BACKGROUND
down's syndrome , caused by the trisomy of human chromosome  <dig> ,  <cit>  is a complex condition characterized by a plethora of phenotypic features, most striking of which are reduced neuron number and synaptic plasticity, early alzheimer-like neurodegeneration, craniofacial dysmorphia, heart development defects, and powerful suppression of the incidence of most solid tumours  <cit> .
in the first few months of life, ds babies display brachycephaly, microcephaly, delayed myelination, reduced growth of frontal lobes, a narrowing of the superior temporal gyrus, diminished size of the brainstem and cerebellum, and up to 50% reduction in numbers of cortical granular neurons  <cit> .
the exact timing of onset of these changes is still unclear.
in a limited study, neuronal progenitor cells from  <dig> foetal ds brains showed a reduced neuron number following further differentiation in vitro, compared to euploid foetal cells  <cit> .
also, ds foetal neural cells showed a reduced propensity for proliferation and survival in vitro compared to euploid controls  <cit> .
tumours of neural tissues, such as neuroblastomas, are comparatively very rarely observed in ds individuals  <cit> .
mouse models for ds display, among other features, behavioural and cognitive defects, synaptic plasticity defects and long term potentiation  deficits in the hippocampus, as well as reduced hippocampal and cerebellar neuron numbers  <cit> .
the reduction of cerebellar granule neuron numbers in mouse models occurs in the first  <dig> days postnatally, due to a defective response to sonic hedgehog   <cit> , secreted by an already reduced number of purkinje neurons  <cit> .
in the hippocampus, the exact timing of the onset of neuronal precursor cell reduction is less clear, but a reduced number of mitotically active granule neuron cell precursors is observed at day p <dig>  <cit> .
at 4– <dig> months of age, mice undergo a neurodegenerative reduction of basal forebrain cholinergic neurons  <cit> , further contributing to the reduction in granule cell neuron numbers  <cit> .
three quarters of human trisomy  <dig> concepti die in utero from developmental arrest  <cit> , and phenotypic features of ds are retained even in mosaic ds subjects  <cit> , as well as in transchromosomic ds mouse models where adult tissues retain <50% trisomic cells, having started from a fully trisomic conceptus  <cit> .
this implies that many phenotypic features of ds must be determined by events occurring very early in development, but the exact nature of these early events, and the role played by increased dosage of individual hsa <dig> genes remain unknown.
in an attempt to study the effects of trisomy  <dig> on the capacity of pluripotent embryonic stem  cells to proliferate and differentiate in vivo, we report here the use of a mouse pluripotent embryonic stem  cell line with a freely segregating hsa <dig> as a single supernumerary chromosome  <cit> , in the generation of transchromosomic teratomas upon subcutaneous injections into syngeneic mice.
RESULTS
generation of teratomas
the cell line , generated by introduction of a single hsa <dig> into the mouse es cell line d <dig>  was found by pcr amplification of human specific markers to contain virtually all of the gene content of hsa <dig>  and was shown not to contain dna from any human chromosome other than hsa <dig>  <cit> .
transchromosomic 47- <dig> and control d <dig> cell lines were cultured under identical conditions and verified at the point of injection to be undifferentiated, having similar proliferation indices, and showing the presence of hsa <dig> in practically all 47- <dig> cells, and absence in d <dig> cells .
integrity of the retained hsa <dig> was verified by human specific pcr of  <dig> markers, and rt-pcr of  <dig> hsa <dig> genes.
in  <dig> independent experiments  and n =  <dig> mice .
sixteen animals developed tumours in both flanks.
no significant difference in size/weight was found between 47- <dig> and d <dig> tumours .
all tumours grew as solitary  spheroid masses in the sub-cutaneous tissue, clearly separated from host tissues by an envelope of connective-like tissue.
histological analysis of the teratomas
qualitative histological analysis of standard h&e stained tumour sections revealed a multitude of different cell types in all tumours.
scattered islands of many tissue types were observed including: keratinized squamous epithelium, ciliated epithelium, glandular epithelium, acini of salivary glands, smooth muscle, cartilage, bone, haematopoietic tissue, fat tissue and undifferentiated tissue.
many tumour slices lacked the presence of differentiated neuroectodermal tissue.
tumour sections were analyzed quantitatively, by a histopathologist blinded to the tumour origin, for the presence and relative abundance  of four types of tissue: neuroectodermal, mesenchymal, epithelial and undifferentiated .
transchromosomic tumours showed an approximately three-fold lower mean percentage of neuroectodermal tissue, both when compared across all tumours , and when compared strictly within pairs of tumours which grew in the same animal , .
the transchromosomic tumours also showed an almost two fold reduction of mrna for mouse tubb <dig>  a neuron specific gene , and a three-fold reduction of mouse gfap mrna, a glia specific gene .
the presence of both neurons and astroglia cells, respectively, within the tumours has been verified by immunofluorescence using antibodies against map <dig> and gfap, which confirmed the observation of the reduced numbers of both types of cells in the transchromosomic tumours, compared to controls .
analysis of a hsa <dig> region causing inhibition of neuroectodermal differentiation
genomic dna and total rna of 47- <dig> tumours were analysed by pcr and rt-pcr, respectively, using the same battery of human sequence specific markers/genes that were verified to be present in the 47- <dig> cell line at the point of injection .
two thirds of all 47- <dig> tumours showed a lack of amplification with multiple primers in the regions distal to prss <dig>  proximal to ttc <dig>  and distal to cstb .
in the deleted segments, both genomic dna and ubiquitously expressed cdna markers failed to amplify.
the percentage of cells within each tumour that might have lost the entire hsa <dig> were estimated by quantitative genomic dna real-time pcr using a single marker within the commonly retained chromosomal segments normalized to a single mouse genomic dna locus.
an average level of 76%  was obtained for retention of hsa <dig> in dna material extracted from the 47- <dig> tumours.
as there were sufficient numbers of cells of many different tissue types in all tumours, and multiple hsa <dig> pcr markers in the deleted regions gave no amplified product, deletions must have occurred before any differentiation of es cells in the tumours took place.
the presence or absence of deletions did not correlate with experimental chronology, or percentages of tissue types including neuroectoderm .
this suggests that deletions most likely occurred during the initial cell divisions in undifferentiated es cells at the very start of tumour growth.
when only the deleted 47- <dig> tumours were compared to either all d <dig> tumours, or only their direct pairs, the statistically significantly reduced percentage of neuroectoderm, and levels of tubb <dig> and gfap, were observed, virtually identical to graphs shown in fig  <dig>  regardless of the cause of the deletions, the data open themselves to interpretation that the segmental trisomy common to all tumours , comprising less than a third of hsa <dig> gene complement, might be sufficient to cause a powerful inhibition of neuroectodermal differentiation  in pluripotent es cells in vivo.
discussion
the undifferentiated es cell inoculates were allowed to develop for  <dig> days, equivalent to the time span of entire intrauterine, and  <dig> postnatal days of mouse development.
this extended period encompasses the critical interval , in which the biggest difference in neuronal precursor cell proliferation was observed between mouse models of ds, and their euploid littermate controls  <cit> .
the use of syngeneic inbred mouse strains also minimizes the individual variation, which is further reduced by the paired analysis of tumours which grow in the same recipient animal.
using this system, we have observed a powerful inhibition of neuroectodermal fate in transchromosomic +hsa <dig> containing pluripotent mouse es cells, compared to their parental control .
this conclusion is not without caveats: the inhibition of neural fate could be specific to the cell line used, or it could be related to the sheer presence of a supernumerary chromosome, not specific to hsa <dig>  though it is impossible to rule out these caveats at this stage, we believe on balance of probabilities, that this system shows a measurable phenotype with potential to map it to a segment of hsa <dig>  the reduced tubb <dig> levels  clearly indicate a reduction in mature neuron numbers in trisomic teratomas, confirmed by neuron specific staining .
the reduction in gfap levels could be partly the result of reduced numbers of mature astrocytes, as well as reduced numbers of neuronal precursors, corresponding to gfap+ cells found in the subgranular zone and hilus  <cit> .
these gfap+ precursors divide and give rise to immature neurons   <cit> .
radial glial cells, which are the main source of precursors for neurogenesis in the dentate gyrus, also express gfap  <cit> .
these very cellular layers are the site of the biggest deficit in mitotic activity at postnatal day  <dig> in mouse models of ds  <cit> .
spontaneous deletions of the supernumerary hsa <dig> were observed in two thirds of transchromosomic tumours .
as there were sufficient numbers of cells of many different tissue types in all tumours, and multiple hsa <dig> pcr reactions  were negative in the deleted regions, deletions must have occurred before any differentiation of es cells in the tumours took place, probably during the early cell divisions at the very start of tumour growth.
alternatively, a subset of cells with a major deletion might have been present within the inoculum of 47- <dig> cells; a less likely explanation, as  <dig> independent cultures gave rise to a similar proportion of tumours with, and without deletions.
we cannot exclude the possibility that the inocula in all  <dig> experiments contained a mixture of two clones, the 47- <dig>  and a segmental trisomy clone.
however, the deleted segments also show subtle variations in the pattern of markers present, making this possibility less likely.
whether the deletions are caused by an instable dna sequence element, and whether or not they provide a selective advantage to the proliferation of cells, remains to be investigated.
regardless of the cause of deletions within the tumours, they had no effect on the statistical significance of any differences shown in fig.
<dig>  though the data are not conclusive on this point, it can be hypothesized that the segmental trisomy of two regions common to all tumours , comprising less than a third of the hsa <dig> gene complement, could be sufficient to cause a powerful inhibition of neuroectodermal differentiation  in pluripotent es cells in vivo.
the telomeric indi region partially overlaps with trisomic regions in mouse segmental trisomy models for ds, such as ts65dn and ts1cje , which both have a reduced number of granular layer neurons in the cerebellum and hippocampus, compared to euploid littermates  <cit> .
the overlap between the telomeric indi segment and the  <dig> mouse models shown in fig.
<dig>  is restricted to  <dig> genes  which are deleted in the third chromosome of the tc <dig> model  <cit> , and therefore remain unchecked.
the telomeric indi segment of  <dig> genes overlapping with trisomic mouse models is fully contained within the ts1rhr, the  <dig> gene-trisomy mouse model, which does not show the reduction in cerebellar volume, or neuron density  <cit> .
this mouse model also shows no hippocampal volume change, and no electrophysiological and behavioural defects associated with hippocampal functions, therefore suggesting that trisomy of this segment is not sufficient to cause the brain pathology in the mouse  <cit> .
however, this  <dig> gene segment was found necessary for most of the cerebellar and hippocampal pathology of ts65dn model, as when its trisomy is reversed to disomy, the pathologies disappeared  <cit> .
this segment also probably contains highly and bi-directionally dose sensitive genes regulating brain development, as a monosomy of this segment  produces striking changes in cerebellar and hippocampal volume and neuron density  <cit> .
the data in our system could differ from those in the ts1rhr model due to the human, rather than mouse, origin of the third chromosome.
interestingly, very recent data in human ds show that duplication of a  <dig>  mb segment, completely contained within the  <dig> gene equivalent segment in the mouse, is sufficient to cause  a range of ds phenotypes, including brachycephaly, intellectual disability, mental retardation, speech learning impairment, and a typical facial gestalt of ds  <cit> .
our data suggest that further studies of individual gene dosage effects within the indi region could reveal major candidate contributors to ds-related hypo-cellularity of the cns, and suppression of neuroblastomas.
CONCLUSIONS
we demonstrate that a supernumerary chromosome  <dig> causes inhibition of neuroectodermal differentiation  of pluripotent es cells.
the data open themselves to interpretation that the trisomy of less than a third of hsa <dig> genes, in two chromosomal regions, could be sufficient to cause this effect.

 down's syndrome, caused by the trisomy of human chromosome, is a complex condition characterized by a plethora of phenotypic features, most striking of which are reduced neuron number and synaptic plasticity, early alzheimer-like neurodegeneration, craniofacial dysmorphia, heart development defects, and powerful suppression of the incidence of most solid tumours. in the first few months of life, ds babies display brachycephaly, microcephaly, delayed myelination, reduced growth of frontal lobes, a narrowing of the superior temporal gyrus, diminished size of the brainstem and cerebellum, and up to 50% reduction in numbers of cortical granular neurons. the exact timing of onset of these changes is still unclear. in a limited study, neuronal progenitor cells from foetal ds brains showed a reduced neuron number following further differentiation in vitro, compared to euploid foetal cells. also, ds foetal neural cells showed a reduced propensity for proliferation and survival in vitro compared to euploid controls. tumours of neural tissues, such as neuroblastomas, are comparatively very rarely observed in ds individuals. mouse models for ds display, among other features, behavioural and cognitive defects, synaptic plasticity defects and long term potentiation deficits in the hippocampus, as well as reduced hippocampal and cerebellar neuron numbers. the reduction of cerebellar granule neuron numbers in mouse models occurs in the first days postnatally, due to a defective response to sonic hedgehog, secreted by an already reduced number of purkinje neurons. in the hippocampus, the exact timing of the onset of neuronal precursor cell reduction is less clear, but a reduced number of mitotically active granule neuron cell precursors is observed at day p. at 4– months of age, mice undergo a neurodegenerative reduction of basal forebrain cholinergic neurons, further contributing to the reduction in granule cell neuron numbers. three quarters of human trisomy concepti die in utero from developmental arrest, and phenotypic features of ds are retained even in mosaic ds subjects, as well as in transchromosomic ds mouse models where adult tissues retain <50% trisomic cells, having started from a fully trisomic conceptus. this implies that many phenotypic features of ds must be determined by events occurring very early in development, but the exact nature of these early events, and the role played by increased dosage of individual hsa genes remain unknown. in an attempt to study the effects of trisomy on the capacity of pluripotent embryonic stem cells to proliferate and differentiate in vivo, we report here the use of a mouse pluripotent embryonic stem cell line with a freely segregating hsa as a single supernumerary chromosome, in the generation of transchromosomic teratomas upon subcutaneous injections into syngeneic mice.  generation of teratomas the cell line, generated by introduction of a single hsa into the mouse es cell line d was found by pcr amplification of human specific markers to contain virtually all of the gene content of hsa and was shown not to contain dna from any human chromosome other than hsa. transchromosomic 47- and control d cell lines were cultured under identical conditions and verified at the point of injection to be undifferentiated, having similar proliferation indices, and showing the presence of hsa in practically all 47- cells, and absence in d cells. integrity of the retained hsa was verified by human specific pcr of markers, and rt-pcr of hsa genes. in independent experiments and n = mice. sixteen animals developed tumours in both flanks. no significant difference in size/weight was found between 47- and d tumours. all tumours grew as solitary spheroid masses in the sub-cutaneous tissue, clearly separated from host tissues by an envelope of connective-like tissue. histological analysis of the teratomas qualitative histological analysis of standard h&e stained tumour sections revealed a multitude of different cell types in all tumours. scattered islands of many tissue types were observed including: keratinized squamous epithelium, ciliated epithelium, glandular epithelium, acini of salivary glands, smooth muscle, cartilage, bone, haematopoietic tissue, fat tissue and undifferentiated tissue. many tumour slices lacked the presence of differentiated neuroectodermal tissue. tumour sections were analyzed quantitatively, by a histopathologist blinded to the tumour origin, for the presence and relative abundance of four types of tissue: neuroectodermal, mesenchymal, epithelial and undifferentiated. transchromosomic tumours showed an approximately three-fold lower mean percentage of neuroectodermal tissue, both when compared across all tumours, and when compared strictly within pairs of tumours which grew in the same animal,. the transchromosomic tumours also showed an almost two fold reduction of mrna for mouse tubb a neuron specific gene, and a three-fold reduction of mouse gfap mrna, a glia specific gene. the presence of both neurons and astroglia cells, respectively, within the tumours has been verified by immunofluorescence using antibodies against map and gfap, which confirmed the observation of the reduced numbers of both types of cells in the transchromosomic tumours, compared to controls. analysis of a hsa region causing inhibition of neuroectodermal differentiation genomic dna and total rna of 47- tumours were analysed by pcr and rt-pcr, respectively, using the same battery of human sequence specific markers/genes that were verified to be present in the 47- cell line at the point of injection. two thirds of all 47- tumours showed a lack of amplification with multiple primers in the regions distal to prss proximal to ttc and distal to cstb. in the deleted segments, both genomic dna and ubiquitously expressed cdna markers failed to amplify. the percentage of cells within each tumour that might have lost the entire hsa were estimated by quantitative genomic dna real-time pcr using a single marker within the commonly retained chromosomal segments normalized to a single mouse genomic dna locus. an average level of 76% was obtained for retention of hsa in dna material extracted from the 47- tumours. as there were sufficient numbers of cells of many different tissue types in all tumours, and multiple hsa pcr markers in the deleted regions gave no amplified product, deletions must have occurred before any differentiation of es cells in the tumours took place. the presence or absence of deletions did not correlate with experimental chronology, or percentages of tissue types including neuroectoderm. this suggests that deletions most likely occurred during the initial cell divisions in undifferentiated es cells at the very start of tumour growth. when only the deleted 47- tumours were compared to either all d tumours, or only their direct pairs, the statistically significantly reduced percentage of neuroectoderm, and levels of tubb and gfap, were observed, virtually identical to graphs shown in fig regardless of the cause of the deletions, the data open themselves to interpretation that the segmental trisomy common to all tumours, comprising less than a third of hsa gene complement, might be sufficient to cause a powerful inhibition of neuroectodermal differentiation in pluripotent es cells in vivo. discussion the undifferentiated es cell inoculates were allowed to develop for days, equivalent to the time span of entire intrauterine, and postnatal days of mouse development. this extended period encompasses the critical interval, in which the biggest difference in neuronal precursor cell proliferation was observed between mouse models of ds, and their euploid littermate controls. the use of syngeneic inbred mouse strains also minimizes the individual variation, which is further reduced by the paired analysis of tumours which grow in the same recipient animal. using this system, we have observed a powerful inhibition of neuroectodermal fate in transchromosomic +hsa containing pluripotent mouse es cells, compared to their parental control. this conclusion is not without caveats: the inhibition of neural fate could be specific to the cell line used, or it could be related to the sheer presence of a supernumerary chromosome, not specific to hsa though it is impossible to rule out these caveats at this stage, we believe on balance of probabilities, that this system shows a measurable phenotype with potential to map it to a segment of hsa the reduced tubb levels clearly indicate a reduction in mature neuron numbers in trisomic teratomas, confirmed by neuron specific staining. the reduction in gfap levels could be partly the result of reduced numbers of mature astrocytes, as well as reduced numbers of neuronal precursors, corresponding to gfap+ cells found in the subgranular zone and hilus. these gfap+ precursors divide and give rise to immature neurons. radial glial cells, which are the main source of precursors for neurogenesis in the dentate gyrus, also express gfap. these very cellular layers are the site of the biggest deficit in mitotic activity at postnatal day in mouse models of ds. spontaneous deletions of the supernumerary hsa were observed in two thirds of transchromosomic tumours. as there were sufficient numbers of cells of many different tissue types in all tumours, and multiple hsa pcr reactions were negative in the deleted regions, deletions must have occurred before any differentiation of es cells in the tumours took place, probably during the early cell divisions at the very start of tumour growth. alternatively, a subset of cells with a major deletion might have been present within the inoculum of 47- cells; a less likely explanation, as independent cultures gave rise to a similar proportion of tumours with, and without deletions. we cannot exclude the possibility that the inocula in all experiments contained a mixture of two clones, the 47- and a segmental trisomy clone. however, the deleted segments also show subtle variations in the pattern of markers present, making this possibility less likely. whether the deletions are caused by an instable dna sequence element, and whether or not they provide a selective advantage to the proliferation of cells, remains to be investigated. regardless of the cause of deletions within the tumours, they had no effect on the statistical significance of any differences shown in fig. though the data are not conclusive on this point, it can be hypothesized that the segmental trisomy of two regions common to all tumours, comprising less than a third of the hsa gene complement, could be sufficient to cause a powerful inhibition of neuroectodermal differentiation in pluripotent es cells in vivo. the telomeric indi region partially overlaps with trisomic regions in mouse segmental trisomy models for ds, such as ts65dn and ts1cje, which both have a reduced number of granular layer neurons in the cerebellum and hippocampus, compared to euploid littermates. the overlap between the telomeric indi segment and the mouse models shown in fig. is restricted to genes which are deleted in the third chromosome of the tc model, and therefore remain unchecked. the telomeric indi segment of genes overlapping with trisomic mouse models is fully contained within the ts1rhr, the gene-trisomy mouse model, which does not show the reduction in cerebellar volume, or neuron density. this mouse model also shows no hippocampal volume change, and no electrophysiological and behavioural defects associated with hippocampal functions, therefore suggesting that trisomy of this segment is not sufficient to cause the brain pathology in the mouse. however, this gene segment was found necessary for most of the cerebellar and hippocampal pathology of ts65dn model, as when its trisomy is reversed to disomy, the pathologies disappeared. this segment also probably contains highly and bi-directionally dose sensitive genes regulating brain development, as a monosomy of this segment produces striking changes in cerebellar and hippocampal volume and neuron density. the data in our system could differ from those in the ts1rhr model due to the human, rather than mouse, origin of the third chromosome. interestingly, very recent data in human ds show that duplication of a mb segment, completely contained within the gene equivalent segment in the mouse, is sufficient to cause a range of ds phenotypes, including brachycephaly, intellectual disability, mental retardation, speech learning impairment, and a typical facial gestalt of ds. our data suggest that further studies of individual gene dosage effects within the indi region could reveal major candidate contributors to ds-related hypo-cellularity of the cns, and suppression of neuroblastomas.  we demonstrate that a supernumerary chromosome causes inhibition of neuroectodermal differentiation of pluripotent es cells. the data open themselves to interpretation that the trisomy of less than a third of hsa genes, in two chromosomal regions, could be sufficient to cause this effect. 
two thirds of transchromosomic tumours also showed a lack of pcr amplification with multiple primers specific for hsa which were present in the es cells at the point of injection, thus restricting a commonly retained trisomy to less than a third of hsa genes. when paired teratomas from the same animals were compared, transchromosomic tumours showed a three-fold lower percentage of neuroectodermal tissue, as well as significantly reduced mrna levels for neuron specific and glia specific genes, relative to euploid controls. we have subcutaneously injected mouse pluripotent es cells containing a single freely segregating supernumerary human chromosome into syngeneic mice, to generate transchromosomic teratomas. transchromosomic cells and parental control cells were injected into opposite flanks of thirty mice in three independent experiments. the data suggest that trisomy of less than a third of hsa genes, in two chromosomal regions, might be sufficient to cause this effect. mouse models for ds show behavioural and cognitive defects, synaptic plasticity defects, and reduced hippocampal and cerebellar neuron numbers. we demonstrate that a supernumerary chromosome causes inhibition of neuroectodermal differentiation of pluripotent es cells. down syndrome, caused by trisomy of human chromosome, is the most common genetic cause of mental retardation in humans.
 down syndrome, caused by trisomy of human chromosome, is the most common genetic cause of mental retardation in humans. among complex phenotypes, it displays a number of neural pathologies including smaller brain size, reduced numbers of neurons, reduced dendritic spine density and plasticity, and early alzheimer-like neurodegeneration. mouse models for ds show behavioural and cognitive defects, synaptic plasticity defects, and reduced hippocampal and cerebellar neuron numbers. early postnatal development of both human and mouse-model ds shows the reduced capability of neuronal precursor cells to generate neurons. the exact molecular cause of this reduction, and the role played by increased dosage of individual hsa genes, remain unknown.  we have subcutaneously injected mouse pluripotent es cells containing a single freely segregating supernumerary human chromosome into syngeneic mice, to generate transchromosomic teratomas. transchromosomic cells and parental control cells were injected into opposite flanks of thirty mice in three independent experiments. tumours were grown for days, a time-span equivalent to combined intra-uterine, and early post-natal mouse development. when paired teratomas from the same animals were compared, transchromosomic tumours showed a three-fold lower percentage of neuroectodermal tissue, as well as significantly reduced mrna levels for neuron specific and glia specific genes, relative to euploid controls. two thirds of transchromosomic tumours also showed a lack of pcr amplification with multiple primers specific for hsa which were present in the es cells at the point of injection, thus restricting a commonly retained trisomy to less than a third of hsa genes.  we demonstrate that a supernumerary chromosome causes inhibition of neuroectodermal differentiation of pluripotent es cells. the data suggest that trisomy of less than a third of hsa genes, in two chromosomal regions, might be sufficient to cause this effect. 