Cryptococcus neoformans is a basidiomycetous yeast ubiquitous in the environment, a model for fungal pathogenesis, and an opportunistic human pathogen of global importance. We have sequenced its ∼20-megabase genome, which contains ∼6500 intron-rich gene structures and encodes a transcriptome abundant in alternatively spliced and antisense messages. The genome is rich in transposons, many of which cluster at candidate centromeric regions. The presence of these transposons may drive karyotype instability and phenotypic variation. C. neoformans encodes unique genes that may contribute to its unusual virulence properties, and comparison of two phenotypically distinct strains reveals variation in gene content in addition to sequence polymorphisms between the genomes.

SummaryPolyploid cells have genomes that contain multiples of the typical diploid chromosome number and are found in many different organisms. Studies in a variety of animal and plant developmental systems have revealed evolutionarily conserved mechanisms that control the generation of polyploidy and have recently begun to provide clues to its physiological function. These studies demonstrate that cellular polyploidy plays important roles during normal development and also contributes to human disease, particularly cancer.Key words: Cancer, Endocycle, Endomitosis, Endoreplication, Genome instability IntroductionPolyploid cells, which contain multiples of the diploid genome equivalent, have been studied for many years, nearly as long as chromosomes themselves have been studied. Now, over a century after the discovery of polyploidy, we know much about the molecular mechanisms that generate cellular polyploidy, but comparably little regarding the physiological function of the polyploid state. This discrepancy exists despite the frequent occurrence of polyploid cells in most multicellular organisms, as well as in human cancers. An important aspect of deciphering the roles for polyploidy lies in understanding the regulation of endoreplication, a cell cycle variation that generates a polyploid genome by repeated rounds of DNA replication in the absence of cell division. Recent advances show that, although some differences exist in the diverse endoreplication programs found from protists to humans, core principles can be applied. New work in genetic model organisms has identified cases in which programmed endoreplication plays key roles in development. Furthermore, recent evidence indicates that endoreplication can confer genome instability, a major cancer-enabling property. In this Primer (see Box), we review such recent discoveries, focusing on work carried out in the past 3 years, and refer the reader to other comprehensive reviews on this topic Lee et al., 2009). From these new insights emerge unifying themes regarding endoreplication that hold promise of elucidating the advantages, as well as the potential disadvantages, of polyploidy. Endoreplication and developmentEndoreplication is typically studied in the context of endopolyploidy, which refers to the presence of polyploid cells in an otherwise diploid organism. Diverse levels of polyploidy occur throughout nature (Table 1). Although polyploidy is well appreciated in plants , many different animal tissues, including the skin, gut, placenta, liver, brain and blood have polyploid cells (Table 1) (Laird et al., 1980;Corash et al., 1989;Fox et al., 2010; Hedgecock and White, 1985;Melaragno et al., 1993;Sherman, 1972;Unhavaithaya and Orr-Weaver, 2012;Zanet et al., 2010). Interestingly, endoreplication is not limited to multi-cellular organisms, with examples described in ciliated protozoa (Yin et al., 2010) and even bacteria (Mendell et al., 2008).Two primary forms of endoreplication have been described: endocycling and endomitosis (Fig. 1). Endocycles are compos...

Summary Background Re-establishing epithelial integrity and biosynthetic capacity is critically important following tissue damage. The adult Drosophila abdominal epithelium provides an attractive new system to address how post-mitotic diploid cells contribute to repair. Results Puncture wounds to the adult Drosophila epidermis close initially by forming a melanized scab. We found that epithelial cells near the wound site fuse to form a giant syncytium, which sends lamellae under the scab to re-epithelialize the damaged site. Other large cells arise more peripherally by initiating endocycles and becoming polyploid, or by cell fusion. Rac GTPase activity is needed for syncytium formation, while the Hippo signaling effector Yorkie modulates both polyploidization and cell fusion. Large cell formation is functionally important because when both polyploidization and fusion are blocked, wounds do not re-epithelialize. Conclusions Our observations indicate that cell mass lost upon wounding can be replaced by polyploidization instead of mitotic proliferation. We propose that large cells generated by polyploidization or cell fusion are essential because they are better able than diploid cells to mechanically stabilize wounds, especially those containing permanent acellular structures, such as scar tissue.

The past decade of research on Drosophila stem cells and niches has provided key insights. Fly stem cells do not occupy a special "state" based on novel "stem cell genes" but resemble transiently arrested tissue progenitors. Moreover, individual stem cells and downstream progenitors are highly dynamic and dispensable, not tissue bulwarks. Niches, rather than fixed cell lineages, ensure tissue health by holding stem cells and repressing cell differentiation inside, but not outside. We review the five best-understood adult Drosophila stem cells and argue that the fundamental biology of stem cells and niches is conserved between Drosophila and mice.

The sexual development and virulence of the fungal pathogen Cryptococcus neoformans is controlled by a bipolar mating system determined by a single locus that exists in two alleles, ␣ and a. The ␣ and a mating-type alleles from two divergent varieties were cloned and sequenced. The C. neoformans mating-type locus is unique, spans >100 kb, and contains more than 20 genes. MAT-encoded products include homologs of regulators of sexual development in other fungi, pheromone and pheromone receptors, divergent components of a MAP kinase cascade, and other proteins with no obvious function in mating. The ␣ and a alleles of the mating-type locus have extensively rearranged during evolution and strain divergence but are stable during genetic crosses and in the population. The C. neoformans mating-type locus is strikingly different from the other known fungal mating-type loci, sharing features with the self-incompatibility systems and sex chromosomes of algae, plants, and animals. Our study establishes a new paradigm for mating-type loci in fungi with implications for the evolution of cell identity and self/nonself recognition.Self/nonself recognition events underlie the function of the major histocompatibility locus in defense against infection and organ transplant rejection, the self-incompatibility systems that prevent inbreeding in plants, and the production of offspring by sexual reproduction. During sexual reproduction, specialized genomic regions promote self/nonself interactions. Sexdetermining regions include the mating-type loci in fungi and the sex chromosomes in plants and animals. Dimorphic sex chromosome systems independently evolved in animals, mosses, and dioecious plants. A related but distinct sexual incompatibility system is found in many lower eukaryotes, including algae, protozoans, monoecious plants, and fungi. In these organisms, multiallelic mating-type (MAT) loci monitor cell interactions for sexual compatibility, and if inbreeding is detected, mating is aborted (17,18,50,56).A common theme of sex determinants is the need to be transmitted as a single unit, and recombination within sexdetermining regions is suppressed to avoid generating selffertile or sterile offspring. Several mechanisms operate to suppress recombination. In the fungal MAT loci, extensive sequence divergence prevents recombination between different alleles. In the case of sex chromosomes, both sequence divergence and chromosomal rearrangements suppress recombination. These rearrangements affect nearly the entire sex chromosome in humans or mice, whereas in lower vertebrates and certain dipterous insects, only a limited region of the sex chromosomes is rearranged. These findings suggest that the dimorphic sex chromosomes evolved via accumulation of chromosomal aberrations.Fungal mating-type loci serve as paradigms for understanding gene regulation during sexual development and the determination of cell fate and identity (7,16,32,34,37,40,54). Sexual development of ascomycetous fungi is commonly controlled by a bipolar mating syste...

SummaryCell wall integrity is crucial for fungal growth, development and stress survival. In the model yeast Saccharomyces cerevisiae , the cell integrity Mpk1/Slt2 MAP kinase and calcineurin pathways monitor cell wall integrity and promote cell wall remodelling under stress conditions. We have identified the Cryptococcus neoformans homologue of the S. cerevisiae b -1,3-glucan synthase inhibitor), or FK506 (a calcineurin inhibitor), and mutants lacking Mpk1 display enhanced sensitivity to nikkomycin Z and caspofungin. Lastly, we show that calcineurin and Mpk1 play complementing roles in regulating cell integrity in C. neoformans . Our studies demonstrate that pharmacological inhibition of the cell integrity pathway would enhance the activity of antifungal drugs that target the cell wall.

SummaryCalcineurin is a Ca 21 ±calmodulin-regulated protein phosphatase that is the target of the immunosuppressive drugs cyclosporin A and FK506. Calcineurin is a heterodimer composed of a catalytic A and a regulatory B subunit. In previous studies, the calcineurin A homologue was identified and shown to be required for growth at 378C and hence for virulence of the pathogenic fungus Cryptococcus neoformans.Here, we identify the gene encoding the calcineurin B regulatory subunit and demonstrate that calcineurin B is also required for growth at elevated temperature and virulence. We show that the FKR1-1 mutation, which confers dominant FK506 resistance, results from a 6 bp duplication generating a two-amino-acid insertion in the latch region of calcineurin B. This mutation was found to reduce FKBP12±FK506 binding to calcineurin both in vivo and in vitro. Molecular modelling based on the FKBP12±FK506±calcineurin crystal structure illustrates how this mutation perturbs drug interactions with the phosphatase target. In summary, our studies reveal a central role for calcineurin B in virulence and antifungal drug action in the human fungal pathogen C. neoformans.

Polyploid cells, which contain more than two genome copies, occur throughout nature. Beyond well-established roles in increasing cell size/metabolic output, polyploidy can also promote nonuniform genome, transcriptome, and metabolome alterations. Polyploidy also frequently confers resistance to environmental stresses not tolerated by diploid cells. Recent progress has begun to unravel how this fascinating phenomenon contributes to normal physiology and disease.