Molecular hierarchy in neurons differentiated from mouse ES cells containing a single human chromosome 21

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Abstract

Defects in neurogenesis and neuronal differentiation in the fetal brain of Down syndrome (DS) patients lead to the apparent neuropathological abnormalities and contribute to the phenotypic characters of mental retardation, and premature development of Alzheimer’s disease, those being the most common phenotype in DS. In order to understand the molecular mechanism underlying the cause of phenotypic abnormalities in the DS brain, we have utilized an in vitro model of TT2F mouse embryonic stem cells containing a single human chromosome 21 (hChr21) to study neuron development and neuronal differentiation by microarray containing 15K developmentally expressed cDNAs. Defective neuronal differentiation in the presence of extra hChr21 manifested primarily the post-transcriptional and translational modification, such as Mrpl10, SNAPC3, Srprb, SF3a60 in the early neuronal stem cell stage, and Mrps18a, Eef1g, and Ubce8 in the late differentiated stage. Hierarchical clustering patterned specific expression of hChr21 gene dosage effects on neuron outgrowth, migration, and differentiation, such as Syngr2, Dncic2, Eif3sf, and Peg3.

Section snippets

Materials and methods

ES cell culture and construction of ES cell lines containing hChr21. Parental TT2F cells and microcell hybrid clones TT2F/hChr21 were used and maintained on mitomycin C (Sigma) treated G418 resistant mouse embryonic fibroblasts (Gibco) as feeder layers in DMEM (Sigma) supplemented with 15% FBS (Hyclone), 1 mM sodium pyruvate (Gibco), 0.1 mM non-essential amino acids (Gibco), 0.1 mM 2-mercaptoethanol (Sigma), 2 mM l-glutamine (Gibco), and 1000 U/ml LIF (Gibco), with or without 300 μg/ml G418 (Gibco)

Neuron differentiation of TT2F and TT2F/hChr21 clones

ES cells plated under serum free conditions on PA6 mouse stromal cell feeder layer differentiate along the default ectodermal pathway and then along the neuronal pathway as directed by SDIA of PA6. Neuron cells induced to differentiate from ES cells by SDIA were reported to contain naı̈ve neural precursors that respond to patterning signals, directing a full dorsal–ventral range of neuroectodermal derivatives [18]. In our experiment, two independently established TT2F/hChr21 clones 21–10 and

Discussion

Mental retardation, as the most common genetic cause of developmental disability in DS, represents a condition characterized by subnormal intellectual functioning and impaired adaptive behavior that become manifest during developmental years. The relationship between mental retardation and the trisomic condition of DS is complex. Conspicuous morphological abnormalities start to be apparent in brains of newborns and older infants with DS. They have shortened basilar dendrites, a decreased number

Conclusions

Defining how an extra copy of hChr21 results in the phenotype of DS is a special case of the more general problem of explaining how chromosomal imbalance produces abnormalities in morphology and function. No single mechanism can explain the deleterious consequences of aneuploidy. In this study, we found the complex network of molecular changes in neuronal stem cells with extra hChr21 for the defective neuron differentiation mechanism by locus-specific disturbance of chromosome balance and

Acknowledgements

We are grateful to Dr. Shin-ichi Hayashi for PA6 cells. This work was supported in part by the grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan; JSPS Postdoctoral Fellowship for Foreign Researcher P01104; JSPS Grant-in-aid for Scientific Research FY2000; and CUHK Direct Grant for Research 2002.2.024.

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      On the other hand, quantitative analyses of normal and trisomic tissues allowed identification of transcriptional alteration in DS. Several whole-genome approaches or transcriptome studies have been performed by microarrays or Serial Analysis of Genome Expression (SAGE), to investigate gene expression variations in trisomic tissues compared to the euploid ones using human tissues or cell lines, and also mouse trisomic model tissues and their non-trisomic littermates.91–99 These transcriptome studies have been particularly focused on brain, cerebellum or neuronal cell lines, considering their major interest in the understanding of the mechanisms involved in the MR pathogenesis in DS.91,93–96,99,100

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    These authors contributed equally to this work.

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