A Cytological F1 RNAi Screen for Defects in Drosophila melanogaster Female Meiosis.

Genetic screens for recessive alleles induce mutations, make the mutated chromosomes homozygous, and then assay those homozygotes for the phenotype of interest. When screening for genes required for female meiosis, the phenotype of interest has typically been nondisjunction from chromosome segregation errors. As this requires that mutant females be viable and fertile, any mutants that are lethal or sterile when homozygous cannot be recovered by this approach. To overcome these limitations, we have screened the VALIUM22 collection of RNAi constructs that target germline-expressing genes in a vector optimized for germline expression by driving RNAi with GAL4 under control of a germline-specific promoter (nanos or mat-alpha4). This allowed us to test genes that would be lethal if knocked down in all cells, and by examining unfertilized metaphase-arrested mature oocytes, we could identify defects in sterile females. After screening >1450 lines of the collection for two different defects (chromosome congression and the hypoxic sequestration of Mps1-GFP to ooplasmic filaments), we obtained multiple hits for both phenotypes, identified novel meiotic phenotypes for genes that had been previously characterized in other processes, and identified the first phenotypes to be associated with several previously uncharacterized genes.

A Cytological F1 RNAi Screen for Defects in Drosophila melanogaster Female Meiosis.

Genetic screens for recessive alleles induce mutations, make the mutated chromosomes homozygous, and then assay those homozygotes for the phenotype of interest. When screening for genes required for female meiosis, the phenotype of interest has typically been nondisjunction from chromosome segregation errors. As this requires that mutant females be viable and fertile, any mutants that are lethal or sterile when homozygous cannot be recovered by this approach. To overcome these limitations, our lab has screened the VALIUM22 collection produced by the Harvard TRiP Project, which contains RNAi constructs targeting genes known to be expressed in the germline in a vector optimized for germline expression. By driving RNAi with GAL4 under control of a germline-specific promoter (nanos or mat-alpha4), we can test genes that would be lethal if knocked down in all cells, and by examining unfertilized metaphase-arrested mature oocytes, we can identify defects associated with genes whose knockdown results in sterility or causes other errors besides nondisjunction. We screened this collection to identify genes that disrupt either of two phenotypes when knocked down: the ability of meiotic chromosomes to congress to a single mass at the end of prometaphase, and the sequestration of Mps1-GFP to ooplasmic filaments in response to hypoxia. After screening >1450 lines of the collection, we obtained multiple hits for both phenotypes, identified novel meiotic phenotypes for genes that had been previously characterized in other processes, and identified the first phenotypes to be associated with several previously uncharacterized genes.

Gradual differentiation uncoupled from cell cycle exit generates heterogeneity in the epidermal stem cell layer.

Highly regenerative tissues continuously produce terminally differentiated cells to replace those that are lost. How they orchestrate the complex transition from undifferentiated stem cells towards post-mitotic, molecularly distinct and often spatially segregated differentiated populations is not well understood. In the adult skin epidermis, the stem cell compartment contains molecularly heterogeneous subpopulations1-4 whose relationship to the complete trajectory of differentiation remains unknown. Here we show that differentiation, from commitment to exit from the stem cell layer, is a multi-day process wherein cells transit through a continuum of transcriptional changes with upregulation of differentiation genes preceding downregulation of typical stemness genes. Differentiation-committed cells remain capable of dividing to produce daughter cells fated to further differentiate, demonstrating that differentiation is uncoupled from cell cycle exit. These cell divisions are not required as part of an obligate transit-amplifying programme but help to buffer the differentiating cell pool during heightened demand. Thus, instead of distinct contributions from multiple progenitors, a continuous gradual differentiation process fuels homeostatic epidermal turnover.

Skin-resident immune cells actively coordinate their distribution with epidermal cells during homeostasis.

Organs consist of multiple cell types that ensure proper architecture and function. How different cell types coexist and interact to maintain their homeostasis in vivo remains elusive. The skin epidermis comprises mostly epithelial cells, but also harbours Langerhans cells (LCs) and dendritic epidermal T cells (DETCs). Whether and how distributions of LCs and DETCs are regulated during homeostasis is unclear. Here, by tracking individual cells in the skin of live adult mice over time, we show that LCs and DETCs actively maintain a non-random spatial distribution despite continuous turnover of neighbouring basal epithelial cells. Moreover, the density of epithelial cells regulates the composition of LCs and DETCs in the epidermis. Finally, LCs require the GTPase Rac1 to maintain their positional stability, density and tiling pattern reminiscent of neuronal self-avoidance. We propose that these cellular mechanisms provide the epidermis with an optimal response to environmental insults.

A Simplified Strategy for Introducing Genetic Variants into Drosophila Compound Autosome Stocks.

Drosophila stocks bearing compound chromosomes, single molecules of DNA that carry the genomic complement of two chromosomes, are useful tools for studying meiosis and mitosis. However, these stocks cannot easily be crossed to stocks with regular chromosomes, due to the lethality of the resulting whole-chromosome aneuploidy. This prevents the examination of interesting genetic variants in a compound chromosome background. Methods to circumvent this difficulty have included the use of triploid females or nondisjunction (caused by either cold-induced microtubule depolymerization or meiotic mutants). Here, we present a new approach for crossing compound chromosomes that takes advantage of the nonhomologous segregations that result when multiple chromosomes in the same genome are prevented from meiotic crossing over by heterozygosity for balancer chromosomes. This approach gives higher yields of the desired progeny in fewer generations of crossing. Using this technique, we have created and validated stocks carrying both a compound-X and compound-2, as well as compound-2 stocks carrying the meiotic mutant nod.