Basal-like breast cancers arising in women carrying mutations in the BRCA1 gene, encoding the tumor suppressor protein BRCA1, are thought to develop from the mammary stem cell. To explore early cellular changes that occur in BRCA1 mutation carriers, we have prospectively isolated distinct epithelial subpopulations from normal mammary tissue and preneoplastic specimens from individuals heterozygous for a BRCA1 mutation. We describe three epithelial subsets including basal stem/progenitor, luminal progenitor and mature luminal cells. Unexpectedly, we found that breast tissue from BRCA1 mutation carriers harbors an expanded luminal progenitor population that shows factor-independent growth in vitro. Moreover, gene expression profiling revealed that breast tissue heterozygous for a BRCA1 mutation and basal breast tumors were more similar to normal luminal progenitor cells than any other subset, including the stem cell-enriched population. The c-KIT tyrosine kinase receptor (encoded by KIT) emerged as a key marker of luminal progenitor cells and was more highly expressed in BRCA1-associated preneoplastic tissue and tumors. Our findings suggest that an aberrant luminal progenitor population is a target for transformation in BRCA1-associated basal tumors .

The ETS gene Fli-1 is involved in the induction of erythroleukemia in mice by Friend murine leukemia virus and Ewings sarcoma in children. Mice with a targeted null mutation in the Fli-1 locus die at day 11.5 of embryogenesis with loss of vascular integrity leading to bleeding within the vascular plexus of the cerebral meninges and specific downregulation of Tek/Tie-2, the receptor for angiopoietin-1. We also show that dysmegakaryopoiesis in Fli-1 null embryos resembles that frequently seen in patients with terminal deletions of 11q (Jacobsen or Paris-Trousseau Syndrome). We map the megakaryocytic defects in 14 Jacobsen patients to a minimal region on 11q that includes the Fli-1 gene and suggest that dysmegakaryopoiesis in these patients may be caused by hemizygous loss of Fli-1.

The murine Nanog gene, a member of the homeobox family of DNA binding transcription factors, has been shown recently to maintain pluripotency of embryonic stem cells. We have used a sequence homology and expression screen to identify and clone the mouse and human Nanog genes and characterized their phylogenetic context and expression patterns. We report here the gene structure and expression patterns of the mouse Nanog gene, the human Nanog and Nanog2 genes, and six processed human Nanog pseudogenes. Mouse Nanog expression is high in undifferentiated embryonic stem cells and is down-regulated during embryonic stem cell differentiation, concomitant with loss of pluripotency. Murine embryonic Nanog expression is detected in the inner cell mass of the blastocyst. After implantation, Nanog is detectable at embryonic day (E) 6 in proximal epiblast in the region of the presumptive primitive streak. Expression extends distally as the streak elongates during gastrulation and remains restricted to epiblast. Nanog RNA is down-regulated in cells ingressing through the streak to form mesoderm and definitive endoderm. Nanog expression also marks the pluripotent germ cells of the nascent gonad at E11.5-E12.5 and is highly expressed in germ cell tumour and teratoma-derived cell lines. Reverse transcriptase-polymerase chain reaction analysis detected mouse Nanog expression at low levels in several adult tissues. The human Nanog genes are expressed in embryonic stem cells and down-regulated in all adult tissues and differentiated cell lines examined. High levels of human Nanog expression were detected by Northern analysis in the undifferentiated N-Tera embryonal carcinoma cell line. The conservation in gene sequence, structure, and expression of mouse and human Nanog and Nanog2 genes may reflect a common role in the maintenance of pluripotency in both species. Developmental

We present single-cell clustering using bifurcation analysis (SCUBA), a novel computational method for extracting lineage relationships from single-cell gene expression data and modeling the dynamic changes associated with cell differentiation. SCUBA draws techniques from nonlinear dynamics and stochastic differential equation theories, providing a systematic framework for modeling complex processes involving multilineage specifications. By applying SCUBA to analyze two complementary, publicly available datasets we successfully reconstructed the cellular hierarchy during early development of mouse embryos, modeled the dynamic changes in gene expression patterns, and predicted the effects of perturbing key transcriptional regulators on inducing lineage biases. The results were robust with respect to experimental platform differences between RT-PCR and RNA sequencing. We selectively tested our predictions in Nanog mutants and found good agreement between SCUBA predictions and the experimental data. We further extended the utility of SCUBA by developing a method to reconstruct missing temporal-order information from a typical single-cell dataset. Analysis of a hematopoietic dataset suggests that our method is effective for reconstructing gene expression dynamics during human B-cell development. In summary, SCUBA provides a useful single-cell data analysis tool that is well-suited for the investigation of developmental processes.single cell | gene expression | bifurcation | differentiation