To examine the role of intercellular interaction on cell differentiation and gene expression in human prostate, we separated the two major epithelial cell populations and studied them in isolation and in combination with stromal cells. The epithelial cells were separated by f low cytometry using antibodies against differentially expressed cell-surface markers CD44 and CD57.

New mutations for Huntington disease (HD) arise from intermediate alleles (IAs) with between 29 and 35 CAG repeats that expand on transmission through the paternal germline to 36 CAGs or greater. Using single sperm analysis, we have assessed CAG mutation frequencies for four IAs in families with sporadic HD (IANM) and IAs ascertained from the general population (IAGP) by analyzing 1161 single sperm from three persons. We show that IANM are more unstable than IAGP with identical size and sequence. Furthermore, comparison of different sized IAs and IAs with different sequences between the CAG and the adjacent CCG tracts indicates that DNA sequence is a major influence on CAG stability. These studies provide estimates of the likelihood of expansion of IANM and IAGP to > or = 36 CAG repeats for these individuals. For an IA with a CAG of 35 in this family with sporadic HD, the likelihood for siblings to inherit a recurrent mutation > or = 36 CAG is approximately 10%. For IAGP of a similar size, the risk of inheriting an expanded allele of > or = 36 CAG through the paternal germline is approximately 6%. These risk estimates are higher than previously reported and provide additional information for counselling in these families. Further studies on persons with IAs will be needed to determine whether these results can be generalized to other families.

Axis formation in Drosophila depends on correct patterning of the follicular epithelium and on signaling between the germ line and soma during oogenesis. We describe a method for identifying genes expressed in the follicle cells with potential roles in axis formation. Follicle cells are purified from whole ovaries by enzymatic digestion, filtration, and f luorescence-activated cell sorting (FACS). Two strategies are used to obtain complementary cell groups. In the first strategy, spatially restricted subpopulations are marked for FACS selection using a green f luorescent protein (GFP) reporter. In the second, cells are purified from animals mutant for the epidermal growth factor receptor ligand gurken (grk) and from their wild-type siblings. cDNA from these samples of spatially restricted or genetically mutant follicle cells is used in differential expression screens employing PCR-based differential display or hybridization to a cDNA microarray. Positives are confirmed by in situ hybridization to whole mounts. These methods are found to be capable of identifying both spatially restricted and grk-dependent transcripts. Results from our pilot screens include (i) the identification of a homologue of the immunophilin FKBP-12 with dorsal anterior expression in egg chambers, (ii) the discovery that the ecdysone-inducible nuclear hormone receptor gene E78 is regulated by grk during oogenesis and is required for proper dorsal appendage formation, and (iii) the identification of a Drosophila homologue of the human SET-binding factor gene SBF1 with elevated transcription in grk mutant egg chambers.Recent years have seen an explosion in tools for analyzing differential gene expression. The development of a PCR-based differential display method by Liang et al. (1, 2) has quickly been followed by array-based methods for monitoring the expression of thousands of defined genes simultaneously (3, 4). Developmental biology, a field whose progress depends heavily on understanding regulated gene expression, stands to benefit from these methods. However, there are technical hurdles to be overcome before molecular screening methods can be widely applied to problems in development. Chief among these hurdles is purification of the tissue of interest from the complex mixture of cell types typically present in a developing system. Whereas some tissues are amenable to microdissection, others will require more sophisticated techniques such as fluorescence-activated cell sorting (FACS) of dissociated tissue (5-7). The molecular screens reported herein rely on a FACS-based approach to purify specialized follicle cells from Drosophila ovaries. The system we have chosen is particularly illustrative of the need for tissue purification: the Drosophila ovary is composed of egg chambers in which a cyst of large germ-line cells is surrounded by a thin epithelium of highly differentiated follicle cells. Patterning of subregions of the follicular epithelium involves intermingled cell groups, each comprising only a tiny fraction of the volume...

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