The role of Drosophila Merlin in spermatogenesis

Background

Drosophila 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 Mer ³ allele, a missense mutation (Met¹⁷⁷→Ile) in the Merlin gene, are viable but sterile; however, the cause of sterility is unknown.

Results

Testis examination reveals that hemizygous Mer ³ mutant males have small seminal vesicles that contain only a few immotile sperm. By cytological and electron microscopy analyses of the Mer ³, Mer ⁴ (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 Mer ⁴ 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.

Conclusion

Merlin 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.     

Merlin Isoforms 1 and 2 Both Act as Tumour Suppressors and Are Required for Optimal Sperm Maturation

The tumour suppressor Merlin, encoded by the gene NF2, is frequently mutated in the autosomal dominant disorder neurofibromatosis type II, characterised primarily by the development of schwannoma and other glial cell tumours. However, NF2 is expressed in virtually all analysed human and rodent organs, and its deletion in mice causes early embryonic lethality. Additionally, NF2 encodes for two major isoforms of Merlin of unknown functionality. Specifically, the tumour suppressor potential of isoform 2 remains controversial. In this study, we used Nf2 isoform-specific knockout mouse models to analyse the function of each isoform during development and organ homeostasis. We found that both isoforms carry full tumour suppressor functionality and can completely compensate the loss of the other isoform during development and in most adult organs. Surprisingly, we discovered that spermatogenesis is strictly dependent on the presence of both isoforms. While the testis primarily expresses isoform 1, we noticed an enrichment of isoform 2 in spermatogonial stem cells. Deletion of either isoform was found to cause decreased sperm quality as observed by maturation defects and head/midpiece abnormalities. These defects led to impaired sperm functionality as assessed by decreased sperm capacitation. Thus, we describe spermatogenesis as a new Nf2-dependent process. Additionally, we provide for the first time in vivo evidence for equal tumour suppressor potentials of Merlin isoform 1 and isoform 2.     

The role of the functional sites of the merlin tumor suppressor in <Emphasis Type="Italic">Drosophila Spermatogenesis</Emphasis>

The Merlin gene of Drosophila is homologous to the human Neurofibromatosis 2 (NF2) gene, an important regulator of proliferation and endocytosis of cell receptors. It was earlier shown that the Thr⁵⁵⁹ residue of the Drosophila Merlin protein was homologous to Ser⁵¹⁸ of the human protein (which was already known to undergo phosphorylation); hence, it was assumed that Thr⁵⁵⁹ of Drosophila also was a substrate of phosphorylation. The mutant Merlin proteins MerT^559D (an analog of the phosphorylated form) and MerT^559A (a nonphosphorylated form) were constructed and tested, under the conditions of ectopic expression, for the ability to correct the spermatogenesis defects induced by the Mer4 mutation. The mutant form MerT^559D was demonstrated to restore the abnormal nebenkern phenotype induced by this mutation, whereas the MerT^559A substituted form did not restore this phenotype. Ectopic expression o the wild-type Merlin protein, MerT^559A mutant form, and mycMer^345–635 truncated protein in a normal genotype resulted in the abnormal nebenkern phenotype, whereas this phenotype was not observed in the case of ectopic expression of the Mer^T559D analog of the phosphorylated form. Ectopic expression of the mycMer³, mycMer^ΔBB, and mycMer^1–379 truncate variants led to disturbance of meiotic cytokinesis.     

Regulation of hedgehog Ligand Expression by the N-End Rule Ubiquitin-Protein Ligase Hyperplastic Discs and the Drosophila GSK3β Homologue,Shaggy

Hedgehog (Hh) morphogen signalling plays an essential role in tissue development and homeostasis. While much is known about the Hh signal transduction pathway, far less is known about the molecules that regulate the expression of the hedgehog (hh) ligand itself. Here we reveal that Shaggy (Sgg), the Drosophila melanogaster orthologue of GSK3β, and the N-end Rule Ubiquitin-protein ligase Hyperplastic Discs (Hyd) act together to co-ordinate Hedgehog signalling through regulating hh ligand expression and Cubitus interruptus (Ci) expression. Increased hh and Ci expression within hyd mutant clones was effectively suppressed by sgg RNAi, placing sgg downstream of hyd. Functionally, sgg RNAi also rescued the adult hyd mutant head phenotype. Consistent with the genetic interactions, we found Hyd to physically interact with Sgg and Ci. Taken together we propose that Hyd and Sgg function to co-ordinate hh ligand and Ci expression, which in turn influences important developmental signalling pathways during imaginal disc development. These findings are important as tight temporal/spatial regulation of hh ligand expression underlies its important roles in animal development and tissue homeostasis. When deregulated, hh ligand family misexpression underlies numerous human diseases (e.g., colorectal, lung, pancreatic and haematological cancers) and developmental defects (e.g., cyclopia and polydactyly). In summary, our Drosophila-based findings highlight an apical role for Hyd and Sgg in initiating Hedgehog signalling, which could also be evolutionarily conserved in mammals.     

In vitro spermatogenesis in Drosophila

Summary In vitro spermatogenesis of isolated single spermatocyte cysts of Drosophila hydei was studied by microscopic observations and time-lapse cinematography. Cysts of spermatocytes isolated during diplotene develop as far as the coiling stage of spermatid differentiation. The existence of an interphase between meiosis I and meiosis II is, for the first time, documented. Meiosis, Nebenkern formation, and elongation of spermatids occur just as in D. melanogaster; however, an individualization cone, as described for D. melanogaster, can not be detected.     

Roles for Drp1, a Dynamin-related Protein,and Milton,a Kinesin-associated Protein,in Mitochondrial Segregation,Unfurling, and Elongation During Drosophila Spermatogenesis

Mitochondria undergo dramatic rearrangement during Drosophila spermatogenesis. In wild type testes, the many small mitochondria present in pre-meiotic spermatocytes later aggregate, fuse, and interwrap in post-meiotic haploid spermatids to form the spherical Nebenkern, whose two giant mitochondrial compartments later unfurl and elongate beside the growing flagellar axoneme. Drp1 encodes a dynamin-related protein whose homologs in many organisms mediate mitochondrial fission and whose Drosophila homolog is known to govern mitochondrial morphology in neurons. The milton gene encodes an adaptor protein that links mitochondria with kinesin and that is required for mitochondrial transport in Drosophila neurons. To determine the roles of Drp1 and Milton in spermatogenesis, we used the FLP-FRT mitotic recombination system to generate spermatocytes homozygous for mutations in either gene in an otherwise heterozygous background. We found that absence of Drp1 leads to abnormal clustering of mitochondria in mature primary spermatocytes and aberrant unfurling of the mitochondrial derivatives in early Drp1 spermatids undergoing axonemal elongation. In milton spermatocytes, mitochondria are distributed normally; however, after meiosis, the Nebenkern is not strongly anchored to the nucleus, and the mitochondrial derivatives do not elongate properly. Our work defines specific functions for Drp1 and Milton in the anchoring, unfurling, and elongation of mitochondria during sperm formation.     

Lipid metabolism and Drosophila sperm development

Lipids are essential membrane structural components and important signal carriers. The major enzymatic metabolisms of various lipids (phospholipid, sphingolipid, cholesterol) are well studied. The developmental function of lipid metabolism has remained, for the most part, elusive. With the help of new techniques and model organisms, the important roles of lipid metabolism in development just start to emerge. Drosophila spermatogenesis is an ideal system for in vivo studies of cytokinesis and membrane remodeling during development. The metabolic regulators of many lipids, including phosphatidylinositol (PI) lipids, fatty acids and cholesterol, are reported to play critical roles in various steps during Drosophila spermatogenesis. In this mini-review, we summarized recent findings supporting a tight link between lipids metabolism and Drosophila sperm development.     

Dll3 pudgy mutation differentially disrupts dynamic expression of somite genes

Mutations in the notch ligand delta-like 3 have been identified in both the pudgy mouse (Dll3(pu); Kusumi et al.: Nat Genet 19:274-278, 1998) and the human disorder spondylocostal dysostosis (SCD; Bulman et al.: Nat Genet 24:438-441, 2000), and a targeted mutation has been generated (Dll3(neo); Dunwoodie et al.: Development 129:1795-1806, 2002). Vertebral and rib malformations deriving from defects in somitic patterning are key features of these disorders. In the mouse, notch pathway genes such as Lfng, Hes1, Hes7, and Hey2 display dynamic patterns of expression in paraxial mesoderm, cycling in synchrony with somite formation (Aulehla and Johnson: Dev Biol 207:49-61, 1999; Forsberg et al.: Curr Biol 8:1027-1030, 1998; Jouve et al.: Development 127:1421-1429, 2000; McGrew et al.: Curr Biol 8:979-982, 1998; Nakagawa et al.: Dev Biol 216:72-84, 1999). We report here that the Dll3(pu) mutation has different effects on the expression of cycling (Lfng and Hes7) and stage-specific genes (Hey3 and Mesp2). This suggests a more complex situation than a single oscillatory mechanism in somitogenesis and provides an explanation for the unique radiological features of the human DLL3-type of SCD.     

Loss of <Emphasis Type="Italic">Tc-arrow</Emphasis> and canonical Wnt signaling alters posterior morphology and pair-rule gene expression in the short-germ insect, <Emphasis Type="Italic">Tribolium castaneum</Emphasis>

Wnt signaling has been implicated in posterior patterning in short-germ insects, including the red flour beetle Tribolium castaneum (Bolognesi et al. Curr Biol 18:1624–1629, 2008b; Angelini and Kaufman Dev Biol 283:409–423, 2005; Miyawaki et al. Mech Dev 121:119–130, 2004). Specifically, depletion of Wnt ligands Tc-Wnt1 and Tc-WntD/8 produces Tribolium embryos lacking abdominal segments. Similar phenotypes are produced by depletion of Tc-porcupine (Tc-porc) or Tc-pangolin (Tc-pan), indicating that the signal is transmitted through the canonical Wnt pathway (Bolognesi et al. Curr Biol 18:1624–1629, 2008b). Here we show that RNAi for the receptor Tc-arrow produced similar truncated phenotypes, providing additional evidence supporting canonical signal transduction. Furthermore, since in Tribolium segments are defined sequentially by a pair-rule gene circuit that, when interrupted, produces truncated phenotypes (Choe et al. Proc Natl Acad Sci U S A 103:6560–6564, 2006), we investigated the relationship between loss of Wnt signaling and this pair-rule gene circuit. After depletion of the receptor Tc-arrow, expression of Tc-Wnt1 was noticeably absent from the growth zone, while Tc-WntD/8 was restricted to a single spot of expression in what remained of the posterior growth zone. The primary pair-rule genes Tc-runt (Tc-run) and Tc-even-skipped (Tc-eve) were expressed normally in the anterior segments, but were reduced to a single spot in the remnants of the posterior growth zone. Thus, expression of pair-rule genes and Tc-WntD/8 are similarly affected by depletion of Wnt signal and disruption of the posterior growth zone.     

Evolution and origin of merlin,the product of the <Emphasis Type="Italic">Neurofibromatosis type 2</Emphasis> (<Emphasis Type="Italic">NF2</Emphasis>) tumor-suppressor gene

Background  

Merlin, the product of the Neurofibromatosis type 2 (NF2) tumor suppressor gene, belongs to the ezrin-radixin-moesin (ERM) subgroup of the protein 4.1 superfamily, which links cell surface glycoproteins to the actin cytoskeleton. While merlin's functional activity has been examined in mammalian and Drosophila models, little is understood about its evolution, diversity, and overall distribution among different taxa.     

The Des-1 protein, required for central spindle assembly and cytokinesis, is associated with mitochondria along the meiotic spindle apparatus and with the contractile ring during male meiosis in Drosophila melanogaster

Spermatogenesis in Drosophila melanogaster serves as an excellent model system for the isolation and analysis of genes required in the control of chromosome segregation and cytokinesis. We report here the isolation and molecular characterization of a novel P-element induced allele of the des-1 gene, which leads to male sterility as a consequence of the failure of central spindle assembly in meiotic spermatocytes and the formation of aberrant meiotic end products characteristic of cytokinesis failure. We have raised affinity-purified antibodies against a Des-1 fusion protein, and localized the Des-1 protein in Drosophila spermatocytes. We show that the Des-1 protein is colocalized with mitochondria throughout male meiosis, becoming intimately associated with mitochondria along the spindle apparatus during anaphase and telophase, and with the Nebenkern, or mitochondrial derivative, of the meiotic end products. In addition, a significant association of Des-1 with the contractile ring is observed during anaphase and telophase of meiosis. These observations, together with the presence of six potential transmembrane domains in the Des-1 protein, raise the possibility that Des-1 may act as part of an anchoring mechanism that links membrane-bounded cellular compartments to components of the cytoskeleton. Received: 3 April 1998 / Accepted: 2 July 1998     

The Drosophila genes CG14593 and CG30106 code for G-protein-coupled receptors specifically activated by the neuropeptides CCHamide-1 and CCHamide-2

Recently, a novel neuropeptide, CCHamide, was discovered in the silkworm Bombyx mori (L. Roller et al., Insect Biochem. Mol. Biol. 38 (2008) 1147–1157). We have now found that all insects with a sequenced genome have two genes, each coding for a different CCHamide, CCHamide-1 and -2. We have also cloned and deorphanized two Drosophila G-protein-coupled receptors (GPCRs) coded for by genes CG14593 and CG30106 that are selectively activated by Drosophila CCH-amide-1 (EC₅₀, 2 × 10⁻⁹ M) and CCH-amide-2 (EC₅₀, 5 × 10⁻⁹ M), respectively. Gene CG30106 (symbol synonym CG14484) has in a previous publication (E.C. Johnson et al., J. Biol. Chem. 278 (2003) 52172–52178) been wrongly assigned to code for an allatostatin-B receptor. This conclusion is based on our findings that the allatostatins-B do not activate the CG30106 receptor and on the recent findings from other research groups that the allatostatins-B activate an unrelated GPCR coded for by gene CG16752. Comparative genomics suggests that a duplication of the CCHamide neuropeptide signalling system occurred after the split of crustaceans and insects, about 410 million years ago, because only one CCHamide neuropeptide gene is found in the water flea Daphnia pulex (Crustacea) and the tick Ixodes scapularis (Chelicerata).     

The cholesterol trafficking protein NPC1 is required for Drosophila spermatogenesis

Niemann–Pick C (NPC) disease is a lethal neurodegenerative disorder affecting cellular sterol trafficking. Besides neurodegeneration, NPC patients also exhibit other pleiotropic conditions, indicating that NPC protein is required for other physiological processes. Previous studies indicated that a sterol shortage that in turn leads to a shortage of steroid hormones (for example, ecdysone in Drosophila) is likely to be the cause of NPC disease pathology. We have shown that mutations in Drosophila npc1, one of the two NPC disease-related genes, leads to larval lethal and male infertility. Here, we reported that npc1 mutants are defective in spermatogenesis and in particular in the membrane-remodeling individualization process. Interestingly, we found that ecdysone, the steroid hormone responsible for the larval lethal phenotype in npc1 mutants, is not required for individualization. However, supplying 7-dehydrocholesterol can partially rescue the male infertility of npc1 mutants, suggesting that a sterol shortage is responsible for the spermatogenesis defects. In addition, the individualization defects of npc1 mutants were enhanced at high temperature, suggesting that the sterol shortage may lead to temperature-sensitive defects in the membrane-remodeling process. Together, our study reveals a sterol-dependent, ecdysone-independent mechanism of NPC1 function in Drosophila spermatogenesis.     

Structural Analysis of Drosophila Merlin Reveals Functional Domains Important for Growth Control and Subcellular Localization

Merlin, the product of the Neurofibromatosis type 2 (NF2) tumor-suppressor gene, is a member of the protein 4.1 superfamily that is most closely related to ezrin, radixin, and moesin (ERM). NF2 is a dominantly inherited disease characterized by the formation of bilateral acoustic schwannomas and other benign tumors associated with the central nervous system. To understand its cellular functions, we are studying a Merlin homologue in Drosophila. As is the case for NF2 tumors, Drosophila cells lacking Merlin function overproliferate relative to their neighbors. Using in vitro mutagenesis, we define functional domains within Merlin required for proper subcellular localization and for genetic rescue of lethal Merlin alleles. Remarkably, the results of these experiments demonstrate that all essential genetic functions reside in the plasma membrane– associated NH₂-terminal 350 amino acids of Merlin. Removal of a seven–amino acid conserved sequence within this domain results in a dominant-negative form of Merlin that is stably associated with the plasma membrane and causes overproliferation when expressed ectopically in the wing. In addition, we provide evidence that the COOH-terminal region of Merlin has a negative regulatory role, as has been shown for ERM proteins. These results provide insights into the functions and functional organization of a novel tumor suppressor gene.