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. Ruth Lehmann 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.
Germ cells are the stem cells for the next generation. Set aside during embryogenesis from the somatic cells that form the body of the organism, germ cells, through the fusion of sperm and egg, generate a new organism. Research in our lab focuses on the biology of germ cells in Drosophila. Despite their critical role, we still know little about how germ cell fate is initiated and maintained and how germ cells evade the ultimately deadly fate of the soma. In contrast to somatic cell fates, no master-regulator transcription factor has been identified that uniquely specifies germ cell fate; instead RNA regulation plays a prominent and highly conserved role in germ cells. As germ cells "make" germ cells, the work in our lab follows the life cycle of the germ line: We are interested in how the complex interactions between RNAs and their binding proteins specify germ cell fate in the early embryo, how germ cells migrate through the embryo to reach the somatic gonad, and how germ cell fate is maintained and protected throughout larval and adult life in order to generate a new organism.
Germ Line–Soma Separation
Drosophila germ cells are formed at the posterior pole of the embryo in a highly specialized cytoplasm, the germ plasm, which is assembled during oogenesis under the control of the maternal genome. The germ plasm provides the primordial germ cells (PGCs) of the embryo with much of the information needed to produce the next generation: the germ plasm harbors specific maternal protein complexes that are required for the localization and translational regulation of a large number of RNAs that encode proteins with functions in germ cell formation, specification, and migration. In a systematic analysis of germ plasm–localized RNAs, we found that, in general, 3'-untranslated region (3'-UTR) sequences fused to reporters can recapitulate the localization and translation pattern of the endogenous RNA. Furthermore, RNAs enriched in the germ plasm are translated at different times during germ cell development. Using single-molecule imaging analysis, we find that germ granules have an unexpected organization. While core germ plasm proteins are uniformly distributed throughout the granule, RNAs assemble with each other and are concentrated in homotypic clusters that occupy RNA-specific positions within the granule (Figure 1). We are now studying how this structure informs translational activity and stability of individual RNAs.
Lehmann Research Abstract Slideshow
Figure 1: Germ plasm is highly enriched in RNA. Posterior pole of an early wild-type Drosophila embryo showing expression of germ cell–less mRNA (gcl) (red) localized to the germ plasm. About 4 million copies of gcl mRNA are deposited into an embryo, where ~2.4 percent of it localizes to the posterior pole, the site of the germ plasm. RNA concentration in the germ plasm is 10-fold higher than in the rest of the embryo.
Image by Tatjana Trcek.
Figure 2: Nuclei associate with germ plasm. Posterior pole of a wild-type Drosophila embryo as nuclei reach the germ plasm to form primordial germ cells. gcl mRNA (red) and nanos mRNA (green) form islands around nuclei organized by centrosomes. Nuclei detected be the DNA stain DAPI stain are shown in blue.
Image by Tatjana Trcek.
Figure 3: Germ cell formation. As nuclei migrate into the germ plasm they are surrounded by membrane to form primordial germ cells. This requires formation of two orthogonal ingressions: a anaphase cytokinesis furrows and a spindle-independent furrow that separates the germ cells from the rest of the embryo. Green, anillin; red, actin; blue, DNA.
Image by Ryan Cinalli. From Cinalli, R.M., and Lehmann R. 2013 Nature Cell Biology 15:839–845.
Figure 4: Germ cell migration. Drosophila primordial germ cells (red, phalloidin) are clustered within the polarized epithelium of the embryonic posterior endoderm (subapical region labeled with aPKC, green). Primordial germ cells are unable to migrate through intact epithelial tissues and instead initiate migration in response to epithelial remodeling (blue, DAPI).
Image by Jessica Seifert.
Figure 5: Mitochondrial maturation during stem cell differentiation. Top: diagram of the germarium at the tip of the ovary. Stem cells (green) are closest to the niche and contain round spectrosomes (red). After stem cell division, daughter cells excluded from the niche begin to differentiate (blue) and their spectrosomes branch into fusomes (red). The differentiating cell undergoes four rounds of amplifying division to form a 16-cell interconnected cyst that matures to an egg chamber (turquoise) composed of 15 nurse cells and an oocyte (white). Bottom left, electron micrograph of wild-type stem cell mitochondria with few cristae; right, from the same germarium, wild-type 16-cell cyst-differentiated cell mitochondria with dense cristae.
Image by Thomas Hurd, with help from Fengxia Liang, Chris Petzold, and Kristen Dancel of the New York University Langone Medical Center, Office of Collaborative Science, Microscopy Core.
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 (Figure 2). The mechanism of PGC cell formation is strikingly different from the process that controls somatic cells: PGC formation relies solely on maternal factors and occurs 1.5 hours and four nuclear divisions earlier than somatic cellularization, which relies on specific factors synthesized by the zygote. Using high-resolution live imaging, we determined that PGCs form via two constrictions: one, the bud-neck constriction, separates a budding nucleus basally from the rest of the embryo; a second constriction, the cytokinetic furrow, separates the dividing nucleus along the anaphase furrow into two cells (Figure 3, Movie 1). This unusual bud-neck constriction occurs even after inhibition of microtubules and thus independent of the triggers that activate anaphase restriction. We found that the maternally supplied germ cell–less (GCL) protein is an instructive and rate-limiting component of bud-neck constriction. GCL encodes a conserved BTB protein.
Unipotent and Highly Specialized
While germ cells are able to generate a new generation, they are also highly specialized cells that have one fate – to develop either into egg or sperm. Soma–germ line interactions are critical for the successful generation of gametes. PGCs are formed at a distance from the somatic gonad and initially associate and then migrate through the posterior midgut primordium (PMG) on their route toward the somatic part of the gonad. We identified a G protein–coupled receptor, Tre1, which is expressed by PGCs and plays an important role in the activation of the migratory program and the directional migration of germ cells (Figure 4, Movie 2). Using two-photon imaging, we found that PGCs do not, as previously believed, move directly through an epithelium but rather they take advantage of the loosening of the epithelium as the future gut stem cells leave the epithelium. These experiments also showed that Tre1 activation is not dependent on a signal from the PMG. Using specific mutations in Tre1, we can distinguish between different models of Tre1 activation and its roles in the initial cellular polarization and directed migration.
Ultimate Stem Cell
To gain a more complete understanding of the regulatory networks governing Drosophila germ line stem cell (GSC) self-renewal and differentiation, we performed an unbiased, transcriptome-wide, in vivo RNA interference (RNAi) screen. Characterization of cellular defects during early stages of ovarian GSC self-renewal and differentiation allowed us to sort about 650 "hits" into phenotypically and functionally meaningful groups. Bioinformatic analysis unveiled a comprehensive set of networks regulating GSC maintenance, survival, or differentiation. This analysis revealed a specific requirement for the mitochondrial ATP synthase in GSC differentiation. Surprisingly, while our screen identified almost every subunit of the ATP synthase, knockdown of other members of the oxidative phosphorylation system did not disrupt differentiation. We found that ATP synthase promotes the maturation of mitochondrial cristae during differentiation through dimerization and specific upregulation of the ATP synthase complex (Figure 5). Our findings suggest that cristae maturation is a key developmental process required for GSC differentiation.
Because of their unique ability to self-renew, our research takes advantage of the opportunities germ cell biology poses to understand, more generally, the cellular mechanisms of totipotency and the challenges associated with immortality.
Studies on germ cell migration are supported by a grant from the National Institutes of Health.
As of March 23, 2016