NORMAL GENOMIC

• Sakurai H, Takai S, Kawamura K, Ogura Y, Yoshioka Y, Kawasaki K
• Drosophila RecQ5 is involved in proper progression of early spermatogenesis.
• Biochem Biophys Res Commun. 2014; 452: 1071-7
• Display abstract

• RecQ5, a member of the conserved RecQ DNA helicase family, is required for the maintenance of genome stability. The human RECQL5 gene is expressed ubiquitously in almost all tissues, with strong expression in the testes (Shimamoto et al., 2000). However, it remains to be elucidated in which cells RecQ5 is expressed and how RecQ5 functions in the testes. In this present study we analyzed the expression of RecQ5 in Drosophila testes. The RecQ5 protein was specifically expressed in germline cells in larval, pupal, and adult testes. Drosophila RecQ5 was localized in nuclei of male germline stem cells, spermatogoniablasts, spermatogonia, and early spermatocytes. As growth of the early spermatocyte proceeded, the amount of RecQ5 increased in the nuclei. However, before maturation of the spermatocyte, the level of RecQ5 declined. Thus, RecQ5 expression was regulated. Furthermore, we compared recq5 mutant testes with the wild-type ones. The most conspicuous alterations were swelling of the apical region of and an increase in the number of spermatocytes in the recq5 testis, suggesting a relative accumulation of spermatocytes in the recq5 mutant testes. Therefore, Drosophila RecQ5 may contribute to the proper progression from germline stem cells to spermatocytes for maintenance of genome stability.

• Davies EL, Lim JG, Joo WJ, Tam CH, Fuller MT
• The transcriptional regulator lola is required for stem cell maintenance and germ cell differentiation in the Drosophila testis.
• Dev Biol. 2013; 373: 310-21
• Display abstract

• Stem cell behavior is regulated by extrinsic signals from specialized microenvironments, or niches, and intrinsic factors required for execution of context-appropriate responses to niche signals. Here we show that function of the transcriptional regulator longitudinals lacking (lola) is required cell autonomously for germline stem cell and somatic cyst stem cell maintenance in the Drosophila testis. In addition, lola is also required for proper execution of key developmental transitions during male germ cell differentiation, including the switch from transit amplifying progenitor to spermatocyte growth and differentiation, as well as meiotic cell cycle progression and spermiogenesis. Different lola isoforms, each having unique C-termini and zinc finger domains, may control different aspects of proliferation and differentiation in the male germline and somatic cyst stem cell lineages.

• Caporilli S, Yu Y, Jiang J, White-Cooper H
• The RNA export factor, Nxt1, is required for tissue specific transcriptional regulation.
• PLoS Genet. 2013; 9: 1003526-1003526
• Display abstract

• The highly conserved, Nxf/Nxt (TAP/p15) RNA nuclear export pathway is important for export of most mRNAs from the nucleus, by interacting with mRNAs and promoting their passage through nuclear pores. Nxt1 is essential for viability; using a partial loss of function allele, we reveal a role for this gene in tissue specific transcription. We show that many Drosophila melanogaster testis-specific mRNAs require Nxt1 for their accumulation. The transcripts that require Nxt1 also depend on a testis-specific transcription complex, tMAC. We show that loss of Nxt1 leads to reduced transcription of tMAC targets. A reporter transcript from a tMAC-dependent promoter is under-expressed in Nxt1 mutants, however the same transcript accumulates in mutants if driven by a tMAC-independent promoter. Thus, in Drosophila primary spermatocytes, the transcription factor used to activate expression of a transcript, rather than the RNA sequence itself or the core transcription machinery, determines whether this expression requires Nxt1. We additionally find that transcripts from intron-less genes are more sensitive to loss of Nxt1 function than those from intron-containing genes and propose a mechanism in which transcript processing feeds back to increase activity of a tissue specific transcription complex.

• Musacchio A, Helin K
• Cell cycle, differentiation and disease.
• Curr Opin Cell Biol. 2013; 25: 673-5

• Lewis PW, Sahoo D, Geng C, Bell M, Lipsick JS, Botchan MR
• Drosophila lin-52 acts in opposition to repressive components of the Myb-MuvB/dREAM complex.
• Mol Cell Biol. 2012; 32: 3218-27
• Display abstract

• The Drosophila melanogaster Myb-MuvB/dREAM complex (MMB/dREAM) participates in both the activation and repression of developmentally regulated genes and origins of DNA replication. Mutants in MMB subunits exhibit diverse phenotypes, including lethality, eye defects, reduced fecundity, and sterility. Here, we used P-element excision to generate mutations in lin-52, which encodes the smallest subunit of the MMB/dREAM complex. lin-52 is required for viability, as null mutants die prior to pupariation. The generation of somatic and germ line mutant clones indicates that lin-52 is required for adult eye development and for early embryogenesis via maternal effects. Interestingly, the maternal-effect embryonic lethality, larval lethality, and adult eye defects could be suppressed by mutations in other subunits of the MMB/dREAM complex. These results suggest that a partial MMB/dREAM complex is responsible for the lethality and eye defects of lin-52 mutants. Furthermore, these findings support a model in which the Lin-52 and Myb proteins counteract the repressive activities of the other members of the MMB/dREAM complex at specific genomic loci in a developmentally controlled manner.

• Doggett K, Jiang J, Aleti G, White-Cooper H
• Wake-up-call, a lin-52 paralogue, and Always early, a lin-9 homologue physically interact, but have opposing functions in regulating testis-specific gene expression.
• Dev Biol. 2011; 355: 381-93
• Display abstract

• A conserved multi-subunit complex (MybMuvB, MMB), regulates transcriptional activity of many different target genes in Drosophila somatic cells. A paralogous complex, tMAC, controls expression of at least 1500 genes in the male germline, and is essential for sperm production. The roles of specific subunits of tMAC, MMB or orthologous complexes in regulating target gene expression are not understood. MMB and orthologous complexes have Lin-52 as a subunit, but Lin-52 did not co-purify with tMAC. We identified wake-up-call (wuc), a lin-52 paralogue, via a physical interaction with the tMAC lin-9-related subunit Aly, and find that Wuc co-localises with known tMAC subunits. We show that wuc, like aly, is required for spermatogenesis. However, despite phenotypic similarities, the role of wuc is very different from that of previously characterised tMAC mutants. Unlike aly, loss of wuc results in only relatively mild defects in testis-specific gene expression. Strikingly, wuc loss of function partially rescues expression of target genes in aly mutant testes. We propose that wuc represses testis-specific gene expression, that this repression is counteracted by aly, and that aly and a testis-specific TF(II)D complex work together to promote high transcriptional activity of spermiogenic genes specifically in primary spermatocytes.

• Moon S, Cho B, Min SH, Lee D, Chung YD
• The THO complex is required for nucleolar integrity in Drosophila spermatocytes.
• Development. 2011; 138: 3835-45
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• The THO complex is a conserved multisubunit protein complex that functions in the formation of export-competent messenger ribonucleoprotein (mRNP). Although the complex has been studied extensively at the single-cell level, its exact role at the multicellular organism level has been poorly understood. Here, we isolated a novel Drosophila male sterile mutant, garmcho (garm). Positional cloning indicated that garm encodes a subunit of the Drosophila THO complex, THOC5. Flies lacking THOC5 showed a meiotic arrest phenotype with severe nucleolar disruption in primary spermatocytes. A functional GFP-tagged fusion protein, THOC5-GFP, revealed a unique pattern of THOC5 localization near the nucleolus. The nucleolar distribution of a testis-specific TATA binding protein (TBP)-associated factor (tTAF), SA, which is required for the expression of genes responsible for sperm differentiation, was severely disrupted in mutant testes lacking THOC5. But THOC5 appeared to be largely dispensable for the expression and nuclear export of either tTAF target mRNAs or tTAF-independent mRNAs. Taken together, our study suggests that the Drosophila THO complex is necessary for proper spermatogenesis by contribution to the establishment or maintenance of nucleolar integrity rather than by nuclear mRNA export in spermatocytes.

• Bonaccorsi S, Giansanti MG, Cenci G, Gatti M
• Preparation of meiotic chromosomes from larval and pupal Drosophila testes.
• Cold Spring Harb Protoc. 2011; 2011: 5579-5579

• Mikhaylova LM, Boutanaev AM, Nurminsky DI
• Transcriptional regulation by Modulo integrates meiosis and spermatid differentiation in male germ line.
• Proc Natl Acad Sci U S A. 2006; 103: 11975-80
• Display abstract

• Transcriptional activation in early spermatocytes involves hundreds of genes, many of which are required for meiosis and spermatid differentiation. A number of the meiotic-arrest genes have been identified as general regulators of transcription; however, the gene-specific transcription factors have remained elusive. To identify such factors, we purified the protein that specifically binds to the promoter of spermatid-differentiation gene Sdic and identified it as Modulo, the Drosophila homologue of nucleolin. Analysis of gene-expression patterns in the male sterile modulo mutant indicates that Modulo supports high expression of the meiotic-arrest genes and is essential for transcription of spermatid-differentiation genes. Expression of Modulo itself is under the control of meiotic-arrest genes and requires the DAZ/DAZL homologue Boule that is involved in the control of G(2)/M transition. Thus, regulatory interactions among Modulo, Boule, and the meiotic-arrest genes integrate meiosis and spermatid differentiation in the male germ line.

• Glasscock E, Singhania A, Tanouye MA
• The mei-P26 gene encodes a RING finger B-box coiled-coil-NHL protein that regulates seizure susceptibility in Drosophilia.
• Genetics. 2005; 170: 1677-89
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• Seizure-suppressor mutations provide unique insight into the genes and mechanisms involved in regulating nervous system excitability. Drosophila bang-sensitive (BS) mutants present a useful tool for identifying seizure suppressors since they are a well-characterized epilepsy model. Here we describe the isolation and characterization of a new Drosophila seizure-suppressor mutant that results from disruption of the meiotic gene mei-P26, which belongs to the RBCC-NHL family of proteins. The mei-P26 mutation reduces seizures in easily shocked (eas) and slamdance (sda) epileptic flies following mechanical stimulation and electroconvulsive shock. In addition, mutant mei-P26 flies exhibit seizure thresholds at least threefold greater than those of wild type. The mei-P26 phenotypes appear to result from missense mutation of a critical residue in the NHL protein-protein interaction domain of the protein. These results reveal a surprising role for mei-P26 outside of the germline as a regulator of seizure susceptibility, possibly by affecting synaptic development as a ubiquitin ligase.

• Sano Y, Akimaru H, Okamura T, Nagao T, Okada M, Ishii S
• Drosophila activating transcription factor-2 is involved in stress response via activation by p38, but not c-Jun NH(2)-terminal kinase.
• Mol Biol Cell. 2005; 16: 2934-46
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• Activating transcription factor (ATF)-2 is a member of the ATF/cAMP response element-binding protein family of transcription factors, and its trans-activating capacity is enhanced by stress-activated protein kinases such as c-Jun NH(2)-terminal kinase (JNK) and p38. However, little is known about the in vivo roles played by ATF-2. Here, we identified the Drosophila homologue of ATF-2 (dATF-2) consisting of 381 amino acids. In response to UV irradiation and osmotic stress, Drosophila p38 (dp38), but not JNK, phosphorylates dATF-2 and enhances dATF-2-dependent transcription. Consistent with this, injection of dATF-2 double-stranded RNA (dsRNA) into embryos did not induce the dorsal closure defects that are commonly observed in the Drosophila JNK mutant. Furthermore, expression of the dominant-negative dp38 enhanced the aberrant wing phenotype caused by expression of a dominant-negative dATF-2. Similar genetic interactions between dATF-2 and the dMEKK1-dp38 signaling pathway also were observed in the osmotic stress-induced lethality of embryos. Loss of dATF-2 in Drosophila S2 cells by using dsRNA abrogated the induction of 40% of the osmotic stress-induced genes, including multiple immune response-related genes. This indicates that dATF-2 is a major transcriptional factor in stress-induced transcription. Thus, dATF-2 is critical for the p38-mediated stress response.

• Bieganowski P, Shilinski K, Tsichlis PN, Brenner C
• Cdc123 and checkpoint forkhead associated with RING proteins control the cell cycle by controlling eIF2gamma abundance.
• J Biol Chem. 2004; 279: 44656-66
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• Eukaryotic initiation factor 2 (eIF2) is a central regulator of translational initiation in times of growth and times of stress. Here we discovered three new conserved regulators of eIF2 in Saccharomyces cerevisiae. cdc123, homolog of mammalian D123, is a new cell division cycle mutant with a G2 delay at permissive temperature and a terminal, mating-proficient G1 arrest point. Cdc123 protein is regulated by nutrient availability. CHF1 and CHF2, homologs of mammalian checkpoint forkhead associated with RING genes, are required for G2 delay and G1 arrest of cdc123-4 and promote G1 delay when over-expressed. Cell cycle delaying activity and the natural instability of Chf1 and Chf2 depend on the integrity of both domains and association with Cdc123. Genetic analysis maps the Chf1 forkhead associated domain-binding site to the conserved Thr-274 of Cdc123, suggesting that mammalian D123 is a key target of Chfr. Gcd11, the gamma subunit of eIF2, is an additional Cdc123-interacting protein that is an essential target of the Cdc123 cell cycle promoting and Chf cell cycle arresting activity whose abundance is regulated by Cdc123, Chf1, and Chf2. Loss of cdc123 activity promotes Chf1 and Chf2 accumulation and Gcd11 depletion, accounting for the essentiality of Cdc123. The data establish the Cdc123-Chf-Gcd11 axis as an essential pathway for nutritional control of START that runs parallel to the Tor-Gcn2-Sui2 system of translational control.

• Garbe D, Doto JB, Sundaram MV
• Caenorhabditis elegans lin-35/Rb, efl-1/E2F and other synthetic multivulva genes negatively regulate the anaphase-promoting complex gene mat-3/APC8.
• Genetics. 2004; 167: 663-72
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• Retinoblastoma (Rb)/E2F complexes repress expression of many genes important for G(1)-to-S transition, but also appear to regulate gene expression at other stages of the cell cycle. In C. elegans, lin-35/Rb and other synthetic Multivulva (SynMuv) group B genes function redundantly with other sets of genes to regulate G(1)/S progression, vulval and pharyngeal differentiation, and other unknown processes required for viability. Here we show that lin-35/Rb, efl-1/E2F, and other SynMuv B genes negatively regulate a component of the anaphase-promoting complex or cyclosome (APC/C). The APC/C is a multisubunit complex that promotes metaphase-to-anaphase progression and G(1) arrest by targeting different substrates for ubiquitination and proteasome-mediated destruction. The C. elegans APC/C gene mat-3/APC8 has been defined by temperature-sensitive embryonic lethal alleles that strongly affect germline meiosis and mitosis but only weakly affect somatic development. We describe severe nonconditional mat-3 alleles and a hypomorphic viable allele (ku233), all of which affect postembryonic cell divisions including those of the vulval lineage. The ku233 lesion is located outside of the mat-3 coding region and reduces mat-3 mRNA expression. Loss-of-function alleles of lin-35/Rb and other SynMuv B genes suppress mat-3(ku233) defects by restoring mat-3 mRNA to wild-type levels. Therefore, Rb/E2F complexes appear to repress mat-3 expression.

• Perez-Alvarado GC, Martinez-Yamout M, Allen MM, Grosschedl R, Dyson HJ, Wright PE
• Structure of the nuclear factor ALY: insights into post-transcriptional regulatory and mRNA nuclear export processes.
• Biochemistry. 2003; 42: 7348-57
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• ALY is a ubiquitously expressed nuclear protein which interacts with proteins such as TAP that are involved in export of mRNA from the nucleus to the cytoplasm, as well as with proteins that bind the T cell receptor alpha gene enhancer. ALY has also been shown to bind mRNA and to co-localize in the nucleus with components of a multiprotein postsplicing complex that is deposited 20-24 nucleotides upstream of exon-exon junctions. ALY has a conserved RNA binding domain (RBD) flanked by Gly-Arg rich N-terminal and C-terminal sequences. We determined the solution structure of the RBD homology region in ALY by nuclear magnetic resonance methods. The RBD motif in ALY has a characteristic beta(1)alpha(1)beta(2)-beta(3)alpha(2)beta(4) fold, consisting of a beta sheet composed of four antiparallel beta strands and two alpha helices that pack on one face of the beta sheet. As in other RBD structures, the beta sheet has an exposed face with hydrophobic and charged residues that could modulate interactions with other molecules. The loop that connects beta strands 2 and 3 is in intermediate motion in the NMR time scale, which is also characteristic of other RBDs. This loop presents side chains close to the exposed surface of the beta sheet and is a primary candidate site for intermolecular interactions. The structure of the conserved RBD of ALY provides insight into the nature of interactions involving this multifunctional protein.

• Spassov DS, Jurecic R
• Mouse Pum1 and Pum2 genes, members of the Pumilio family of RNA-binding proteins, show differential expression in fetal and adult hematopoietic stem cells and progenitors.
• Blood Cells Mol Dis. 2003; 30: 55-69
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• Self-renewal is the common functional property of all types of stem cells and is thought to be regulated by unknown conserved intrinsic and extrinsic molecular mechanisms. Recently, an evolutionarily conserved Pumilio family of RNA-binding proteins that regulate asymmetric cell division was found to be essential for stem cell maintenance and self-renewal in Drosophila and Caenorhabditis elegans. Based on conserved function in invertebrates and lower vertebrates it was recently proposed that an ancestral function of Pumilio proteins is to support proliferation and self-renewal of stem cells. This raises an interesting possibility that Pumilio could be part of evolutionarily conserved intrinsic molecular mechanism that regulates self-renewal of mammalian stem cells. Here we describe cloning and comparative sequence analysis of Pum1 and Pum2 genes, mouse members of the Pumilio family, and for the first time demonstrate expression of Pumilio genes in mammalian hematopoietic stem cells (HSC). Pum1 and Pum2 share 51 and 55% overall similarity with the fly Pum, whereas their RNA-binding domains show a very high degree of evolutionary conservation (86-88% homology). Both genes are expressed in a variety of tissues suggesting that they have widespread function. During blood cell development Pum1 and Pum2 exhibit differential expression in cell populations enriched for HSC and progenitors. Both genes are highly transcribed in populations of adult HSC (Rho-123(low)Sca-1(+)c-kit(+)Lin(-) cells). In a more heterogeneous population of HSC (Lin(-)Sca-1(+)) and in progenitors (Lin(-)Sca-1(-) cells) Pum1 is not transcribed, whereas Pum2 expression is significantly down-regulated. Ongoing in vitro and in vivo functional analysis of mouse Pumilio genes will help to elucidate the biological role of mammalian Pumilio genes and determine whether they play any role in maintenance of mammalian stem cells, such as HSC.

• Yuasa Y et al.
• Drosophila homeodomain protein REPO controls glial differentiation by cooperating with ETS and BTB transcription factors.
• Development. 2003; 130: 2419-28
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• In Drosophila, cell-fate determination of all neuroectoderm-derived glial cells depends on the transcription factor Glial cells missing (GCM), which serves as a binary switch between the neuronal and glial cell fates. Because the expression of GCM is restricted to the early phase of glial development, other factors must be responsible for the terminal differentiation of glial cells. Expression of three transcription factors, Reversed Polarity (REPO), Tramtrack p69 (TTK69) and PointedP1 (PNTP1), is induced by GCM in glial cells. REPO is a paired-like homeodomain protein, expressed exclusively in glial cells, and is required for the migration and differentiation of embryonic glial cells. To understand how REPO functions in glial terminal differentiation, we have analyzed the mechanism of gene regulation by REPO. We show that REPO can act as a transcriptional activator through the CAATTA motif in glial cells, and define three genes whose expression in vivo depends on REPO function. In different types of glial cells, REPO can act alone, or cooperate with either TTK69 or PNTP1 to regulate different target genes. Coordination of target gene expression by these three transcription factors may contribute to the diversity of glial cell types. In addition to promoting glial differentiation, we found that REPO is also necessary to suppress neuronal development, cooperating with TTK69. We propose that REPO plays a key role in both glial development and diversification.

• Yu F, Morin X, Kaushik R, Bahri S, Yang X, Chia W
• A mouse homologue of Drosophila pins can asymmetrically localize and substitute for pins function in Drosophila neuroblasts.
• J Cell Sci. 2003; 116: 887-96
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• Asymmetric cell division is a fundamental mechanism used to generate cellular diversity in invertebrates and vertebrates. In Drosophila, asymmetric division of neuroblasts is achieved by the asymmetric segregation of cell fate determinants Prospero and Numb into the basal daughter cell. Asymmetric segregation of cell fate determinants requires an apically localized protein complex that includes Inscuteable, Pins, Bazooka, DmPar-6, DaPKC and Galphai. Pins acts to stabilize the apical complex during neuroblast divisions. Pins interacts and colocalizes with Inscuteable, as well as maintaining its apical localization. We have isolated a mouse homologue of pins (Pins) and characterized its expression profile. Mouse PINS shares high similarity in sequence and structure with Pins and other Pins-like proteins from mammals. Pins is expressed in many mouse tissues but its expression is enriched in the ventricular zone of the developing central nervous systems. PINS localizes asymmetrically to the apical cortex of mitotic neuroblasts when ectopically expressed in Drosophila embryos. Like Pins, its N-terminal tetratricopeptide repeats can directly interact with the asymmetric localization domain of Insc, and its C-terminal GoLoco-containing region can direct localization to the neuroblast cortex. We further show that Pins can fulfill all aspects of pins function in Drosophila neuroblast asymmetric cell divisions. Our results suggest a conservation of function between the fly and mammalian Pins homologues.

• Ashraf SI, Ip YT
• The Snail protein family regulates neuroblast expression of inscuteable and string, genes involved in asymmetry and cell division in Drosophila.
• Development. 2001; 128: 4757-67
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• Delaminated neuroblasts in Drosophila function as stem cells during embryonic central nervous system development. They go through repeated asymmetric divisions to generate multiple ganglion mother cells, which divide only once more to produce postmitotic neurons. Snail, a zinc-finger transcriptional repressor, is a pan-neural protein, based on its extensive expression in neuroblasts. Previous results have demonstrated that Snail and related proteins, Worniu and Escargot, have redundant and essential functions in the nervous system. We show that the Snail family of proteins control central nervous system development by regulating genes involved in asymmetry and cell division of neuroblasts. In mutant embryos that have the three genes deleted, the expression of inscuteable is significantly lowered, while the expression of other genes that participate in asymmetric division, including miranda, staufen and prospero, appears normal. The deletion mutants also have much reduced expression of string, suggesting that a key component that drives neuroblast cell division is abnormal. Consistent with the gene expression defects, the mutant embryos lose the asymmetric localization of prospero RNA in neuroblasts and lose the staining of Prospero protein that is normally present in ganglion mother cells. Simultaneous expression of inscuteable and string in the snail family deletion mutant efficiently restores Prospero expression in ganglion mother cells, demonstrating that the two genes are key targets of Snail in neuroblasts. Mutation of the dCtBP co-repressor interaction motifs in the Snail protein leads to reduction of the Snail function in central nervous system. These results suggest that the Snail family of proteins control both asymmetry and cell division of neuroblasts by activating, probably indirectly, the expression of inscuteable and string.

• Chu T, Henrion G, Haegeli V, Strickland S
• Cortex, a Drosophila gene required to complete oocyte meiosis, is a member of the Cdc20/fizzy protein family.
• Genesis. 2001; 29: 141-52
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• Mutations in cortex and grauzone cause abnormal arrest in Drosophila female meiosis. cortex was mapped to a 14 kb interval in 26F-27A by the male recombination mapping method. While these experiments mapped the gene accurately, they also illustrated some complexities of this method. Rescue results showed that a 2.8 kb genomic fragment from this interval was able to fully rescue the cortex phenotype. The 2.8 kb rescuing fragment contains a single open reading frame. The predicted amino acid sequence indicates that cortex encodes a WD-repeat protein and is a distant member of the Cdc20 protein family. Results from a developmental Northern analysis showed that the cortex transcript is expressed at high levels during oogenesis and early embryogenesis. Interestingly, the meiotic metaphase-anaphase II arrest defect in embryos laid by cortex homozygous females resembles the mitotic metaphase-anaphase defects observed in yeast cdc20 mutants. The predicted nature of the Cortex protein, together with the observed meiotic phenotype in cortex mutants, suggest that a similar pathway to the cdc20 dependent APC-mediated proteolysis pathway, which governs the metaphase-anaphase transition in mitosis, is also important in regulating oocyte meiosis.

• Panzera Y, Esteban MR, de la Hera A, Goday C
• Meics, a novel zinc-finger protein which relocates from nuclei to the central meiotic spindle during Drosophila spermatogenesis.
• Mech Dev. 2001; 106: 151-4
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• A Drosophila gene encoding a novel zinc-finger protein, Meics, was cloned using a monoclonal antibody. The predicted amino acid sequence contains 12 zinc-finger motifs of the C2H2-type. During spermatogenesis, Meics distributes intranuclearly at pre- and post-meiotic stages whereas it relocates to central-spindle microtubules at both meiotic divisions.

• Wittwer F, van der Straten A, Keleman K, Dickson BJ, Hafen E
• Lilliputian: an AF4/FMR2-related protein that controls cell identity and cell growth.
• Development. 2001; 128: 791-800
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• Members of the AF4/FMR2 family of nuclear proteins are involved in human diseases such as acute lymphoblastic leukemia and mental retardation. Here we report the identification and characterization of the Drosophila lilliputian (lilli) gene, which encodes a nuclear protein related to mammalian AF4 and FMR2. Mutations in lilli suppress excessive neuronal differentiation in response to a constitutively active form of Raf in the eye. In the wild type, Lilli has a partially redundant function in the Ras/MAPK pathway in differentiation but it is essential for normal growth. Loss of Lilli function causes an autonomous reduction in cell size and partially suppresses the increased growth associated with loss of PTEN function. These results suggest that Lilli acts in parallel with the Ras/MAPK and the PI3K/PKB pathways in the control of cell identity and cellular growth.

• Liu H, Jang JK, Graham J, Nycz K, McKim KS
• Two genes required for meiotic recombination in Drosophila are expressed from a dicistronic message.
• Genetics. 2000; 154: 1735-46
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• We have isolated two alleles of a previously unidentified meiotic recombination gene, mei-217. Genetic analysis of these mutants shows that mei-217 is a typical "precondition" gene. The phenotypes of the mutants are meiosis specific. The strongest allele has <10% of the normal level of crossing over, and the residual events are distributed abnormally. We have used double mutant analysis to position mei-217 in the meiotic recombination pathway. In general, mutations causing defects in the initiation of meiotic recombination are epistatic to mutations in mei-41 and spnB. These two mutations, however, are epistatic to mei-217, suggesting that recombination is initiated normally in mei-217 mutants. It is likely that mei-217 mutants are able to make Holliday junction intermediates but are defective in the production of crossovers. These phenotypes are most similar to mutants of the mei-218 gene. This is striking because mei-217 and mei-218 are part of the same transcription unit and are most likely produced from a dicistronic message.

• Maines JZ, Wasserman SA
• Post-transcriptional regulation of the meiotic Cdc25 protein Twine by the Dazl orthologue Boule.
• Nat Cell Biol. 1999; 1: 171-4
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• Boule, a Drosophila orthologue of the vertebrate Dazl fertility factors, is a testis-specific regulator of meiotic entry and germline differentiation. Mutations inactivating either Boule, which is an RNA-binding protein, or Twine, which is a Cdc25-type phosphatase, block meiotic entry in males. Here we show that twine and boule interact genetically. We also find that protein expression from twine messenger RNA correlates with cytoplasmic accumulation of Boule and is markedly reduced by boule mutations. Remarkably, heterologous expression of Twine rescues the boule meiotic-entry defect, indicating that the essential function of Boule at the transition from G2 to M phase during meiosis is in the control of Twine translation.

• Johansen KM, Johansen J, Jin Y, Walker DL, Wang D, Wang Y
• Chromatin structure and nuclear remodeling.
• Crit Rev Eukaryot Gene Expr. 1999; 9: 267-77
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• Nuclear architecture is remodeled during interphase in response to changes in gene activity as well as to changing structural and functional requirements during cell division. Using the monoclonal antibody mAb2A, we have identified two proteins that appear to play important roles in these processes: JIL-1 is a tandem serine-threonine kinase implicated in the regulation of chromatin structure, whereas Skeletor is a novel protein participating in structural nuclear remodeling during the cell cycle. Antibody staining and live imaging of JIL-1-GFP transgenic flies show that JIL-1 localizes to the gene-rich interband regions of larval polytene chromosomes and is upregulated almost twofold on the hypertranscribed male X chromosome compared with autosomes. We propose that JIL-1 may play a role in transcriptional control potentially by regulating chromatin structure. The other mAb2A antigen, Skeletor, is distributed in a nuclear meshwork pattern that can be observed in stereo pair images to reorganize during the cell cycle to form a spindle-like structure at prometaphase that is distinct from the microtubule spindle apparatus. Taking advantage of the powerful molecular and genetic approaches offered in Drosophila, the study of these two proteins promises to yield new insight into what defines nuclear architecture at the molecular level and how its remodeling is regulated.

• Crow JF
• Unmasking a cheating gene.
• Science. 1999; 283: 1651-2

• Fuller MT
• Genetic control of cell proliferation and differentiation in Drosophila spermatogenesis.
• Semin Cell Dev Biol. 1998; 9: 433-44
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• Outlines of the genetic circuitry regulating male gametogenesis in Drosophila have begun to appear. Cessation of mitotic proliferation and onset of the meiotic program is regulated by the bam and bgcn genes acting within male germ cells and a TGF-beta class signaling cascade in surrounding somatic cells. Onset of spermatid differentiation is regulated by a stage- and tissue-specific transcriptional program controlled by the aly, can, mia and sa genes. A cross-regulatory mechanism might act, in part by controlling expression of the twine cell cycle phosphatase, to delay the G2/M transition of meiosis I until genes required for spermatid differentiation have been transcribed.

• Wilson PG, Zheng Y, Oakley CE, Oakley BR, Borisy GG, Fuller MT
• Differential expression of two gamma-tubulin isoforms during gametogenesis and development in Drosophila.
• Dev Biol. 1997; 184: 207-21
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• Previous work identified a gamma-tubulin gene, gamma Tub23C, in Drosophila (Zheng et al., 1991). We now report identification of a second gamma-tubulin gene, gamma Tub37CD. Immunoblot analysis and immunolocalization show that gamma Tub37CD and gamma Tub23C are differentially expressed during gametogenesis and development. During oogenesis, gamma Tub23C was detected at centrosomes and in the cytoplasm of mitotic germ cells, but was not detected in germ cells following completion of mitosis. Conversely, gamma Tub37CD was not detected in proliferating germ cells, but appeared to accumulate in germ cells during egg chamber development. Neither gamma-tubulin isoform was detected at the anterior or posterior poles of developing oocytes. During spermatogenesis, only gamma Tub23C was detected at centrosomes, where it showed cell cycle- and differentiation-dependent organization. During the transition into the first meiotic division, gamma Tub23C became organized as a corpuscular focus at centrioles until completion of meiosis II. During postmeiotic spermatid differentiation, gamma Tub23C was detected first as a rod and then as a collar-like structure near the juncture of the nucleus and the elongating flagellum, but was not detected in bundles of mature sperm. The germline-specific CDC25 encoded by twine is required for organization of gamma Tub23C into corpuscular focus in spermatocytes, but not for separation of centriole pairs in M-phase or postmeiotic organization of gamma Tub23C at centrioles. Following reconstitution of a canonical centrosome at fertilization, only gamma Tub37CD was detected at centrosomes in syncytial embryos, but both gamma Tub37CD and gamma Tub23C were detected at centrosomes in cellularized embryos. Colocalization of these two isoforms suggests that gamma Tub23C and gamma Tub37CD both contain structural features of gamma-tubulins essential for localization to centrosomes.

• Hennig W
• Spermatogenesis in Drosophila.
• Int J Dev Biol. 1996; 40: 167-76
• Display abstract

• A short summary on the present knowledge on spermatogenesis in Drosophila is given which also points out particular questions of interest in the context of this morphogenetic process. Such points of interest are the formation of lampbrush loops in primary spermatocytes, the chromosomal events during meiosis, the occurrence of chromatin rearrangements and the regulation of gene activities at the posttranscriptional level. The activities and some major conclusions from my laboratory are subsequently described. They include studies of the expression of histone variants, the structure and function of lampbrush loops and the expression of genes participating in sperm morphogenesis.

• Lin TY, Viswanathan S, Wood C, Wilson PG, Wolf N, Fuller MT
• Coordinate developmental control of the meiotic cell cycle and spermatid differentiation in Drosophila males.
• Development. 1996; 122: 1331-41
• Display abstract

• Wild-type function of four Drosophila genes, spermatocyte arrest, cannonball, always early and meiosis I arrest, is required both for cell-cycle progression through the G2/M transition of meiosis I in males and for onset of spermatid differentiation. In males mutant for any one of these meiotic arrest genes, mature primary spermatocytes with partially condensed chromosomes accumulate and postmeiotic cells are lacking. The arrest in cell-cycle progression occurs prior to degradation of cyclin A protein. The block in spermatogenesis in these mutants is not simply a secondary consequence of meiotic cell-cycle arrest, as spermatid differentiation proceeds in males mutant for the cell cycle activating phosphatase twine. Instead, the arrest of both meiosis and spermiogenesis suggests a control point that may serve to coordinate the male meiotic cell cycle with the spermatid differentiation program. The phenotype of the Drosophila meiotic arrest mutants is strikingly similar to the histopathological features of meiosis I maturation arrest infertility in human males, suggesting that the control point may be conserved from flies to man.

• Wolgemuth DJ, Rhee K, Wu S, Ravnik SE
• Genetic control of mitosis, meiosis and cellular differentiation during mammalian spermatogenesis.
• Reprod Fertil Dev. 1995; 7: 669-83
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• Gametogenesis in both the male and female mammal represents a specialized and highly regulated series of cell cycle events, involving both mitosis and meiosis as well as subsequent differentiation. Recent advances in our understanding of the genetic control of the eukaryotic cell cycle have underscored the evolutionarily-conserved nature of these regulatory processes. However, most of the data have been obtained from yeast model systems and mammalian cell lines. Furthermore, most of the observations focus on regulation of mitotic cell cycles. In the present paper: (i) aspects of gametogenesis in mammals that represent unique cell-cycle control points are highlighted; (ii) current knowledge on the regulation of the germ cell cycle, in the context of what is known in yeast and other model eukaryotic systems, is summarized; and (iii) strategies that can be used to identify additional cell cycle regulating genes are outlined.

• Ozaki T, Irie K, Sakiyama S
• Molecular cloning and cell cycle-dependent expression of a novel gene that is homologous to cdc37.
• DNA Cell Biol. 1995; 14: 1017-23
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• A cDNA clone, termed N17, was isolated from a rat fibroblast 3Y1 cDNA library by a differential screening technique. The expression of the N17 gene was significantly increased in a variety of rapidly growing cells, including v-src-, v-Ha-ras-, or v-mos-transformed 3Y1 cells, when compared with parental 3Y1 cells. The N17 gene is present as a single copy in rat genome and is evolutionarily conserved among higher eukaryotes. The predicted open reading frame (ORF) encodes a polypeptide of 379 amino acids that exhibits a significant similarity with those of the cell cycle control protein Cdc37. The amount of N17 mRNA starts to be increased in the late G1 phase and the same level was retained until just before the G2/M phase. Taken together, these results suggest that N17 gene product may play a crucial role in the cell cycle control of 3Y1 cells.

• Sjakste NI
• [Changes in the macromolecular composition and organization of the cell nucleus during gameto-, embryo- and histogenesis].
• Ontogenez. 1993; 24: 5-21
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• Data about changes in the molecular organization of the cell nucleus during the organism development and tissue differentiation are summarized. The peculiarities of the nucleus organization of gametes, its changes in the course of fertilization and early embryogenesis are discussed. Data concerning different objects of developmental biology are reviewed separately. The data about tissue specificity of the chromatin structure and characteristic features of nuclei of nervous, muscle, epithelial, and connective tissue cells are also presented. Different levels of chromatin organization, i.e., the primary and secondary DNA structure, protein composition, nucleosomal and supranucleosomal structures, DNA supercoiling in chromatin domains, nuclear skeleton structures, are specifically concerned in each particular case.

• Giroux CN
• Meiosis: components and process in nuclear differentiation.
• Dev Genet. 1992; 13: 387-91

• Riggs CD, Hasenkampf CA
• Antibodies directed against a meiosis-specific, chromatin-associated protein identify conserved meiotic epitopes.
• Chromosoma. 1991; 101: 92-8
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• The molecular mechanisms by which meiotic events are regulated are at present unknown. To approach this problem, we have exploited the natural synchrony of Lilium meiocytes to compare the nuclear protein profiles of a variety of stages of meiosis. This approach has facilitated the identification of a number of nuclear proteins that appear and disappear in a stage-specific fashion. Here we report the presence of an abundant nuclear protein that first appears during premeiotic interphase, a period during which the irreversible commitment to meiosis occurs. Antibodies directed against this protein demonstrate its meiosis specificity as well as conservation of the epitope(s) in both mono- and dicotyledonous plant species. Chromatin fractionation studies indicate that this protein, which we have termed meiotin-1, is associated with strings of nucleosomes. Implications for meiotic chromatin packaging and chromosome structure are discussed.

• Spence AM, Coulson A, Hodgkin J
• The product of fem-1, a nematode sex-determining gene, contains a motif found in cell cycle control proteins and receptors for cell-cell interactions.
• Cell. 1990; 60: 981-90
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• We report the cloning and sequencing of fem-1, a gene required for sex determination in both germline and somatic tissues in the nematode C. elegans. Clones carrying a 5.5 kb fragment are able to rescue the progeny of a fem-1 mutant when injected into its oocytes. The major fem-1 transcript in both sexes is 2.4 kb and comprises 11 exons. It encodes a soluble, intracellular protein of 656 amino acids that includes near its N-terminus six contiguous copies of a motif found in the products of the cdc10 gene of S. pombe, the SWI6 gene of S. cerevisiae, the Notch gene of Drosophila, and the lin-12 and glp-1 genes of C. elegans.

• Newlon C, Gussin G
• Molecular information in developmental genetics.
• Cell. 1975; 5: 213-25