ANALYSIS OF GENE-EXPRESSION IN A COMPLEX DIFFERENTIATION HIERARCHY BYGLOBAL AMPLIFICATION OF CDNA FROM SINGLE CELLS

Citation
G. Brady et al., ANALYSIS OF GENE-EXPRESSION IN A COMPLEX DIFFERENTIATION HIERARCHY BYGLOBAL AMPLIFICATION OF CDNA FROM SINGLE CELLS, Current biology, 5(8), 1995, pp. 909-922
Citations number
66
Categorie Soggetti
Biology,Biology
Journal title
ISSN journal
09609822
Volume
5
Issue
8
Year of publication
1995
Pages
909 - 922
Database
ISI
SICI code
0960-9822(1995)5:8<909:AOGIAC>2.0.ZU;2-M
Abstract
Background: Many differentiating tissues contain progenitor cells that differ in their commitment states but cannot be readily distinguished or segregated. Molecular analysis is therefore restricted to mixed po pulations or cell lines which may also be heterogeneous, and the criti cal differences in gene expression that might determine divergent deve lopment are obscured. In this study, we combined global amplification of mRNA transcripts in single cells with identification of the develop mental potential of processed cells on the basis of the fates of their sibling cells from clonal starts. Results: We analyzed clones of from four to eight hemopoietic precursor cells which had a variety of diff erentiative potentials; sibling cells generally each formed clones of identical composition in secondary culture. Globally amplified cDNA wa s prepared from individual precursors whose developmental potential wa s identified by tracking sibling fates. Further cDNA samples were prep ared from terminally maturing, homogeneous hemopoietic cell population s. Together, the samples represented 16 positions in the hemopoietic d evelopmental hierarchy. Expression patterns in the sample set were det ermined for 29 genes known to be involved in hemopoietic cell growth, differentiation or function. The cDNAs from a bipotent erythroid/megak aryocyte precursor and a bipotent neutrophil/macrophage precursor were subtractively hybridized, yielding numerous differentially expressed cDNA clones. Hybridization of such clones to the entire precursor samp le set identified transcripts with consistent patterns of differential expression in the precursor hierarchy. Conclusions: Tracking of sibli ng fates reliably identifies the differentiative potential of a single cell taken for PCR analysis, and demonstrates the existence of a vari ety of distinct and stable states of differentiative commitment. Globa l amplification of cDNA from single precursor cells, identified by sib ling fates, yields a true representation of lineage- and stage-specifi c gene expression, as confirmed by hybridization to a broad panel of p robes. The results provide the first expression mapping of these genes that distinguishes between progenitors in different commitment states , generate new insights and predictions relevant to mechanism, and int roduce a powerful set of tools for unravelling the genetic basis of li neage divergence.