of foetuses with ds and in mouse models.
using different markers of proliferation the authors showed that both ds foetuses and p <dig> ts65dn mice have a higher number of proliferating cells in g <dig> and a smaller number of cells in the m phase of the cell cycle  <cit> .
a similar result was reported in the forebrain of ts65dn embryos  <cit>  where delayed expansion of neocortical layers and reduced growth of the hippocampus were found to be correlated with a slower cell cycle.
interestingly, their results suggest that the cell cycle abnormality, occurring in the trisomic ventricular zone of the neocortex but not in the hippocampus, might be developmentally regulated or compensated during neurogenesis.
thus, neurogenesis impairment occurs in different areas of the developing brain in trisomy  <dig> but not continuously, suggesting that genes involved might be different depending on the brain structure and the developmental stage.
CONCLUSIONS
high throughput gene expression analysis in the cerebellum of a large number of ts1cje and euploid mice demonstrates a prevailing gene dosage effect of trisomy and a limited secondary effect across postnatal development.
moreover by using an enriched cell population involved in cerebellar hypoplasia in ds we were not able to detect a global change in gene expression.
80% of gene expression differences were attributed to dosage imbalance, suggesting that the three-copy genes are likely to be directly responsible for the phenotype present in cerebellum.
by correlating the effects of trisomy and those of postnatal development on gene expression, we identified only  <dig> three-copy genes that are constantly over-expressed in ts1cje mice during early postnatal development of the cerebellum, and only  <dig> three-copy genes that are regulated during normal postnatal development of the cerebellum and are over-expressed in the ts1cje mice.
only girk <dig> is common to both of these groups suggesting that this inwardly rectifying potassium channel expressed in granule cells in the cerebellum could be involved in cerebellar hypoplasia in ds.
we propose that reducing the expression of girk <dig> in the ts1cje mice could help to recover a normal cerebellum phenotype.

 down syndrome results from the presence in three copies of human chromosome the smallest human autosome containing about known protein-coding genes. the mechanisms by which this aneuploidy produces the complex and variable phenotype observed in ds patients are still under discussion. the use of large scale gene expression methods such as microarrays were expected to shed light on which genes contribute to the ds phenotype as well as to the phenotypic variability. for the genes on chromosome all studies have confirmed a general increase of transcription following the chromosomal imbalance, or "primary gene dosage effect". rna samples prepared from cells or tissues of ds patients or mouse models showed a global over-expression of the three-copy genes. however, even if the mean over-expression was generally reported to be close to the expected value of, recent studies in ds cell lines have reported that about 70% of the three-copy genes were significantly below the ratio. in these particular cell lines at least, a large proportion of the chromosome transcripts were compensated for the primary gene dosage effect. as for non-chromosome genes, results are less consistent. the aneuploidy of an entire chromosome could affect the expression of either a limited number genes or a large number in a more random and extensive way. this question is still debated and more comprehensive studies assessing the variability among samples, tissues and development stages are needed. we thus designed a large scale gene expression study in which we could measure the effects of trisomy on a large number of samples in a tissue that is affected in ds where we could quantify the defect during postnatal development in order to correlate gene expression changes with the phenotype observed. the ts1cje mouse model of ds is a segmental trisomy of mouse chromosome with many genes orthologous to human chromosome present in three copies. this mouse model has the advantage of being available as large colonies of mice on b6c3snf1/orl mixed genetic background and rapidly screened. we focused our study on cerebellum since adult ts1cje mice show a reduction in cerebellar volume that parallel the observations in ds patients and in another mouse model of ds. the reduced size of the cerebellum and the reduced cerebellar granule cell number in ts65dn adults originate around birth because of a defect in granule cell precursor proliferation. in the present study four early postnatal time points that are crucial for cerebellar development were investigated which could provide a read-out of genes involved in cerebellar hypoplasia in ds. these four time points correspond to birth and postnatal days, and. during this time period granule cells proliferate and migrate from the external to the internal granule cell layer and purkinje cells start differentiating and growing their highly dense dendritic tree. we quantified the proliferation of granule cell precursors on fixed cerebellum slices of ts1cje and euploid mice at p p and p using immunohistochemistry and histology. a significant 30% decrease of their mitotic index was observed at p but not at p and p in agreement with the results obtained in ts65dn mice. finally and in order to find gene expression variations in granule cell precursor rich cerebellar regions, external granule cell layers of newborn ts1cje and euploid mice were dissected and analyzed on microarrays.  the cerebellar volume in ts1cje adult mice is reduced using magnetic resonance imaging we measured the volumes of whole brain and cerebellum of six male adult ts1cje and nine euploid littermates with a very high-resolution of μm. total brain volumes were not different between trisomic and euploid mice. however we found a significant 16% decrease in the cerebellum volume of ts1cje mice as compared to euploids. average volumes are given in cubic mm; cb = cerebellum; brain volume includes cb. normalized cb refers to the cb volume divided by the total brain volume. granule cell proliferation is decreased in newborn ts1cje mice granule cell proliferation was measured in the cerebellum of ts1cje and euploid mice at p p and p at a time when granule cell precursors proliferate in the external granule cell layer. cryostat sections of cerebellar cortex were immunostained using an antibody against ki, a specific cellular proliferation marker. mitotic index was calculated as the number of ki67-positive cells divided by the total cell number determined by cresyl violet counterstaining. we observed a significant % reduction of the mitotic index in the external granule cell layer of ts1cje mice at p but not at p and p. this result is similar to the 22% decrease found in the cerebellum of ts65dn mice at birth but not at p using hematoxylin and cresyl violet stainings. altogether these data suggest a similar early event occurring around birth that affects granule cell proliferation in the cerebellum of ts1cje and ts65dn mice. we did not observe any change in the number of pyknotic nuclei between ts1cje and euploid mice. gene expression changes in the cerebellum of ts1cje and wild type mice between p and p10 in order to find genes or pathways involved in the cerebellar hypoplasia, we analyzed gene expression in the cerebellum of ts1cje and euploid mice at p p p and p using pangenomic two-color microarrays containing probes representing approximately mouse genes. the main objective was to detect differentially expressed genes between ts1cje and euploid samples. thus we hybridized ts1cje versus euploid samples at each postnatal developmental stage. in order to estimate the impact of postnatal development on gene expression while still evaluating the difference between ts1cje and euploid samples with a minimum of experiments, we performed an additional series of comparisons between ts1cje and euploid samples at two successive stages. fifty-six samples from individual cerebella were hybridized on microarrays. to detect genes differentially expressed during cerebellar postnatal development and/or in ts1cje mice, we performed an analysis of variance with two factors: the postnatal developmental stage and the genotype. in addition we compared the expression of previously selected genes in ts1cje and euploid samples at p using quantitative real-time pcr. our model considers the effects of factors: the development and the trisomy effect. μ represent the mean gene expression. effect of trisomy on gene expression at p p p and p10 in ts1cje mice at p p p and p between and genes were found to be significantly differentially expressed. the differentially expressed genes represented between % to % of the expressed genes present on the microarray. these results indicate that trisomy does not lead to a significant global change in gene expression during early cerebellar postnatal development. number of differentially expressed genes in ts1cje mice at p p p and p and during normal cerebelelar development p0–p p0–p and p0–p obtained by anova. we then listed genes that were dysregulated in ts1cje cerebella at all four postnatal development stages studied and found only with up-regulated genes. among them, are triplicated genes. one of them, son or'son cell proliferation protein', had been previously reported as a candidate gene for ds cerebellar phenotype. all ratios have a p-value <. in bold are three-copy genes. effect of development on gene expression between p and p p and p and p and p the following number of genes were differentially expressed, respectively, and representing about to % of expressed genes. these results demonstrate that postnatal development impacts on the expression of a greater number of genes than trisomy does, as already seen in previous analysis. combined effects of trisomy and postnatal development a high proportion of genes whose expression is regulated during cerebellar postnatal development were also differentially expressed in ts1cje mice at least at one time point. among these, are triplicated genes: girk olig and dscam. girk is the only triplicated gene differentially expressed during cerebellar postnatal development and constantly over-expressed in ts1cje mice between p and p girk also know as kcnj is a voltage-gated potassium channel mainly expressed in granule cells of the cerebellum. a point mutation of girk in weaver mice leads to granule cell degeneration. olig is a transcription factor involved in neuron fate commitment and dscam is a cell-surface receptor implicated in cell adhesion. expression ratio between trisomic and euploid mice and betwen two time points.. ns: not significant. effect of trisomy on the expression of genes involved in neuron proliferation in order to identify specific pathways involved in cerebellar postnatal development and disrupted in ts1cje mice, we performed qpcr experiments on a selection of genes using the cerebellum of ts1cje mice and euploid mice from three litters at birth. the list contained genes belonging to pathways involved in cell proliferation and differentiation of the cerebellum or directly in the ds cerebellar phenotype: the notch signalling pathway, numb, gli gli and ptch involved in the shh signaling pathway, nfat and nfat and rcan regulating ca2+ signalling and son and hmgn two triplicated genes that have been proposed to be candidate genes for the cerebellar phenotype. expression ratios between ts1cje and euploid cerebellum are shown in figure the six triplicated genes tested were significantly over-expressed with a mean ratio greater than. surprisingly, of the euploid genes tested, were found to be slightly but significantly over-expressed: shh and its receptor ptch. major primary effect of trisomy exists in the external granule cell layer in order to find gene expression variations in specific regions of the cerebellum where proliferation is impaired in ts1cje mice, external granule cell layers of newborn ts1cje and euploid mice from three litters were dissected and analyzed individually on microarrays. we used illumina pangenomic microarrays containing more than transcripts corresponding to about genes. gene expression data were analyzed using t-test. of the expressed genes only 4% were found to be significantly differentially expressed, with ratios between and. this low percentage was similar to those observed in the entire cerebellum at p p p and p as shown on figure 3a, the distribution of expression ratios for euploid genes was approximately while the distribution of the three-copy genes was shifted towards higher values corresponding to the gene dosage effect. figure shows the mean expression ratios for each individual chromosome. only the part of mmu present in three copies in ts1cje gave a mean expression ratio that was significantly different from. no difference was observed in the expression ratio variances, particularly between the mmu16b and all other chromosomes. the distributions of p-values were also very different for the euploid and the three-copy genes. for the euploid genes the p-value distribution was uniform indicating that very few genes were differentially expressed. however for the three-copy genes, all p-values were below with a higher proportion under. all three-copy genes expressed in the external granule cell layer at p were found to be significantly over-expressed. in order to reduce the number of false positives that occur during multiple testing, we applied the false discovery rate controlling procedure of benjamini and hochberg on the t-test p-values. as reported in table we found only significantly dysregulated genes. of these genes, are three-copy genes, representing more than 70% of the dysregulated genes. the other nine are located on eight other chromosomes and are related to ion transport, signal transduction, cell proliferation or dna processes. altogether these results show a major primary gene dosage effect on the three-copy genes. this effect was also clearly detected using principal component analysis. pca is a technique used to represent multidimensional data sets along principal component axes that reffect the degree of variance in the data. ts1cje and euploid samples were grouped separately only when considering the three-copy genes. genes were found significantly differentially expressed by t-test followed by false discovery rate controlling procedure of benjamini and hochberg. among them, are three-copy genes, indicated in bold. twelve three-copy genes were found significantly differentially expressed by student t-test but failed the false discovery rate controlling procedure of benjamini and hochberg. a partial monosomy of mmu is present in ts1cje mice among the genes whose expression was down-regulated in the external granule cell layer of ts1cje mice, were located on the telomeric part of mmu12: cdca7l and sp. their expression ratios were and respectively. since ts1cje mice are the result of the translocation of the distal part of mmu from sod to znf to the telomeric part of mmu we suspected a deletion of these two down-regulated genes. to confirm this chromosomal rearrangement, a comparative genomic hybridization using high resolution agilent genomic microarrays was performed. a 50% reduction of the genomic content corresponding to the chromosomal segment on mmu located between positions and was found. the deleted part represented about mb and contained genes: dnahc sp sp abcb and itgb all of them are related to either neuronal proliferation or maturation, embryogenesis or peripheral and central nervous system development. the additional translocated mmu segment started at position just proximal to the sod gene and spanned to position at the telomeric end of mmu this genomic interval represented about mb. discussion ds is caused by an aneuploidy corresponding to the trisomy or partial trisomy of chromosome the publication of the dna sequence of the human genome revealed the presence of about genes on chromosome although this number is low as compared to other human autosomes, it is still too large to develop transgenic mouse models for all of them. moreover transgenic mouse models for single genes are not adequate to address fundamental questions on gene interactions. thus in order to find the genes that contribute to the ds phenotype and to the phenotypic variability, gene expression profiling has been applied to mouse models with segmental duplications of dna segments orthologous to human chromosome the studies published so far were conducted in different mouse models, on different tissues and at various developmental stages. in addition the technology and the statistical tools were different thereby reducing the possibility of grouping the analysis and making insights into the genotype-phenotype status of ds. we thus designed a gene expression study in which we could measure the effects of trisomy in the ts1cje mouse model, on a large number of samples, in a tissue that is affected in ds where we could quantify the effetcs during postnatal development in order to correlate gene expression changes to the phenotype observed. ds affects many aspects of brain development and represents the most common cause of inherited human mental retardation. motor deficits are among the most frequently occurring features of ds. individuals with ds exhibit hypotonia and motor dysfunction that could be related to cerebellar dysfunction. ds patients present a consistent reduction of the total brain volume with a disproportionally greater reduction in the cerebellum. one of the major findings in ts65dn and ts1cje mouse models of ds carrying segmental duplications, is the significant reduction in cerebellar volume paralleling that observed in ds patients. this cerebellar hypoplasia likely results from a decrease in the density of granule cells, the most abundant neuronal cell population. moreover, behavioural studies have shown motor dysfunctions in ts65dn that are more severely affected in tasks involving a high degree of balance and coordination. this hypoplasia occurs during cerebellar postnatal development in the first weeks after birth and does not result from a neurodegenerative process. more recently, a reduction of about 20% in the number of granule cell precursors has been reported at birth and days after birth in the cerebellar external granule cell layer of ts65dn mice. this reduction is much more significant at p than at p. in the present study we have shown a 17% significant decrease of volume in the cerebellum of adult ts1cje mice and a % significant decrease of granule cell precursor proliferation only at birth and not at p or p we analyzed global gene expression during postnatal development of the cerebellum in ts1cje mice at the time when major developmental changes occur: at birth at p p and p during this period granule cells proliferate, migrate from the external to the internal layer of the cerebellum, and differentiate. our previous study performed at p p and p showed a clear over expression of triplicated genes in the cerebellum of ts1cje mice and a small effect on the expression of euploid genes. however since the proliferative external granule cell layer disappears by p, this first study was not designed to detect gene expression changes between p and p to increase the statistical power of gene expression analysis, we used a large number of samples: cerebella from ts1cje and euploid mice bred on the mixed genetic background. we performed an anova with a type i error risk of 5% and we found between % to % of genes regulated at each time point including some three-copy genes. then we applied a false discovery rate with a threshold on the gene list and found no significant gene at all time points, not even three-copy genes. by increasing the fdr threshold up to we found only significant genes among which are three-copy genes. these results suggest a limited secondary effect of trisomy on euploid genes. most three-copy genes were not found significantly differentially expressed suggesting that compensatory mechanisms take place during postnatal development. only three-copy genes were constantly over-expressed in the cerebellum of ts1cje mice at p p p and p using qpcr, we confirmed the increased expression of six three-copy genes at p four of these genes were also found over-expressed in the study of mao et al. conducted on ds foetal tissues, which included three cerebella. we then performed a second transcriptome analysis using dissected cerebellar regions enriched in granule cell precursors from ts1cje and nine euploid mice at p at the time when we observed a significant decrease of cell proliferation. about 4% of all genes were significantly differentially expressed in ts1cje samples as compared to euploid samples. this value was close the ones obtained in the entire cerebellum at p p p or p all the expressed three-copy genes were significantly over-expressed in ts1cje with 12genes showing expression ratio significantly lower than. after multi-test correction using the fdr procedure, genes were still significantly modulated in ts1cje mice. among them are three-copy genes, indicating a major primary effect of trisomy on the three-copy genes. from the euploid genes, map to a chromosomal deletion and are under-expressed. the other seven are not well characterized and could be new gene candidates for cerebellar ds phenotypes and cerebellum postnatal development. by using an enriched and more homogeneous cell population, we hoped to clarify the secondary effects of trisomy. surprisingly, not only did we observed a main primary gene dosage effect but we also identified for the first time a deletion in the ts1cje mice. alternatively it is possible that the secondary effect of trisomy is variable among cells or since we demonstrated a proliferation defect in the granule cell precursors, it is plausible that we failed to identify cell cycle-regulated genes because cells from tissue are not synchronized. in addition to the three-copy genes, we were able to detect a gene dosage effect on two genes mapping to mouse chromosome, close to the translocation breakpoint of the trisomic segment. a mb segment of mmu was found to be deleted in ts1cje mice. this segment contains genes: dnahc sp sp abcb and itgb sp is a member of a family of zinc finger transcription factors and is highly expressed in the developing hippocampus and cerebellum, in particular in granule cells. knock-down of sp leads to an increased number of highly branched dendrites during maturation of granule cells in the cerebellar cortex. a reduced cell proliferation in hippocampus but not cerebellum has also been reported in sp null mutant newborns. thus, sp the only gene found expressed in the external granule cell layer is more likely to affect neuron maturation than proliferation. however it is unlikely that the cerebellum phenotype observed in ts1cje mice results from the mmu deletion since a very similar phenotype is present in the ts65dn mouse model that do not show any rearrangement on mmu we found a decrease in cdca7l gene expression, a transcription factor involved in cell apoptosis and medulloblastoma transformation. since cdca7l is located approximately kb proximal to the translocation breakpoint on mmu we concluded that the gene dosage effect extends to regions that are close to the chromosomal rearrangement. we then looked for genes that were regulated during normal postnatal development of the cerebellum and that were also differentially expressed between ts1cje and euploid cerebellum at one of the postnatal stages tested. among the genes found, only three were triplicated: olig dscam and girk olig encodes a basic helix-loop-helix transcription factor that is expressed in both the developing and mature vertebrate central nervous system. olig has critical function during the formation of motor neurons and oligodendrocytes of the ventral neural tube. dscam encodes an axon guidance molecule that is expressed by neurons in the central nervous system during development and throughout adult life. its expression in the developing cerebellum is stronger in purkinje neurons. a dscam mutation in mice leads to a subtle defect in the caudal folium of the cerebellum. girk is the only gene over-expressed in ts1cje across postnatal development of the cerebellum. it is also over-expressed in the external granule cell layer of ts1cje newborns. girk is a member of the g protein-gated inwardly rectifying potassium channel family that regulates cellular excitability and neurotransmission. it is mainly expressed in the cerebellum granule cells. a missense mutation in girk leads to abnormalities of the cell cycle and apoptosis in the external granule cell layer of the cerebellum in the weaver mice. the girk mutated gene encodes channels that exhibit loss of potassium selectivity. in contrast, girk knock-out mice are morphologically indistinguishable from wild-type mice, suggesting that the weaver phenotype is likely due to abnormal girk function. ion channels can regulate cell cycle and implication of transmembrane potassium fluxes via inward rectifier channels in the regulation of cell cycle has been proposed. moreover it has been hypothesized that over-expression of the girk subunit in trisomic mice will likely produce a hyperpolarization of the membrane. depolarization enhances calcium entry via voltage-sensitive ca2+ channels and activates calmodulin kinase and calcineurin phophatase. the activation of calcineurin induces many genes encoding extracellular and intracellular signalling molecules involved in granule cell development.. thus, hyperpolarization of the membrane may prevent or reduce calcium entry in cells and decrease cell proliferation. to further identify gene candidates for the ds cerebellar phenotype, we selected gene candidates involved in genetic regulation of the proliferation of the granule cell precursors and looked at their expression in the cerebellum of ts1cje and euploid mice at birth. sonic hedgehog is secreted by purkinje cells and regulates proliferation of the granule cell progenitors. the mitotic response of cultured granule cell precursors to shh was shown to be reduced in ts65dn mice as compared to euploids. systemic treatment of newborn ts65dn mice with an agonist of the sonic hedgehog pathway increased the mitotic index of trisomic granule cell precursors and even restored the granule cell population in one week. however, to date, genes involved in the shh pathway have not been assigned to human chromosome nor have they been shown to be differentially expressed in trisomic mice or in people with ds. thus the positive effect of treatment with agonists of the shh pathway could result from an over-stimulation of shh receptors that are present on granule cell precursors. it remains to be shown whether this positive effect will normalize the cerebellar volume and improve the cerebellar-related behavioural deficits observed in ts65dn mice. other genes have been reported to regulate the proliferation of granule cell progenitors. some interact with the shh pathway, such as numb which is a suppressor of shh signaling. numb also regulates notch pathway. notch signalling is another crucial development-regulated pathway that appears to maintain cells in an undifferentiated state in the early mammalian central nervous system. in addition, we tested other genes related to granule cell proliferation or neural proliferation including igf-i, bfgf, pax, olig and olig. we were not able to show any consistent difference of expression for any of these genes except a slight but significant over-expression of shh. functional profiling of the differentially expressed genes from microarray experiments didn't reveal any enrichment in gene ontology categories related to cell proliferation. two genes over-expressed in the external granule cell layer of ts1cje mice at birth, s100a and dlk encode calcium ion binding proteins. s100a belongs to the s family proteins that play an important role in cell growth, differentiation, and motility through calcium-dependent signalling pathways. s100a encodes the calcyclin protein whose expression is associated with neuronal differentiation. this gene is thus a candidate for the cerebellar hypoplasia. dlk is a transmembrane and secreted protein from the epidermal growth factor-like homeotic family. dlk expression is increased in gliomas and over-expression in transfected cells promotes cell proliferation by inducing the expression of cyclin d cdk and e2f. however, we could not detect any change in expression of any of these genes suggesting that either dlk has no effect on granule cell proliferation or that the effect is not visible because of lack of cell synchronization in tissues. recently, the generation of a collection of haploid yeast strains that each bear an extra copy of one or more of almost all of the yeast chromosomes has been published. their characterization revealed that aneuploid strains share a number of phenotypes, including defects in cell cycle progression, that are independent of the identity of the individual extra chromosomes. it was thus proposed that disruption of cell homeostasy in ds could be due to the additional chromosomal material rather than the gene content. however aneuploidy is a condition frequently found in tumor cells which often display high rate of proliferation, suggesting that yeast and mammalian cells can respond differently to aneuploidy. moreover in ds, reduced proliferation rates have not been observed in all proliferative cells across development and in adulthood. we show here a reduced proliferation of granule cell precursors only at birth. several studies have reported evidence of neurogenesis impairment in the developing neocortex, in the dentate gyrus of ds foetuses and in mouse models. in the dentate gyrus, cell proliferation is decreased in aged but not young adult ts65dn mice suggesting a neurodegenerative related process occurring in trisomic mice. another study has reported a lower cell proliferation due to cell cycle alteration in the dentate gyrus of foetuses with ds and in mouse models. using different markers of proliferation the authors showed that both ds foetuses and p ts65dn mice have a higher number of proliferating cells in g and a smaller number of cells in the m phase of the cell cycle. a similar result was reported in the forebrain of ts65dn embryos where delayed expansion of neocortical layers and reduced growth of the hippocampus were found to be correlated with a slower cell cycle. interestingly, their results suggest that the cell cycle abnormality, occurring in the trisomic ventricular zone of the neocortex but not in the hippocampus, might be developmentally regulated or compensated during neurogenesis. thus, neurogenesis impairment occurs in different areas of the developing brain in trisomy but not continuously, suggesting that genes involved might be different depending on the brain structure and the developmental stage.  high throughput gene expression analysis in the cerebellum of a large number of ts1cje and euploid mice demonstrates a prevailing gene dosage effect of trisomy and a limited secondary effect across postnatal development. moreover by using an enriched cell population involved in cerebellar hypoplasia in ds we were not able to detect a global change in gene expression. 80% of gene expression differences were attributed to dosage imbalance, suggesting that the three-copy genes are likely to be directly responsible for the phenotype present in cerebellum. by correlating the effects of trisomy and those of postnatal development on gene expression, we identified only three-copy genes that are constantly over-expressed in ts1cje mice during early postnatal development of the cerebellum, and only three-copy genes that are regulated during normal postnatal development of the cerebellum and are over-expressed in the ts1cje mice. only girk is common to both of these groups suggesting that this inwardly rectifying potassium channel expressed in granule cells in the cerebellum could be involved in cerebellar hypoplasia in ds. we propose that reducing the expression of girk in the ts1cje mice could help to recover a normal cerebellum phenotype. 
the impact of this gene dosage effect on the whole transcriptome is still debated and longitudinal studies assessing the variability among samples, tissues and developmental stages are needed. statistical analysis of microarray data revealed a major gene dosage effect: for the three-copy genes as well as for a mb segment from mouse chromosome that we show for the first time as being deleted in the ts1cje mice. this gene dosage effect impacts moderately on the expression of euploid genes. the decrease in granule cell proliferation demonstrated in newborn ts1cje cerebellum was correlated with a major gene dosage effect on the transcriptome in dissected cerebellar external granule cell layer. high throughput gene expression analysis in the cerebellum of a large number of samples of ts1cje and euploid mice has revealed a prevailing gene dosage effect on triplicated genes. altogether these results strongly suggest that the three-copy genes are directly responsible for the phenotype present in cerebellum. we thus designed a large scale gene expression study in mice in which we could measure the effects of trisomy on a large number of samples in a tissue that is affected in down syndrome and where we could quantify the defect during postnatal development in order to correlate gene expression changes to the phenotype observed. we provide here a short list of candidate genes. only genes were significantly dysregulated in ts1cje mice at all four postnatal development stages studied from birth to days after birth, and among them are three-copy genes. moreover using an enriched cell population that is thought responsible for the cerebellar hypoplasia in down syndrome, a global destabilization of gene expression was not detected. recent studies have demonstrated an increased transcript level of the three-copy genes with some dosage compensation or amplification for a subset of them. down syndrome is a chromosomal disorder caused by the presence of three copies of chromosome the mechanisms by which this aneuploidy produces the complex and variable phenotype observed in people with down syndrome are still under discussion.
 down syndrome is a chromosomal disorder caused by the presence of three copies of chromosome the mechanisms by which this aneuploidy produces the complex and variable phenotype observed in people with down syndrome are still under discussion. recent studies have demonstrated an increased transcript level of the three-copy genes with some dosage compensation or amplification for a subset of them. the impact of this gene dosage effect on the whole transcriptome is still debated and longitudinal studies assessing the variability among samples, tissues and developmental stages are needed.  we thus designed a large scale gene expression study in mice in which we could measure the effects of trisomy on a large number of samples in a tissue that is affected in down syndrome and where we could quantify the defect during postnatal development in order to correlate gene expression changes to the phenotype observed. statistical analysis of microarray data revealed a major gene dosage effect: for the three-copy genes as well as for a mb segment from mouse chromosome that we show for the first time as being deleted in the ts1cje mice. this gene dosage effect impacts moderately on the expression of euploid genes. only genes were significantly dysregulated in ts1cje mice at all four postnatal development stages studied from birth to days after birth, and among them are three-copy genes. the decrease in granule cell proliferation demonstrated in newborn ts1cje cerebellum was correlated with a major gene dosage effect on the transcriptome in dissected cerebellar external granule cell layer.  high throughput gene expression analysis in the cerebellum of a large number of samples of ts1cje and euploid mice has revealed a prevailing gene dosage effect on triplicated genes. moreover using an enriched cell population that is thought responsible for the cerebellar hypoplasia in down syndrome, a global destabilization of gene expression was not detected. altogether these results strongly suggest that the three-copy genes are directly responsible for the phenotype present in cerebellum. we provide here a short list of candidate genes. 