The switch from precursor cell proliferation to onset of differentiation in adult stem cell lineages must be carefully regulated to produce sufficient progeny to maintain and repair tissues, yet prevent overproliferation that may enable oncogenesis. In the Drosophila male germ cell lineage, spermatogonia produced by germ line stem cells undergo a limited number of transit amplifying mitotic divisions before switching to the spermatocyte program that sets up meiosis and eventual spermatid differentiation. The number of transit amplifying divisions is set by accumulation of the bag-of-marbles (Bam) protein to a critical threshold. In bam mutants, spermatogonia proliferate through several extra rounds of mitosis and then die without becoming spermatocytes. Here, we show that a key role of Bam for the mitosis to differentiation switch is repressing expression of Held Out Wings (how), homolog of mammalian Quaking. Knockdown of how in germ cells was sufficient to allow spermatogonia mutant for bam or its partner benign gonial cell neoplasm to differentiate, while forced expression of nuclear-targeted How protein in spermatogonia wild-type for bam resulted in continued proliferation at the expense of differentiation. Our findings suggest that Bam targets how RNA for degradation by acting as an adapter to recruit the CCR4-NOT deadenylation complex via binding its subunit, Caf40. As How is itself an RNA-binding protein with roles in RNA processing, our findings reveal that the switch from proliferation to meiosis and differentiation in the Drosophila male germ line adult stem cell lineage is regulated by a cascade of RNA-binding proteins.

During meiosis, germ cell and stage-specific components impose additional layers of regulation on the core cell cycle machinery to set up an extended G2 period termed meiotic prophase. In Drosophila males, meiotic prophase lasts 3.5 days, during which spermatocytes turn up expression of over 3000 genes and grow 25-fold in volume. Previous work showed that the core cell cycle regulator Cyclin B (CycB) is subject to translational repression in immature Drosophila spermatocytes, mediated by the RNA-binding protein Rbp4 and its partner Fest. Here we show that another spermatocyte-specific protein, Lut, is required for translational repression of cycB in an 8-hour window just before spermatocytes are fully mature. In males mutant for rbp4 or lut, spermatocytes enter and exit the meiotic divisions 6-8 hours earlier than in wild-type. In addition, we show that spermatocyte-specific isoforms of Syncrip (Syp) are required for expression of CycB protein and normal entry into the meiotic divisions. Both Lut and Syp interact with Fest in an RNA-independent manner. Thus a complex of spermatocyte-specific regulators choreograph the timing of expression of CycB protein during male meiotic prophase. SUMMARY STATEMENT Expression of a conserved cell cycle component, Cyclin B, is regulated by multiple mechanisms in the Drosophila male germline to dictate the correct timing of meiotic division.

ABSTRACT During meiosis, germ cell and stage-specific components impose additional layers of regulation on the core cell cycle machinery to set up an extended G2 period termed meiotic prophase. In Drosophila males, meiotic prophase lasts 3.5 days, during which spermatocytes upregulate over 1800 genes and grow 25-fold. Previous work has shown that the cell cycle regulator Cyclin B (CycB) is subject to translational repression in immature spermatocytes, mediated by the RNA-binding protein Rbp4 and its partner Fest. Here, we show that the spermatocyte-specific protein Lut is required for translational repression of cycB in an 8-h window just before spermatocytes are fully mature. In males mutant for rbp4 or lut, spermatocytes enter and exit meiotic division 6-8 h earlier than in wild type. In addition, spermatocyte-specific isoforms of Syncrip (Syp) are required for expression of CycB protein in mature spermatocytes and normal entry into the meiotic divisions. Lut and Syp interact with Fest independent of RNA. Thus, a set of spermatocyte-specific regulators choreograph the timing of expression of CycB protein during male meiotic prophase.

For more than 100 years, the fruit fly Drosophila melanogaster has been one of the most studied model organisms. Here, we present a single-cell atlas of the adult fly, Tabula Drosophilae, that includes 580,000 nuclei from 15 individually dissected sexed tissues as well as the entire head and body, annotated to >250 distinct cell types. We provide an in-depth analysis of cell type-related gene signatures and transcription factor markers, as well as sexual dimorphism, across the whole animal. Analysis of common cell types between tissues, such as blood and muscle cells, reveals rare cell types and tissue-specific subtypes. This atlas provides a valuable resource for the Drosophila community and serves as a reference to study genetic perturbations and disease models at single-cell resolution.

The unique cell cycle dynamics of meiosis are controlled by layers of regulation imposed on core mitotic cell cycle machinery components by the program of germ cell development. Although the mechanisms that regulate Cdk1/Cyclin B activity in meiosis in oocytes have been well studied, little is known about the transacting factors responsible for developmental control of these factors in male gametogenesis. During meiotic prophase in Drosophila males, transcript for the core cell cycle protein Cyclin B1 (CycB) is expressed in spermatocytes, but the protein does not accumulate in spermatocytes until just before the meiotic divisions. Here, we show that two interacting proteins, Rbp4 and Fest, expressed at the onset of spermatocyte differentiation under control of the developmental program of male gametogenesis, function to direct cell type- and stage-specific repression of translation of the core G2/M cell cycle component cycB during the specialized cell cycle of male meiosis. Binding of Fest to Rbp4 requires a 31-amino acid region within Rbp4. Rbp4 and Fest are required for translational repression of cycB in immature spermatocytes, with Rbp4 binding sequences in a cell type-specific shortened form of the cycB 3'UTR. Finally, we show that Fest is required for proper execution of meiosis I.

Intercellular signaling is essential for the coordination of growth and development in higher plants. Although hundreds of putative receptors have been identified in Arabidopsis (Arabidopsis thaliana), only a few families of extracellular signaling molecules have been discovered, and their biological roles are largely unknown. To expand our insight into the developmental processes potentially regulated by ligand-mediated signal transduction pathways, we undertook a systematic expression analysis of the members of the Arabidopsis CLAVATA3/ESR-RELATED (CLE) small signaling polypeptide family. Using reporter constructs, we show that the CLE genes have distinct and specific patterns of promoter activity. We find that each Arabidopsis tissue expresses at least one CLE gene, indicating that CLE-mediated signaling pathways are likely to play roles in many biological processes during the plant life cycle. Some CLE genes that are closely related in sequence have dissimilar expression profiles, yet in many tissues multiple CLE genes have overlapping patterns of promoter-driven reporter activity. This observation, plus the general absence of detectable morphological phenotypes in cle null mutants, suggest that a high degree of functional redundancy exists among CLE gene family members. Our work establishes a community resource of CLE-related biological materials and provides a platform for understanding and ultimately manipulating many different plant signaling systems.

Intercellular signaling is essential for the coordination of growth and development in higher plants. Although hundreds of putative receptors have been identified in Arabidopsis (Arabidopsis thaliana), only a few families of extracellular signaling molecules have been discovered, and their biological roles are largely unknown. To expand our insight into the developmental processes potentially regulated by ligand-mediated signal transduction pathways, we undertook a systematic expression analysis of the members of the Arabidopsis CLAVATA3/ESR-RELATED (CLE) small signaling polypeptide family. Using reporter constructs, we show that the CLE genes have distinct and specific patterns of promoter activity. We find that each Arabidopsis tissue expresses at least one CLE gene, indicating that CLE-mediated signaling pathways are likely to play roles in many biological processes during the plant life cycle. Some CLE genes that are closely related in sequence have dissimilar expression profiles, yet in many tissues multiple CLE genes have overlapping patterns of promoter-driven reporter activity. This observation, plus the general absence of detectable morphological phenotypes in cle null mutants, suggest that a high degree of functional redundancy exists among CLE gene family members. Our work establishes a community resource of CLE-related biological materials and provides a platform for understanding and ultimately manipulating many different plant signaling systems.

Translational control is crucial for proper timing of developmental events that take place in the absence of transcription, as in meiotic activation in oocytes, early embryogenesis in many organisms, and spermatogenesis. Here we show that a novel form of the translation initiation complex component eIF4G in Drosophila, eIF4G2, is required specifically for male germ cells to undergo meiotic division and proper spermatid differentiation. Flies mutant for eIF4G2 are viable and female fertile but male sterile. Spermatocytes form, but the germ cells in mutant males skip the major events of the meiotic divisions and form aberrant spermatids with large nuclei. Consistent with the failure to undergo the meiotic divisions, function of eIF4G2 is required post-transcriptionally for normal accumulation of the core cell cycle regulatory proteins Twine and CycB in mature spermatocytes. Loss of eIF4G2 function also causes widespread defects in spermatid differentiation. Although differentiation markers Dj and Fzo are expressed in late-stage eIF4G2 mutant germ cells, several key steps of spermatid differentiation fail, including formation of a compact mitochondrial derivative and full elongation. Our results suggest that an alternate form of the translation initiation machinery may be required for regulation and execution of key steps in male germ cell differentiation.

In this workshop, guest speakers from several fields including developmental biology, signal transduction and behavioral research, will highlight recent advances in our understanding of endocrine regulation of growth and metabolism. Discussions will emphasize emerging parallels between Drosophila endocrine models and their potential significance to human endocrine biology and related diseases. One goal of this session is to show how areas of endocrine research dominated by studies of mammalian models are increasingly available for study in Drosophila. Areas of research to be covered include development of cells producing insulin, differentiation of cells that sense and regulate glucose and other metabolites, neuropeptide signals that regulate response to environmental stress, and discovery of signaling pathways that mediate responses to these endocrine signals. Speakers include Seung Kim (Stanford University), Ping Shen (Univ. of Georgia, Athens), Volker Hartenstein (UCLA), Eric Rulifson (Univ. of Pennsylvania), R.J. Wessels (Burnham Institute, La Jolla, California), Pierre Léopold (CNRS, Nice), and Sean Oldham (Burnham Institute).

Background: MicroRNAs (miRNAs) are small 20-25 nucleoticle non-protein-coding RNAs that negatively regulate expression of genes in many organisms, ranging from plants to humans. The MIR164 family of miRNAs in Arabidopsis consists of three members that share sequence complementarity to transcripts of NAC family transcription factors, including CUP-SHAPED COTYLE-DON1 (CUC1) and CUC2. CUC1 and CUC2 are redundantly required for the formation of boundaries between organ primordia. The analysis of transgenic plants that either overexpress miR164a or miR164b or express a miRNA-resistant version of CUC1 or CUC2 has shown that miRNA regulation of CUC1 and CUC2 is necessary for normal flower development. A loss-of-function allele of MIR164b did not result in a mutant phenotype, possibly because of functional redundancy among the three members of the MIR164 family.Results: In this study, we describe the characterization of the early extra petals1 (eep1) Arabidopsis mutant, whose predominant phenotype is the formation of extra petals in early-arising flowers. We demonstrate that eep1 is a loss-of-function allele of MIR164c, one of three known members of the MIR164 family. Our analyses of miR164c function and eep1 mir164b double mutants reveal that miR164c controls petal number in a nonreclunclant manner by regulating the transcript accumulation of the transcription factors CUC1 and CUC2.Conclusions: The data presented in this study indicate that closely related miRNA family members that are predicted to target the same set of genes can have different functions during development, possibly because of nonoverlapping expression patterns.

This thesis is organized around the theme of modulation of transcriptional states in Arabidopsis thaliana. The two particular mechanisms on which this work focuses are (1) microRNA-mediated negative regulation of protein levels (either by mRNA cleavage or by repression of translation) and (2) transduction of extracellular signals into the cell to affect the transcription program. Chapter 2 characterizes the role of the EARLY EXTRA PETALS (EEP1) microRNA in the regulation of organ formation in the flower and shoot. The eep1 loss-of-function mutant has extra petals, and it enhances the shoot phenotype of the pinoid mutant, which has defects in auxin signaling and organ formation. EEP1 is nearly identical to a pair of published miRNAs (MIR164a and b); all three are predicted to target the mRNAs of six genes in the NAC family of transcription factors. Two of these genes, CUPSHAPED COTYLEDONS1 and 2 (CUC1 and 2), are redundantly required in flower development. Phenotypic and molecular analysis of lines overexpressing EEP1 are consistent with (1) negative regulation of CUC1 and CUC2 by EEP1 and (2) cleavage of the CUC2 mRNA promoted by EEP1. Chapter 3 describes the investigation, by reverse genetics, of five proteins encoded by genes in the CLV3/ERS (CLE) family. Due to the similarity of these proteins to CLAVATA3 (CLV3), the likely secreted ligand for the CLAVATA1 receptor-like kinase, functional analyses were performed in order to determine whether these proteins might also function as ligands for CLV1 or other receptor-like kinases. The results presented here derive from experiments using overexpression, double-stranded RNA interference (dsRNAi), and promoter-glucuronidase (GUS) reporter expression.

Genomic imprinting is an epigenetic process that results in the preferential silencing of one of the two parental copies of a gene. Although the precise mechanisms by which genomic imprinting occurs are unknown, the tendency of imprinted genes to exist in chromosomal clusters suggests long-range regulation through shared regulatory elements. We characterize a 800-kb region on the distal end of mouse chromosome 7 that contains a cluster of four maternally expressed genes, H19, Mash2, Kvlqt1, and p57(Kip2), as well as two paternally expressed genes, Igf2 and Ins2, and assess the expression and imprinting of Mash2, Kvlqt1, and p57(Kip2) during development in embryonic and extraembryonic tissues. Unlike Igf2 and Ins2, which depend on H19 for their imprinting, Mash2, p57(Kip2), and Kvlqt1 are unaffected by a deletion of the H19 gene region, suggesting that these more telomeric genes are not regulated by the mechanism that controls H19, Igf2, and Ins2. Mutations in human p57(Kip2) have been implicated in Beckwith-Wiedemann syndrome, a disease that has also been associated with loss of imprinting of IGF2. We find, however, that a deletion of the gene has no effect on imprinting within the cluster. Surprisingly, the three maternally expressed genes are regulated very differently by DNA methylation; p57(Kip2) is activated, Kvlqt1 is silenced, and Mash2 is unaffected in mice lacking DNA methyltransferase. We conclude that H19 is not a global regulator of imprinting on distal chromosome 7 and that the telomeric genes are imprinted by a separate mechanism(s).