Research
Germ Cells are Forever
Germ cells transmit the potential to build a completely new organism to their offspring. The mechanisms that endow germ cells with the ability to propagate totipotency across generations are poorly understood. Using Drosophila melanogaster as a model organism, the Lehmann lab studies how germ cells are set apart from somatic cells during embryogenesis, how early germ cells are guided to the gonad and how germ cell fate is maintained and protected throughout life in order to generate egg and sperm.
STRUCTURAL AND FUNCTIONAL ORGANIZATION OF GERM PLASM CONDENSATES
In Drosophila germ cells form in a specialized cytoplasmic region, the germplasm, at the posterior pole of the embryo. Our long-standing interest in germ cell development began with identification of maternal effect genes that control the formation and specification of germ cell fate. Molecular characterization of these genes demonstrated that phylogenetically conserved, germ line-specific RNA regulators play an extensive regulatory role during all stages of the germ line life cycle. We found that germplasm RNAs are translationally repressed in the somatic regions and that localization to germ granules is linked to their translational activation. The critical sequences for both RNA localization to the germ plasm and translational regulation in germ granules are located in the 3’UTR of germplasm RNAs. Recently, we applied single-molecule FISH and super resolution microscopy to show that mRNAs are spatially organized within the granule, whereas core germ plasm proteins are distributed evenly throughout the granule. We found that multiple mRNA copies of the same gene organize into “homotypic clusters” that occupy defined positions within the granule and that RNAs sort within the granules in a 3’UTR-independent, yet transcript-specific manner. Among the protein products that are enriched in the germplasm, we identified Oskar protein as the seed factor for germ plasm assembly and showed that Oskar protein, at a high concentration, condenses into phase-separated granules, independent of any other germplasm component. In vivo, Oskar is instructive for the localization of all other germplasm proteins and germ granule localized RNAs. How mRNAs are spatially organized within a germ granule and whether their position defines functional properties has been a central question in the RNA localization field. Germ granules have a conserved and well-defined function and are thus ideal to study the structure and function of RNP biocondensates, a topic of intense present study in the lab. We have developed tools to do so in a controlled in vivo system, while also making use of in vitro reconstitution and heterologous tissue culture systems as well as structural studies at the EM and super-resolution resolution level.
Relevant Publications:
Trcek T, Douglas T.E., Grosch M, Shroff H, Rothenberg E, Lehmann R. Sequence-Independent Self-Assembly of Germ Granule mRNAs into Homotypic Clusters. Mol Cell. 2020. Abstract.
Kistler KE, Trcek T, Hurd TR, Chen R, Liang FX, Sall J, Kato M, Lehmann R. Phase transitioned nuclear Oskar promotes cell division of Drosophila primordial germ cells. Elife. 2018 Sep 27;7. pii: e37949. Abstract
Trcek T, Grosch M, York A, Shroff H, Lionnet T, and Lehmann R, Drosophila germ granules are structured and contain homotypic mRNA clusters Nature Communications (Aug 2015) 6:1-12 Abstract
Yang N, Yu Z, Hu M, Wang M, Lehmann R, Xu RM, Structure of Drosophila Oskar reveals a novel RNA binding protein. PNAS (Aug 2015) 201515568:1-8 Abstract
Liu H, Wang JY, Huang Y, Li Z, Gong W, Lehmann R, Xu RM., Structural basis for methylarginine-dependent recognition of Aubergine by Tudor. Genes and Development (Sep 2010) 24:1876-81 Abstract
Image by Tatjana Trcek
Image by Ruoyu Chen
Germline-Soma Dichotomy
The entire Drosophila embryo initially develops as a syncytium of synchronously dividing nuclei. However, when a subset of these nuclei reaches the germ plasm, they become surrounded by cell membranes and are destined for the primordial germ cell (PGC) fate. The mechanisms by which PGC form are strikingly different from the process that controls somatic cells. For examples, PGC form about an hour earlier during embryogenesis than the cells that will give rise to all somatic structures and the process occurs independent of new (embryonic) transcription relying entirely on maternal RNAs and proteins deposited in the egg. We are interested in the molecular switches leading to this divergence in cell lineage and have characterized three, largely independent pathways that establish the soma–germline dichotomy as PGCs are specified. Two pathways are dedicated to suppress somatic programs, one by inhibiting zygotic transcription globally in germ cells and the other by specifically degrading the tyrosine kinase receptor Torso that drives somatic differentiation in germ cells. A third pathway, mediated by Rho1-activation and myosin contraction, promotes formation of PGCs via a constriction furrow that is independent of the mitotic spindle apparatus. We are using genetic, genomic and biochemical approaches as well as high-resolution live-imaging and optogenetics to identify the cellular mechanisms that generate the immortal germ cell fate.
Relevant Publications:
Pae J, Cinalli RM, Marzio A, Pagano M, Lehmann R. GCL and CUL3 Control the Switch between Cell Lineages by Mediating Localized Degradation of an RTK. Dev Cell. 2017 Jul 24;42(2):130-142.e7. Abstract
Coux RX, Teixeira FK, Lehmann R. L(3)mbt and the LINT complex safeguard cellular identity in the Drosophila ovary. Development. 2018 Apr 4;145(7). pii: dev160721. Abstract
Cinalli RM and Lehmann R , A spindle-independent cleavage pathway controls germ cell formation in Drosophila Nature Cell Biology (Jul 2013) 15:839-45 Abstract
Image by Marty Alani; Drosophila embryo, lateral view
Image by Mariyah Saiduddin; Drosophila embryo, posterior view
Germ Cell Migration
Primordial germ cells (PGCs) form at the posterior pole of the Drosophila embryo and are carried inside the embryo during gastrulation in juxtaposition to the posterior midgut. Subsequently, PGCs migrate actively through the midgut epithelium and navigate along the midgut toward the mesoderm, where they associate with somatic gonadal precursors to form the embryonic gonad. At least three signaling pathways regulate germ cell migration: a) The G protein coupled receptor Tre1 controls transepithelial migration through the posterior midgut. b) Wunen and Wunen 2, two lipid phosphate phosphatase, whose action repels PGCs and can lead to PGC death. c) Isoprenylation via the HMGCoA reductase pathway spatially regulates the production of a germ cell attractant. Complementary to genetic and biochemical studies we are using high-resolution live imaging to develop a dynamic, cellular view of germ cell chemoattraction and repulsion and have developed in vitro migration assays to identify the molecules involved in germ cell chemotaxis and survival. With these tools, we recently found that stable polarization of germ cells creates tension needed for germ cells to separate and propel from each other at the onset of migration and identified a conserved family of isoprenoids that leads PGCs to the gonad.
Relevant Publications:
Slaidina M, Lehmann R. Quantitative Differences in a Single Maternal Factor Determine Survival Probabilities among Drosophila Germ Cells. Curr Biol. 2017 Jan 23;27(2):291-297. Abstract
LeBlanc M, Lehmann R. Domain-specific control of germ cell polarity and migration by multifunction Tre1 GPCR. J Cell Biol.2017:216(9):2945-2958. Abstract
Seifert JRK, Lehmann R. Drosophila Primordial Germ Cell Migration Requires Epithelial Remodeling of the Endoderm. Dev. 2012. 139: 2101-2106. Abstract.
NETWORKS THAT REGULATE GERM CELL DEVELOPMENT AND ORGAN HOMEOSTASIS.
Germ cells have to refrain from developing along any somatic program, yet they rely on somatic cells as support during gonad morphogenesis and for stem cell self-renewal and differentiation in the adult. Once primordial germ cells (PGCs) reach the somatic gonad, they proliferate but do not differentiate until late larval-pupal stages. During the proliferation phase in the early stages of larval gonad development, a negative feedback loop triggered by EGFR ligand expressed by PGCs coordinates PGC proliferation and somatic cell growth such that gonads which received too few germ cells can catch up. To identify all major networks required for GSC self-renewal and differentiation we conducted a comprehensive genetic study using germline-specific RNAi in collaboration with the Hannon lab. These functional studies were recently complemented by single cell RNA sequencing to uncover all known cell types of the developing larval ovary and in the adult. With these comprehensive and large-scale data sets we can now assemble developmental networks for germ cell differentiation and gonad morphogenesis.
Relevant Publications:
Slaidina M., Banisch T.U., Gupta S., and Lehmann, R. A single-cell atlas of the developing Drosophila ovary identifies follicle stem cell progenitors. Genes Dev. 2020. Abstract.
Sanchez CG, Teixeira FK, Czech B, Preall JB, Zamparini AL, Seifert JR, Malone CD, Hannon GJ, Lehmann R. Regulation of Ribosome Biogenesis and Protein Synthesis Controls Germline Stem Cell Differentiation. Cell Stem Cell. 2016 Feb 4;18(2):276-90. Abstract
Gilboa L and Lehmann R, Soma-germline interactions coordinate homeostasis and growth in the Drosophila gonad Nature (Sep 2006) 443:97-100 Abstract
Image by Torsten Banisch
MITOCHONDRIA: GERMLINE TRANSMISSION AND SELECTION
Mitochondria are passed to the next generation exclusively through the female germline. As mitochondria (mt) carry their own genome, and in contrast to the nuclear genome, are not equipped with robust recombination or proof-reading mechanisms, mutations accumulate in mt DNA through generations, a process described as Muller’s ratchet. In the germline, processes such as the mitochondrial bottleneck and purifying selection for mitochondria ‘fitness’ can protect against Muller’s ratchet, but the molecular mechanisms underlying these processes remain largely unclear. Of the thousands of mitochondria in the egg cell only a few hundred end up in primordial germ cells (PGCs). We found that mitochondria are specifically enriched at the posterior pole of the growing oocyte and that this process influences the size of the bottleneck by increasing the number of mt genomes inherited by PGCs. Furthermore, we found that developmentally-regulated mitochondrial fragmentation during early stages of germ cells differentiation in the adult triggers selection of defective mitochondria via mitophagy and selective mtDNA replication. To better understand the cellular mechanisms that support mitochondrial transmission, we are developing new techniques that will allow us to target and follow individual mitochondria and their genomes directly in vivo.
Relevant Publications:
Lieber T, Jeedigunta SP, Palozzi JM, Lehmann R, Hurd TR. Mitochondrial fragmentation drives selective removal of deleterious mtDNA in the germline. Nature. 2019. Abstract
Hurd TR, Herrmann B, Sauerwald J, Sanny J, Grosch M, Lehmann R. Long Oskar Controls Mitochondrial Inheritance in Drosophila melanogaster. Dev Cell. 2016 Dec 5;39(5):560-571. Abstract
Teixeira FK, Sanchez CG, Hurd TR, Seifert JRK, Czech B, Preall JB, Hannon GJ, and Lehmann R, ATP synthase promotes germ cell differentiation independent of oxidative phosphorylation Nature Cell Biology (May 2015) 7:689-96 Abstract
Image by Melissa Pamula; Mitochondria (green) and nuclei (blue) in Drosophila egg chamber
DEFENSE AND ADAPTATION MECHANISMS
Host-intruder interactions are powerful means to manipulate and evolve. Intruders can either be located within the genome, in the form of “transposable elements” (TE), or be acquired in the form of intracellular pathogens, such as bacteria of the Wolbachia family, that are passed on from generation to generation. Common to both TE and Wolbachia is that a) they are required to be transmitted though the germline, b) they can have deleterious effects on reproduction (in the form of hybrid dysgenesis and cytoplasmic incompatibility, respectively), and c) they can be beneficial for the host (by facilitating genomic change/evolution and protection against viruses).
TE activity in the Drosophila germline is regulated by chromatin-based as well as small RNA-mediated (piRNA) mechanisms. Components of the piRNA biogenesis machinery interfere with normal germline development. Similarly, activation of TEs such as the P-element hybrid dysgenesis causes infertility. However, TE activity and reproductive defects are not necessarily correlated. Using classical example of TE activity we have been interested in identifying the systemic connection between TE activity and germ line development.
More recently we have started to explore Wolbachia, an intracellular bacterium that is capable of infecting a remarkably large range of insect hosts. We are using the Wolbachia/Drosophila model as a genetically tractable system for studying host-pathogen interactions. Wolbachia infection has been correlated with protection against a variety of diseases caused by viruses, such as dengue fever and Zika. The effectiveness of the antiviral response is closely linked to Wolbachia density, however increased Wolbachia titers can have deleterious effects on host viability and reproduction. We have used a Wolbachia-infected Drosophila cell line to perform an unbiased high-throughput, whole genome RNAi screen to quantitatively assess changes in Wolbachia levels. We found that mutually beneficial interactions between Wolbachia and its host rely on regulation of host translation. Like mitochondria, Wolbachia are only transmitted via the female germ line, however we still know little about how Wolbachia successfully colonizes the host and propagates between cells and within the germline. To learn more about the fascinating biology of Wolbachia and the underlying mechanisms of host adaptation and manipulation, we are using EM three-dimensional reconstructions to identify intracellular niches where Wolbachia reside and are developing tools to visualize and manipulate Wolbachia transmission live.
Relevant Publications:
Teixeira FK, Okuniewska M, Malone CD, Coux RX, Rio DC, Lehmann R. piRNA-mediated regulation of transposon alternative splicing in the soma and germ line. Nature. 2017 Dec 14;552(7684):268-272. Abstract
Rangan P, Malone CD, Navarro C, Newbold SP, Hayes PS, Sachidanandam R, Hannon GJ, Lehmann R, piRNA Production Requires Heterochromatin Formation in Drosophila. Curr Biol (Aug 2011) 21:1373-9 Abstract
Grobler Y, Yun CY, Kahler DJ, Bergman CM, Lee H, Oliver B, Lehmann R. Whole genome screen reveals a novel relationship between Wolbachia levels and Drosophila host translation. PLoS Pathog. 2018 Nov 13;14(11):e1007445. Abstract
