Genetic Analysis of Viable Hsp90 Alleles Reveals a Critical Role in Drosophila Spermatogenesis (original) (raw)
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PLOS One, 2010
The molecular chaperone Hsp90 has been found to be essential for viability in all tested eukaryotes, from the budding yeast to Drosophila. In mammals, two genes encode the two highly similar and functionally largely redundant isoforms Hsp90a and Hsp90b. Although they are co-expressed in most if not all cells, their relative levels vary between tissues and during development. Since mouse embryos lacking Hsp90b die at implantation, and despite the fact that Hsp90 inhibitors being tested as anti-cancer agents are relatively well tolerated, the organismic functions of Hsp90 in mammals remain largely unknown. We have generated mouse lines carrying gene trap insertions in the Hsp90a gene to investigate the global functions of this isoform. Surprisingly, mice without Hsp90a are apparently normal, with one major exception. Mutant male mice, whose Hsp90b levels are unchanged, are sterile because of a complete failure to produce sperm. While the development of the male reproductive system appears to be normal, spermatogenesis arrests specifically at the pachytene stage of meiosis I. Over time, the number of spermatocytes and the levels of the meiotic regulators and Hsp90 interactors Hsp70-2, NASP and Cdc2 are reduced. We speculate that Hsp90a may be required to maintain and to activate these regulators and/or to disassemble the synaptonemal complex that holds homologous chromosomes together. The link between fertility and Hsp90 is further supported by our finding that an Hsp90 inhibitor that can cross the blood-testis barrier can partially phenocopy the genetic defects.
Journal of cell science, 1997
The developmental and heat-shock-induced expression of two small heat-shock proteins (Hsp23 and Hsp27) was investigated during spermatogenesis in Drosophila melanogaster. Both of these Hsps were expressed in unstressed and stressed male gonads as shown by immunoblotting. Immunostaining of whole-mount organs and thin sections of testes showed that an anti-Hsp23 antibody specifically decorated cells of the somatic lineage, such as the cyst cells and the epithelial cells of the testis and of the seminal vesicle. Hsp27 was expressed in some somatic cells (cyst cells and epithelial cells of the accessory glands) and, in addition, was also visible in the maturing spermatocytes of the germline. The same cell-specific pattern of expression was observed after heat shock, and cells which did not express Hsp23 and Hsp27 in the absence of stress were similarly unable to mount a heat shock response for these s-Hsps. However other Hsps such as Hsp70 and Hsp22 were induced under heat-shock conditi...
Hsp60C is required in follicle as well as germline cells during oogenesis inDrosophila melanogaster
Developmental Dynamics, 2008
Hsp60C gene of Drosophila melanogaster shows a dynamic spatiotemporal expression during oogenesis and seems to contribute bulk of the Hsp60 family proteins in ovarioles. Hsp60 distribution overlaps with that of F-actin–rich membranes/structures in follicle, nurse, and egg cells throughout oogenesis. Skeletal muscle fibers associated with ovarioles and in other parts of the body show patterned location of Hsp60 in A-bands. During stages 11–12, Hsp60 accumulates at junctions of nurse cells and oocyte, where a new microtubule organizing center is known to develop. A recessive hypomorph allele, Hsp60C1 causes complete sterility of the rare surviving homozygous adults. Their egg chambers show very little Hsp60C transcripts or Hsp60 protein. Beginning at stages 6–7, Hsp60C1 chambers show a disorganized follicle cell layer with poor cell adhesion in addition to abnormal organization of F-actin and other cytoskeletal structures in follicle, nurse, and egg cells. Additionally, expression and localizations of Hrb98DE, Squid, and Gurken proteins in nurse cells and oocyte are also severely affected. Hsp60C1 homozygous follicle cell clones in Hsp60C1/+ ovarioles show disruptions in follicle epithelial and cytoskeleton arrangements. Likewise, egg chambers with Hsp60C1 homozygous germline clones in Hsp60C1/+ flies show abnormal oogenesis. Our results provide the first evidence for an essential role of Hsp60C in Drosophila oogenesis, especially in organization and maintenance of cytoskeletal and cell adhesion components. Developmental Dynamics 237:1334-1347, 2008. © 2008 Wiley-Liss, Inc.
A Drosophila toolkit for defining gene function in spermatogenesis
Reproduction, 2017
Expression profiling and genomic sequencing methods enable the accumulation of vast quantities of data that relate to the expression of genes during the maturation of male germ cells from primordial germ cells to spermatozoa and potential mutations that underlie male infertility. However, the determination of gene function in specific aspects of spermatogenesis or linking abnormal gene function with infertility remain rate limiting, as even in an era of CRISPR analysis of gene function in mammalian models, this still requires considerable resources and time. Comparative developmental biology studies have shown the remarkable conservation of spermatogenic developmental processes from insects to vertebrates and provide an avenue of rapid assessment of gene function to inform the potential roles of specific genes in rodent and human spermatogenesis. The vinegar fly,Drosophila melanogaster, has been used as a model organism for developmental genetic studies for over one hundred years, a...
Functional Ecology, 2004
The relationship between heat shock protein Hsp70 expression level and the duration of heat-induced male sterility was investigated in four populations of Drosophila buzzatii Patterson and Wheeler. The effect of heat hardening on the duration of sterility was further examined after flies developed at either 25 or 31 ° C. In addition, Hsp70 expression was measured in testes after development at three different thermal regimes. 2. Four main hypotheses were tested: (i) Hsp70 is expressed in testes of D. buzzatii males even at non-stressful temperatures, and the level of expression increases with increasing rearing temperature. (ii) Hsp70 expression level differs between populations and is negatively correlated with the duration of poststress sterility. (iii) Experimentally induced Hsp70 expression at the pupal stage shortens the sterility period of flies reared above the temperature threshold of sterility. (iv) In contrast, a hardening treatment during the pupal stage of flies reared at 25 ° C results in a longer time to fertility. 3. The results matched the hypotheses, leading to the conclusion that higher Hsp70 expression reduces the duration of heat-induced male sterility.
FEBS Letters, 1999
The chaperonins are evolutionarily conserved essential cellular proteins that help folding newly synthesized or translocated proteins, spending ATP. We present here the molecular analysis of the hsp60 gene promoter region and of two Drosophila hsp60 ethyl methane sulfonate embryonic lethal alleles that have an identical phenotype. No heat shock element sequences were found in the 5P P region, supporting previous data (Kozlova, T. et al., 1997) which suggests that mitochondrial Drosophila melanogaster HSP60.1 is not heat inducible. By sequencing the lethal allele's entire open reading frame (ORF), we found a C-T transition in the hsp60 F409 allele that produces a serine to leucine change, apparently distorting the protein equatorial domain structure. No changes were found in the hsp60 G93 ORF. However, an analysis of the heterogeneous nuclear RNA levels showed a reduction of the hsp60 transcript in hsp60 G93 flies as compared to the wild-type. These data suggest that although the defects in the hsp60 gene produced by these alleles are at different levels, both behave as null mutations.
Developmental Biology, 2003
Spermatogenesis in Drosophila is maintained by germ-line stem cells. These cells undergo self-renewing divisions and also generate daughter gonial cells, whose function is to amplify the germ cell pool. Gonial cells subsequently differentiate into spermatocytes that undergo meiosis and generate haploid gametes. To elucidate the circuitry that controls progression through spermatogenic stem cell lineages, we are identifying mutations that lead to either excess germ cells or germ cell loss. From a collection of male sterile mutants, we identified P-element-induced hypomorphic alleles of nop60B, a gene encoding a pseudouridine synthase. Although null mutations are lethal, our P element-induced alleles generate viable, but sterile flies, exhibiting severe testicular atrophy. Sterility is reversed by P-element excision, and the atrophy is rescued by a Nop60B transgene, confirming identity of the gene. Using cell-type-specific markers, we find that testicular atrophy is due to severe loss of germ cells, including stem cells, but much milder effects on the somatic cells, which are themselves maintained by a stem cell lineage. We show that Nop60B activity is required intrinsically for the maintenance of germ-line stem cells. The relationship of these phenotypes to the human syndrome Dyskeratosis congenita, caused by mutations in a Nop60B homolog, is discussed.