S. A. Kopyl

The role of Drosophila Merlin in spermatogenesis

BackgroundDrosophila Merlin, the homolog of the human Neurofibromatosis 2 (NF2) gene, is important for the regulation of cell proliferation and receptor endocytosis. Male flies carrying a Mer3 allele, a missense mutation (Met177→Ile) in the Merlin gene, are viable but sterile; however, the cause of sterility is unknown.ResultsTestis examination reveals that hemizygous Mer3 mutant males have small seminal vesicles that contain only a few immotile sperm. By cytological and electron microscopy analyses of the Mer3, Mer4 (Gln170→stop), and control testes at various stages of spermatogenesis, we show that Merlin mutations affect meiotic cytokinesis of spermatocytes, cyst polarization and nuclear shaping during spermatid elongation, and spermatid individualization. We also demonstrate that the lethality and sterility phenotype of the Mer4 mutant is rescued by the introduction of a wild-type Merlin gene. Immunostaining demonstrates that the Merlin protein is redistributed to the area associated with the microtubules of the central spindle in telophase and its staining is less in the region of the contractile ring during meiotic cytokinesis. At the onion stage, Merlin is concentrated in the Nebenkern of spermatids, and this mitochondrial localization is maintained throughout sperm formation. Also, Merlin exhibits punctate staining in the acrosomal region of mature sperm.ConclusionMerlin mutations affect spermatogenesis at multiple stages. The Merlin protein is dynamically redistributed during meiosis of spermatocytes and is concentrated in the Nebenkern of spermatids. Our results demonstrated for the first time the mitochondrial localization of Merlin and suggest that Merlin may play a role in mitochondria formation and function during spermatogenesis.

Genes Controlling Development: Morphoses, Phenocopies, Dimorphs, and Other Visible Expressions of Mutant Genes

We studied facultative dominant lethal mutations obtained earlier inDrosophila melanogaster. In some genotypes, these mutations were expressed as lethals, but in other genotypes they lacked this expression. The mutations were maintained in the following cultures: (1) females Muller-5 heterozygous for the mutation; (2) males crossed to attached-Xfemales; and (3) females and males homozygous for the mutation. During culturing, many mutations were found to give rise to phenotypically abnormal progeny. Generally, these abnormalities were morphoses involving various body parts; they were mostly asymmetric and nonheritable. Maternal and paternal effects in the formation of morphoses were observed. In four cases, dimorphic mutations were recorded: a female homozygous for the mutation had mutant phenotype whereas its male counterpart was phenotypically normal. The mutations were recessive with regard to the norm. New phenotypes behaving as mutations with incomplete penetrance arose during culturing. In cultures of mutant homozygotes phenocopies would appear en masse; they would persist for one or two generations and disappear. One wave of phenocopies succeeded another. Visible phenotypes appeared, which further behaved as ordinary recessive mutations. We concluded that these visible manifestations are characteristic for regulatory mutations controlling ontogeny. Their appearance is explained by the activation of new regulatory scenarios caused by blocking standard regulatory pathways.

From Genetics of Intraspecific Differences to Genetics of Intraspecific Similarity

Based on the Mendelian approach to heredity, modern genetics describes inheritance of characters belonging to the category of intraspecific difference. the other large category of characters,intraspecific similarity, stays out of investigation. In this review, the genome part responsible for intraspecific similarity is considered as invariant and regulatory. An approach to studying the invariant part of the Drosophila melanogaster genome is formulated and the results of examining this genome part are presented. The expression of mutations at genes in the invariant genome part is different from that of Mendelian genes. We conclude that these genes are present in the genome in multiple copies and they are functionally haploid in the diploid genome. Severe abnormalities of development appearing in the progeny of mutant parents suggest that the mutant genes are genes regulating ontogeny. A hypothesis on an elementary ontogenetic event is advanced and the general scheme of ontogeny is presented. A concept on two types of gene allelism (cis- and trans-allelism) is formulated. This approach opens a possibility for studying genetic material responsible for the formation of intraspecific similarity characters at different taxonomic levels on the basis of crossing individuals of the same species.

Role of the porcupine gene in the development of the wing imaginal disk of Drosophila melanogaster

A search for the genes interacting with the Merlin tumor suppressor gene revealed a Merlin-porcupine interaction during wing morphogenesis. Ectopic expression of the porcupine gene in the wing imaginal disk reduced the adult wing, while addition of an UAS construct with a full-length or truncated copy of the Merlin gene partly restored the wing phenotype. The highest restoration level was observed upon adding the fragments coding for the C end of the Merlin protein. In addition, the porcupine gene was shown to mediate the wingless gene autoregulation, which occurs at two ontogenetic stages, segmentation during embryo development and determination of the wg expression band at the boundary between the dorsal and ventral compartments of the wing imaginal disk.

The influence of morphogene Wg on the formation of an ectopic eye in Drosophila melanogaster

Due to the ectopic expression of the ey gene in the wing imaginal disc under the action of the 1096-Gal4 driver, a part of the wing disc cells change their fate and become eye cells. Ectopic eyes are induced in definite regions of the wing disc and form a stable pattern on the wing of an adult fly. Here, we have shown that the ectopic expression of Wg inhibits the formation of ectopic eyes, and conversely the expression of Wg is reduced in the sites of ectopic Ey expression. Experiments with overexpression of the vesicular traffic protein Hrs capable of inhibiting the Wg signaling agree with the notion on antagonism of Wg and Ey in ectopic eyes. Our results confirm that the processes of formation of normal and ectopic eyes are principally similar with regard to genetic control.

Role of the Drosophila melanogaster hrs gene in wing formation

The Hrs (hepatocyte growth factor receptor tyrosine kinase substrate) protein is an endosomal protein whose function is to transport receptor tyrosine kinases from early endosomes to lysosomes. Since receptor tyrosine kinases are involved in various signaling pathways, HSR defects lead to various malformations. A study of the role of the hrs gene in wing development in Drosophila confirmed that the gene is involved in the formation of the D/V boundary of the wing imaginal disk and suggested a new role in wing vein refinement for the gene. Structural analysis of the hrs gene transcripts indicated that transcript B is responsible for vein refinement.

Drosophila melanogaster gene Merlin interacts with the clathrin adaptor protein gene lap

The protein Merlin is involved in the regulation of cell proliferation and differentiation in the eyes and wings of Drosophila and is a homolog of the human protein encoded by the Neurofibromatosis 2 (NF2) gene whose mutations cause auricular nerve tumors. Recent studies show that Merlin and Expanded cooperatively regulate the recycling of membrane receptors, such as the epidermal growth factor receptor (EGFR). By performing a search for potential genetic interactions between Merlin (Mer) and the genes important for vesicular trafficking, we found that ectopic expression in the wing pouch of the clathrin adapter protein Lap involved in clathrin-mediated receptor endocytosis resulted in the formation of extra vein materials. On the one hand, coexpression of wild-type Merlin and lap in the wing pouch restored normal venation, while overexpression of a dominant-negative mutant MerDBB together with lap enhanced ectopic vein formation. Using various constructs with Merlin truncated copies, we showed the C-terminal portion of the Merlin protein to be responsible for the Merlin-lap genetic interaction. Furthermore, we showed that the Merlin and Lap proteins colocalized at the cortex of the wing imaginal disc cells.

A study of cellular localization of HRS and other endosome markers during spermatogenesis in Drosophila melanogaster using chimeric GFP constructs

Acrosome is a specialized organelle in spermatozoids necessary for fertilization of oocyte. Acrosome is formed, according to various theories, either from vesicles of the Golgi apparatus or from endosomes and lysosomes. Hepatocyte growth factor regulated tyrosine kinase substrate (Hrs) is a multidomain protein component of ESCRT-0 complex, that participates in sorting of proteins absorbed during endocytosis. It has been shown that, in mammals, Hgs protein (Hrs homologue) possesses the ability to bind the acrosome, but whether Drosophila Hrs has a similar capacity has not been determined. We found that two forms of Hrs protein are expressed in Drosophila testes: a long form, Hrs-B, and a truncated form, Hrs-A. The later lacks a VHS domain and a portion of FYVE domain, both directly involved in Hrs anchoring to endosomes. We also determined that, unlike mammal Hgs, Drosophila Hrs-B isoform and an almost identical truncated form, Hrs8 ([290–760]), are localized not to acrosome, but to spermatocyte fusomes. This localization requires a portion of the protein molecule located between amino acid residues 383 and 472, which presumably correspond to NF2- and/or Stam-binding domains. In situ hybridization for hrs mRNA showed that the gene is expressed during early spermatogenesis. This is consistent with our data that the Hrs protein binds to spermatocyte fusomes and is absent during later spermatogenesis stages. In addition, we have shown that Hrs is not involved in regulation or implementation of cytokinesis in spermatocytes. Finally, despite the absence of Hrs on Drosophila acrosomes, we detected endosome markers Rab4, Rab7, and Rab11 on this organelle. Thus, our data support the endosomal hypothesis of acrosome biogenesis.

Genetic modifiers of ectopic eye formation on wings of Drosophila melanogaster

The ectopic expression of the master ey gene by the GAL4-UAS system can induce ectopic eye formation in different organs. The formation of ectopic eyes takes place in certain regions of imaginal discs, which partially overlap with the regions responsible for the transdetermination of differentiated cells (essentially meaning the alteration of the cell fate). In this way, ectopic eye induction could be considered as a model for cellular plasticity studies. In the present work, we performed a search for transgenes, the ectopic coexpression of which with the master ey gene induced morphologic changes in the ectopic eyes on the wing compared to the sole ey expression. Most of the transgenes found to affect the size of ectopic eyes belonged to the class of vesicular trafficking genes capable of affecting different signaling pathways. The ectopic expression of the revealed transgenes in the wing and eye discs altered the morphology of both normal wings and normal eyes. We argue that the effect of these genes may be that they change the size of the region responsible for cell fate transdetermination.

A dose-sensitive modifier of the of Drosophila melanogaster ectopic eye

Ectopic eyes induced in a wing serve as a system for studying developmental plasticity in Drosophila. We used a set of chromosome deficiencies covering the second chromosome to ask whether there are dose-sensitive modifiers of the process. We identified three overlapping deletions showing the enlargement of ectopic eyes. The study of the genes localized in the region of interest suggests that the mutation in the sxc (super sex combs) gene (PcG group) is responsible for the observed phenotype.