Allan C. Spradling

Stem Cells and Niches: Mechanisms That Promote Stem Cell Maintenance throughout Life

Niches are local tissue microenvironments that maintain and regulate stem cells. Long-predicted from mammalian studies, these structures have recently been characterized within several invertebrate tissues using methods that reliably identify individual stem cells and their functional requirements. Although similar single-cell resolution has usually not been achieved in mammalian tissues, principles likely to govern the behavior of niches in diverse organisms are emerging. Considerable progress has been made in elucidating how the microenvironment promotes stem cell maintenance. Mechanisms of stem cell maintenance are key to the regulation of homeostasis and likely contribute to aging and tumorigenesis when altered during adulthood.

The adult Drosophila posterior midgut is maintained by pluripotent stem cells.

Vertebrate and invertebrate digestive systems show extensive similarities in their development, cellular makeup and genetic control. The Drosophila midgut is typical: enterocytes make up the majority of the intestinal epithelial monolayer, but are interspersed with hormone-producing enteroendocrine cells. Human (and mouse) intestinal cells are continuously replenished by stem cells, the misregulation of which may underlie some common digestive diseases and cancer. In contrast, stem cells have not been described in the intestines of flies, and Drosophila intestinal cells have been thought to be relatively stable. Here we use lineage labelling to show that adult Drosophila posterior midgut cells are continuously replenished by a distinctive population of intestinal stem cells (ISCs). As in vertebrates, ISCs are multipotent, and Notch signalling is required to produce an appropriate fraction of enteroendocrine cells. Notch is also required for the differentiation of ISC daughter cells, a role that has not been addressed in vertebrates. Unlike previously characterized stem cells, which reside in niches containing a specific partner stromal cell, ISCs adjoin only the basement membrane, differentiated enterocytes and their most recent daughters. The identification of Drosophila intestinal stem cells with striking similarities to their vertebrate counterparts will facilitate the genetic analysis of normal and abnormal intestinal function.

decapentaplegic Is Essential for the Maintenance and Division of Germline Stem Cells in the Drosophila Ovary

Stem cells are thought to occupy special local environments, or niches, established by neighboring cells that give them the capability for self-renewal. Each ovariole in the Drosophila ovary contains two germline stem cells surrounded by a group of differentiated somatic cells that express hedgehog and wingless. Here we show that the BMP2/4 homolog decapentaplegic (dpp) is specifically required to maintain female germline stem cells and promote their division. Overexpression of dpp blocks germline stem cell differentiation. Conversely, mutations in dpp or its receptor (saxophone) accelerate stem cell loss and retard stem cell division. We constructed mutant germline stem cell clones to show that the dpp signal is directly received by germline stem cells. Thus, dpp signaling helps define a niche that controls germline stem cell proliferation.

The carnegie protein trap library: a versatile tool for Drosophila developmental studies.

Metazoan physiology depends on intricate patterns of gene expression that remain poorly known. Using transposon mutagenesis in Drosophila, we constructed a library of 7404 protein trap and enhancer trap lines, the Carnegie collection, to facilitate gene expression mapping at single-cell resolution. By sequencing the genomic insertion sites, determining splicing patterns downstream of the enhanced green fluorescent protein (EGFP) exon, and analyzing expression patterns in the ovary and salivary gland, we found that 600–900 different genes are trapped in our collection. A core set of 244 lines trapped different identifiable protein isoforms, while insertions likely to act as GFP-enhancer traps were found in 256 additional genes. At least 8 novel genes were also identified. Our results demonstrate that the Carnegie collection will be useful as a discovery tool in diverse areas of cell and developmental biology and suggest new strategies for greatly increasing the coverage of the Drosophila proteome with protein trap insertions.

Differentiating germ cells can revert into functional stem cells in Drosophila melanogaster ovaries

Many tissues including blood, skin, gut and germ cells are continuously maintained by tissue stem cells. Under certain conditions, however, other organs can undergo repair using stem-cell-like progenitors generated by cell de-differentiation. Cell fates have been broadened experimentally, but mechanisms allowing de-differentiation to a stem cell state are poorly known. Germline stem cells begin to differentiate by forming interconnected germ cell cysts (cystocytes), and under certain conditions male mouse cystocytes have been postulated to revert into functional progenitors. Here we report that four- and eight-cell Drosophila germline cystocytes generated either in second instar larval ovaries or in adults over-producing the BMP4-like stem cell signal Decapentaplegic efficiently convert into single stem-like cells. These de-differentiated cells can develop into functional germline stem cells and support normal fertility. Our results show that cystocytes represent a relatively abundant source of regenerative precursors that might help replenish germ cells after depletion by genotoxic chemicals, radiation or normal ageing. More generally, Drosophila cystocytes now provide a system for studying de-differentiation and its potential as a source of functional stem cells.

The effect of chromosomal position on the expression of the drosophila xanthine dehydrogenase gene

Thirty-six isogenic D. melanogaster strains that differed only in the chromosomal location of a 7.2 or an 8.1 kb DNA segment containing the (autosomal) rosy gene were constructed by P-element-mediated gene transfer. Since the flies were homozygous for a rosy- allele, rosy gene function in these indicated the influence of flanking sequences on gene expression. The tissue distribution of XDH activity in all the strains was normal. Each line exhibited a characteristic level of adult XDH-specific activity. The majority of these values were close to wild-type levels; however, the total variation in specific activity among the lines was nearly fivefold. Thus position effects influence expression of the rosy gene quantitatively but do not detectably alter tissue specificity. X-linked rosy insertions were expressed on average 1.6 times more activity in males than in females. Hence the gene acquires at least partial dosage compensation upon insertion into the X chromosome.

MiMIC: a highly versatile transposon insertion resource for engineering Drosophila melanogaster genes

We demonstrate the versatility of a collection of insertions of the transposon Minos-mediated integration cassette (MiMIC), in Drosophila melanogaster. MiMIC contains a gene-trap cassette and the yellow+ marker flanked by two inverted bacteriophage ΦC31 integrase attP sites. MiMIC integrates almost at random in the genome to create sites for DNA manipulation. The attP sites allow the replacement of the intervening sequence of the transposon with any other sequence through recombinase-mediated cassette exchange (RMCE). We can revert insertions that function as gene traps and cause mutant phenotypes to revert to wild type by RMCE and modify insertions to control GAL4 or QF overexpression systems or perform lineage analysis using the Flp recombinase system. Insertions in coding introns can be exchanged with protein-tag cassettes to create fusion proteins to follow protein expression and perform biochemical experiments. The applications of MiMIC vastly extend the D. melanogaster toolkit.

slow border cells, a locus required for a developmentally regulated cell migration during oogenesis, encodes Drosophila C/EBP.

During Drosophila oogenesis six to ten follicle cells, the border cells, undergo a dramatic and stereotypic migration through the developing egg chamber. We identified four independent P element insertion mutations that specifically blocked border cell migration. They defined a single, novel locus that was named slow border cells (slbo), because hypomorphic alleles caused delayed onset of the migration. Laser ablation of the border cells, or failure of their migration, caused improper morphogenesis of the micropyle, the egg-shell structure through which the sperm enters at fertilization. The slbo locus was found to encode a product homologous to the CCAAT/enhancer-binding protein (C/EBP), a basic region-leucine zipper transcription factor. Drosophila C/EBP may be required for the expression of gene products mediating border cell migration.

An empty Drosophila stem cell niche reactivates the proliferation of ectopic cells

Stem cells are thought to reside in regulatory microenvironments (“niches”) generated by stable stromal neighbors. To investigate the significance of empty niches vacated by stem cell loss, we studied Drosophila ovarioles, which maintain two to three germ-line stem cells in a niche requiring adhesive stromal cap cells and Decapentaplegic signals. After experimentally emptying the germ-line stem cell niche, cap cell activity persists for several weeks. Initially, somatic inner germarium sheath cells enter the empty niche, respond to Dpp, but fail to divide. Subsequently, follicle cell progenitors, including somatic stem cells enter the niche, respond to Dpp, and proliferate as long as cap cells remain. Proliferation requires the normal hedgehog signal of the somatic stem cells as well as proximity to the niche. Thus, empty niches can persist, signal incoming cells, and support ectopic proliferation. Similar events may underlie some disease states.

The organization and amplification of two chromosomal domains containing drosophila chorion genes

Drosophila chorion genes are located in two clusters, one on the X chromosome at 7F1-2 of the polytene chromosome map and a second on the third chromosome at 66D11-15. Genes in both regions undergo amplification in ovarian follicle cells prior to their expression late in oogenesis. Analysis of cloned genomic segments derived from these chromosomal sites revealed that each cluster contains two tandemly transcribed chorion protein genes separated by only 1-2 kb. At least two other regions complementary to ovary RNA are located within 5 kb of these genes. During oogenesis, the transcribed sequences within each cluster, as well as the spacer sequences that separate them, are amplified equally. Sequences adjacent to the transcribed regions also replicate differentially but to a lesser extent, giving rise to gradients of decreasing amplification involving 40-50 kb of flanking chromosomal sequences. Differences between the restriction maps of unamplified and amplified DNA could not be detected in genomic DNA within either of the 90-100 kb domains of amplification. These observations suggest a model of amplification in which additional rounds of replication are specifically initiated within the central gene-containing regions, followed by bidirectional replication in the absence of discrete termination sites.