Open access peer-reviewed chapter
Abstract
Animals specify primordial germ cells (PGCs) in two alternate modes: preformation and epigenesis. Epigenesis relies on signal transduction from the surrounding tissues to instruct a group of cells to acquire PGC identity. Preformation, thought to be the more derived PGC specification mode, is instead based on the maternal inheritance of germ cell-determining factors. We use the zebrafish as a model system, in which PGCs are specified through maternal inheritance of germ plasm, to study this process in vertebrates. In zebrafish, maternally inherited germ plasm ribonucleoparticles (RNPs) have co-opted the cytoskeletal machinery to reach progressive levels of multimerization, resulting in the formation of four large masses of aggregated germ plasm RNPs. At later stages, germ plasm masses continue to use components of the cell division machinery, such as the spindles, centrosomes, and/or subcellular organelles to segregate asymmetrically during cell division and subsequently induce germ cell fate. This chapter discusses the current knowledge of germ cell specification focusing on the zebrafish as a model system. We also provide a comparative analysis of the mechanism for germ plasm RNP segregation in zebrafish versus other known vertebrate systems of germ cell preformation, such as in amphibian and avian models.
Keywords
- Germ cells
- germ plasm
- zebrafish
- cell division
- cytoskeleton
- RNP segregation
1. Introduction
1.1. Fundamentals of germ cell specification: Epigenesis vs. preformation
One of the most fundamental early cell fate decisions in animal embryos is the specification of primordial germ cells (PGCs) from the somatic tissue. PGCs are the precursors to gametes and thus hold the information to recreate the species in each generation. Consequently, individuals from every population must employ a robust mechanism for PGC induction. Specification occurs by one of two mechanistic modes:
Figure 1.
Mechanisms of primordial germ cell specification. Species employing preformation deposit germ plasm in oocytes. Throughout development, germ plasm localizes into select cells that eventually become the germ cells. In species employing epigenesis, cells receive inductive signals from neighboring tissue to become the primordial germ cells.
When comparing epigenesis and preformation as PGC-determining mechanisms, it was originally pointed out that epigenetic germ cell determination is an exception, and most animals use germ plasm [1, 2]. Subsequently, the analysis of distribution of these mechanisms within the evolutionary tree has led to the hypothesis that epigenesis is an ancestral PGC-determining mechanism and that preformation has arisen multiple times from this basal mode. Many clades employ both mechanisms, for example, within amphibians axolotls (urodeles) [3] use epigenesis and frogs (anurans) employ preformation [4]. Similarly, species in the group reptilia, such as turtles, use epigenesis [5], and species of the related group aves (birds) use preformation [6].
When protein-coding sequences of vertebrate species using epigenesis are compared with sister taxa employing preformation, genes of the latter evolve more rapidly. For example, when comparing protein-coding sequences from species in the amphibian and actinopterygian (ray-finned fishes) taxons, in all cases, species using preformation grouped further away from mammals than those using epigenesis. Evans et al. [7] found that no biological factor, including genome size, longevity, and generation time, correlates with sequence rate changes as optimally as the mode of PGC induction. This has led to the suggestion that the use of germ plasm relieves constraints on somatic development by dissociating PGC development from somatic development during early embryogenesis. This is because in animals using preformation mechanisms, changes in gene networks involved in somatic tissue development can occur without causing deleterious effects on the germ line, which would cause sterility and would therefore be selected against. This developmental flexibility allows for a faster rate of evolution of developmental programs. For example, ancestral gene regulatory networks (GRNs) for pluripotency and mesoderm specification found in axolotls are conserved through the evolution of mammals, whereas in frogs, pluripotency GRNs have been lost and the number of key regulators (i.e. multiple copies of Nodal and Mix) have increased in the mesoderm specification GRN [2]. Since axolotls specify PGCs through epigenesis within the mesoderm, the GRN is under constraints preventing variation—constraints that are not present in frogs. However, frogs carry preformed germ plasm, whose direct role in PGC induction allows the embryo to undergo drastic changes to the mesoderm specification GRN, such as Nodal expansion [8]. Consistent with this hypothesis, the use of germ plasm coincides with increased morphological variation and enhanced speciation within a clade [7].
Although these two PGC-determining mechanisms differ, many of the factors involved in PGC development are highly conserved regardless of the mechanism employed to differentiate PGCs. For example, the gene product of
1.1. Epigenesis, a mechanism by which cells receive signals from their surroundings
Most animal clades include lineages that use epigenesis to specify PGCs. Within the amphibian clade, axolotl (salamander) PGC specification occurs in primitive ectoderm (animal cap) cells in response to ventral mesoderm-inducing signals [12], including fibroblast growth factor (FGF) and BMP4 [13]. PGC precursors arise in the ventral marginal zone and migrate over the blastopore during gastrulation and by tailbud stage are detected in the posterior of the dorsal–lateral mesoderm expressing PGC-specific genes, such as
All mammals specify PGCs through inductive signaling, and this mechanism is most well studied in mouse. In these organisms, extraembryonic tissue signals the most proximal region of the postimplantation epiblast to become the PGCs, whereas cells that do not receive the signals give rise to somatic tissue [15]. The mouse PGC precursors are detected just prior to primitive streak formation, and by the end of primitive streak formation, a population of around 40 PGCs are generated. Key instructive signals are the bone morphogenic proteins (BMP2, BMP4, and BMP8B), which act through SMAD1 and SMAD5 [16, 17,18]. The population of PGCs is heavily reliant on BMP dosage as loss of BMP4 reduces their induction [18]. Some RNAs present in the germ plasm in organisms with a preformative mechanism, such as
1.2. Preformation: Germ plasm deposits determine the germ cell fate
Preformation describes a mechanism whereby differentiation as germ cells is decided by the acquisition of maternally inherited determinants following fertilization [24]. The common term to describe such determinants is germ plasm, which is considered as a specialized cytoplasm enriched for factors that function in PGC determination. Germ plasm contains RNAs (both coding and noncoding) and proteins, which in many organisms appear to assemble together in punctate structures known as ribonucleoparticles (RNPs). In many organisms, germ plasm closely associates with the cytoskeleton and/or mitochondria. Proposed functions of germ plasm include the translational regulation of germ plasm RNAs, the establishment of a partially repressive chromatin in the germ line and the prevention of activation of somatic development by repressing mRNA transcription (reviewed in [25, 26]).
In
P-granules are another type of RNA-rich cytoplasm structures involved in the specification of the
Although research on PGC specification is sparse for colonial ascidians, studies of a single species,
2. Germ cell determinant segregation in zebrafish
2.1. Maternal inheritance of germ plasm RNPs
The fish model,
2.1.1. Animal region germ plasm factors
Known animal region germ plasm factors include
Another animal germ plasm RNA is that of the gene
Other germline-specific RNAs found at the animal pole include
For tested animal germ plasm RNAs (
2.1.2. Vegetal region germ plasm factors
Vegetally localized germ plasm RNPs contain RNAs for the genes
Recent studies have shown that vegetal RNPs appear to be differentially distributed in the vegetal cortex, specifically at a cortical depth that is deeper than that of factors that also localize to the animal pole but are required for axis induction [78]. This differential distribution along cortical depth, with germ plasm RNPs at deeper levels and dorsal factors at more superficial ones, likely facilitates the symmetric animally directed movement of germ plasm RNPs [75, 79] in spite of the asymmetric movement of the bulk of the cortex required for axis specification [80, 81].
2.2. Repackaging germ plasm: Gradual multimerization and recruitment
As mentioned above, after egg activation and prior to the first cell division, animally localized RNPs are present in single particles, which we refer to as singletons, spread throughout the developing blastodisc. Germ plasm RNP singletons aggregate in a wave-like fashion, where the wave of aggregation emanates from the center of the blastodisc outwards, toward the edge of the blastodisc.
Germ plasm RNP multimerization at this stage depends on the interplay between microtubule and microfilament networks during blastomere cell division (Figure 2A). After fertilization in zebrafish, paternally derived centrioles act as a template to reconstruct the centrosome using maternally derived components. This newly formed centrosome nucleates a sperm aster or monoaster prior to initiation of the first cell division cycle. In this structure, plus ends of the astral microtubules interact with germ plasm RNPs at the cortex and help direct germ plasm RNP multimerization in a process of
Figure 2.
Germ plasm accumulation during the first zebrafish cell cycles. A) In the egg, animal pole (AP) germ plasm RNPs are dispersed at the blastodisc cortex, whereas vegetal pole (VP) germ plasm is localized at the vegetal pole. B) Prior to the initiation of cell cycling, during pronuclear fusion, astral microtubule growth moves AP germ plasm RNPs outward, whereas VP germ plasm begins to travel toward the AP. During the next several cell cycles, aggregated RNPs are collected in the (C) first furrow, (D) second furrow and (E) third furrow. F) Germ plasm RNPs in the first and second furrows are stabilized, whereas all other RNPs are cleared. Figure adapted from [
Another maternal factor important for germ plasm RNP recruitment during these early stages is Buc. In addition to a role for Buc to assemble the Bb during oogenesis, this factor is also required for recruitment of germ plasm during early embryogenesis, and its overexpression in the early embryo leads to an increase in PGCs [49]. Recently, a microtubule motor protein, Kif5Ba (kinesin), was found to bind to Buc and mediate its recruitment (thereby recruitment of other germ plasm molecules) to the cleavage furrow. Germ plasm in
Unlike the first cell cycle, which contains a monoaster involved in pronuclear fusion, blastomere cell cycle divisions contain bipolar microtubule asters nucleated by the pair of centrosomes at spindle poles. When spindle microtubules originating from opposing asters overlap at the spindle midzone, they signal furrow formation along the length of the blastomere. The furrow initiates as a microtubule-free zone that forms at the region of overlap between asters from opposite sides of the furrow. During this process, germ plasm RNP multimers and associated F-actin continue their outward movement, which in the midzone between spindle poles coincides with the microtubule-free zone at the site of furrow formation. This movement results in the accumulation of both F-actin and germ plasm RNPs along the forming furrow, the latter forming a rod-shaped structure [72, 79, 85, 86] composed of individual multimeric groups of RNPs. We refer to this accumulation of germ plasm RNPs at the furrow as the process of
During cell division associated with the first several cell cycles, recruitment of germ plasm RNPs to the forming furrow occurs from both sides due to the bipolar nature of the asters. In the furrow, germ plasm RNPs are still connected to the tips microtubules, which form parallel to one another and perpendicular to the furrow forming a structure known as the furrow microtubule array (FMA). Both pre-aggregation and recruitment may facilitate germ plasm RNP multimerization using the same basic mechanism: in both cases, radially expanding microtubule growth (from a monoaster during the first cell cycle and bipolar asters in subsequent cell cycles) facilitates germ plasm RNP multimerization. Because of their intrinsic arrangements, the monoaster does not result in furrow formation and therefore can only contribute to pre-aggregation, whereas asters from bipolar spindles contribute to both continued pre-aggregation and implement recruitment. Thus, germ plasm furrow recruitment employs the normal cell division machinery, in particular astral microtubules, to mediate the aggregation and local gathering of RNP multimers to the forming furrow [86]. This simple mechanism normally couples furrow induction and germ plasm RNP furrow recruitment. Under certain mutant conditions, however, germ plasm RNP furrow recruitment is partially dissociated from furrow formation, as occurs in mutant embryos that fail to initiate a furrow and that nevertheless show
2.3. Insuring a tight fit: High-order RNP multimerization
2.3.1. Compaction of germ plasm in a modified midbody
As the contractile ring forms and leads to the division of cytokinesis, the FMA tilts distally and moves to the edge of the blastodisc [89, 90]. During this process, the rod-shaped RNP arrangement compacts into tight masses at the edges of the blastodisc, in a process of
Through distal compaction, animal germ plasm RNPs acquire a distal position at the furrow, and their aggregate is transformed from a rod-like structure to a round and more compact mass, possibly driven by an increase in neighbor-to-neighbor RNP contact and concomitant reduction in germ plasm mass volume. The extent by which this process is driven through cytoskeletal rearrangements, RNP–RNP interactions, or both remains to be determined. The subcellular cues that result in the redistribution of FMA microtubules and germ plasm RNPs to the distal end of the furrow also remain largely unknown, although, as mentioned above, a reduction in myosin activity results in FMA stabilization and a lack of proximodistal reorganization [91]. Analysis of
The process of aggregation, recruitment, and distal compaction of germ plasm RNPs that occurs in the furrow for the first cell cycle becomes repeated during furrow formation for the second and third cell cycles (Figure 2E,F). The observed pattern of germ plasm RNP recruitment supports a model in which astral microtubules of the spindle apparatus mediate the local gathering of cortical germ plasm RNPs. Since in each subsequent spindle apparatus covers half the cortex as in the previous one, this predicts the accumulation of germ plasm RNPs of about half each subsequent cell cycle. This prediction is indeed observed during the first three cell cycles [86]. Thus, local germ plasm RNP furrow recruitment, coupled to the alternating (by 90 degrees) cleavage orientation pattern, gradually allows the gathering of germ plasm RNPs from the blastodisc cortex into the forming furrows. The adaptation of the cell division machinery for germ plasm RNP recruitment constitutes a simple and effective system to amass inherited single germ plasm RNP aggregates.
A consequence of this mechanism is that germ plasm continues to undergo recruitment to forming furrows for as long as there are germ plasm RNP aggregates in the cortex. This manifests in recruitment of germ plasm RNPs to the third furrow, temporarily generating embryos with eight visible germ plasm masses. However, the four aggregates collected during the third cell cycle do not undergo the subsequent step of ingression and instead appear to become degraded. At the same time, the outward movement of germ plasm RNPs remaining at the cortex that do not become recruited to the furrows, which is also mediated by the cycles of growing astral microtubules, result in the accumulation of these RNPs to the periphery of the blastodisc, where they similarly appear to become degraded. After the first several cycles, only the four larger germ plasm masses remain, corresponding to those recruited during the first and second cell cycles and which encompassed larger regions of the cortex and therefore amassed the largest numbers of germ plasm RNPs. The underlying basis for the selective stabilization of the first four aggregates is not known, but it is possible that these aggregates contain an amount of germ plasm RNPs above a certain threshold that allows their stabilization or further routing into the germ plasm segregation pathway [86].
Studies have shown that when RNA constructs containing a green fluorescent protein (GFP)-coding region coupled to a
2.3.2. Animal meet vegetal RNPs: Generating a full-complement of zebrafish germ plasm
During formation of the first furrows, only animal pole germ plasm RNPs are found in the forming germ plasm masses; however, at the end of the distal compaction phase as the first furrow is completed, vegetally localized RNPs, such as
2.4. Maintaining germ plasm potential during cell division
2.4.1. Ingression into cells and asymmetric segregation
At about the 16-cell stage, the four germ plasm masses, which formed during the first two cell cycles and do not undergo degradation, translocate from their location at the blastomere–yolk cell boundary in each of four corners of the blastodisc into four cells [75, 79, 85, 92], a process that roughly coincides with cellularization of the blastomeres (Figure 3). Although this process of germ plasm ingression has not been yet characterized in the zebrafish, one might hypothesize that it is similar to
Figure 3.
Germ plasm to primordial germ cell transition in zebrafish. A) Compact, yet separate animal and vegetal germ plasm aggregates at 16-cell stage are found at the edge of the first two furrows. B) At approximately the 32-cell stage, germ plasm aggregates ingress into four cells (aggregates are depicted with a lighter color to indicate that they are likely in bottom tier blastomeres, closest to their original location at the blastodisc–yolk cell boundary). C) Before PGC-specific expression, germ plasm asymmetrically segregates so that only one cell receives the aggregate (germ plasm masses are diagrammed at the various stages of the cell cycle that is represented as proceeding counterclockwise, indicating association of the germ plasm mass to one of the spindle poles to generate two asymmetric daughter cells, one with and one without the germ plasm mass—in this diagram, the counterclockwise progression is for the representation of the cell cycle only, and in reality each PGC is undergoing cell cycling independently). In (C) and (D), blue color depicts DNA. D) At approximately the 1K-cell stage, germ plasm aggregates break down into smaller particles, and daughter cells inherit these particles symmetrically. E) Cells from these previous founders begin to exhibit PGC-specific expression. F) After migration in the 24-hour embryo, PGCs accumulate in the prospective gonad just above the yolk.
Once the germ plasm has ingressed into four PGCs, these cells continue to divide and during the cell division process their germ plasm segregates asymmetrically (Figure 3). Although the mechanism by which asymmetric segregation of germ plasm occurs is not completely understood, the germ plasm aggregates form a cup-shape structure that associates with one of the two spindle poles [92, 98], suggesting that segregation might rely on the spindle apparatus as proposed in
During late blastula stage, the pattern of germ plasm segregation changes: now germ plasm distributes in a perinuclear arrangement and is inherited by both daughter cells [92]. At the same time, zygotic expression of the germ line-specific gene
Interestingly, DNA replication-inhibited embryos display a transition between asymmetric and symmetric segregation patterns that occurs at a developmental time similar to that in control embryos. This suggests that this transition in modes of germ cell determinant segregation does not rely on nucleo/cytoplasmic ratio, which has been proposed to regulate transcriptional activation at MBT [101, 102] or zygotic transcription initiation itself, but instead relies on a DNA-independent maternal temporal mechanism, possibly an intrinsic developmental timer or, alternatively, the counting of cell divisions [92].
2.4.2. Activation of the germ cell program
Little is known about the activation of PGC program in zebrafish. In mouse, activation of the PGC program involves the expression of three interdependent proteins:
Even though Vasa protein does not colocalize to the germ plasm during the early cleavage period [92, 106, 107], it is found in perinuclear patches around the germinal vesicle during oogenesis and is uniformly distributed in all embryonic cells prior to MBT [92, 107]. At around 3-4 hours post-fertilization (hpf), when the zygotic genome is activated, Vasa protein levels increase [92]. The bulk of this increase in Vasa protein is dependent on the presence of a nucleus in the PGCs, suggesting that a large part of translated Vasa is derived from new zygotic expression. However, a small amount of Vasa does accumulate in embryos whose cells lack a nucleus, suggestive of translation of Vasa protein from maternal transcripts. This finding has led to the hypothesis that maternally inherited
An important hallmark of the activation of the germ cell program in animal systems is their subsequent migration (reviewed in [108, 109]). Two components found in the zebrafish germ plasm are required for PGC migration and maintenance in this organism:
3. Comparative analysis of germ plasm aggregation in vertebrates: Independent yet similar solutions
3.1. Other fish species
The mode of PGC induction within teleost fish is not fully conserved. Fish in the ostariophysan lineage, such as carp, Fegrade’s danio, tetra, and zebrafish, localize
Embryos from sturgeon species, considered a primitive fish that acts as a basal outgroup for the teleost lineage, show many similarities to anurans including holoblastic cleavage, forming a distinct blastocoel and archenteron and undergoing primary neurulation. Two studies had varying conclusions on whether sturgeon embryos employ epigenesis or preformation [8, 114]. One group found that
3.2. Amphibians (X. laevis )
Figure 4.
Comparison of vertebrate species employing germ plasm. All three species, zebrafish, frog and chicken, localize germ plasm during oogenesis. At the two-cell stage, two sites of localization of germ plasm (animal and vegetal) are found in zebrafish, only one vegetal site in frog and one site localizing at the basement of the first cleavage furrow in the chick. Both zebrafish and frog embryos have been shown to multimerize germ plasm RNPs into four large aggregates during the first several cycles, which during early embryogenesis are inherited by four separate sets of PGCs in zebrafish and one main PGC cluster in frogs. Chick PGCs are found in a crescent-shaped distribution at the anterior edge of the primitive streak. After PGC migration, each organism contains bilaterally situated groupings of PGCs at the site of the prospective gonads.
While the early/METRO pathway does not involve microtubules [117, 118],
The segregation of germ plasm in
Although there are many similarities in the pathways of germ plasm segregation in
3.3. Other vertebrate species (chick)
Until relatively recently, studies suggested that the chick used epigenesis [125, 126], although these studies relied on in vitro culture [127]. However, Vasa protein (CVH in chick) was found to accumulate at the base of the membrane furrows in the early cleavage stage chick embryo, a location strikingly similar to that for
4. Conclusions
Epigenesis describes an inductive mechanism used notably by mammals in which tissues signal for a set of cells to become the PGCs. Preformation describes a mechanism using germ granules placed in the oocyte, which are collected into a set of cells to become the PGCs. Even though mechanisms differ, many of the RNAs and proteins that specify the germ line are conserved between animals using preformation and epigenesis.
Developmental biology studies have focused on genetic models to decipher the molecules and mechanisms for germ line establishment. Zebrafish use preformed germ granules known as germ plasm RNPs, which aggregate together, recruit to the furrow and distally compact into tight masses that ingress into only four cells. Throughout the remainder of maternal stage cell divisions, these four cells asymmetrically segregate the germ plasm aggregate so as only one of the dividing cells keeps the mass. When the zygotic genome is activated, these cells divide and generate the PGC population. It is tempting to speculate that the maternal process of germ plasm inheritance is designed to optimize the gathering of germ plasm material into large masses capable of influencing cell fate and that their subsequent asymmetric segregation during the cleavage stages preserves their full inductive potential until activation of the zygotic genome.
Understanding the mechanisms of germ cell determination will contribute to our ability to interpret cases of impaired fertility and will facilitate the promotion of healthy reproduction and assisted reproductive methods. In addition, recent studies in various biological systems have identified common links between germ cell gene expression programs, and those of stem and cancer cells [128–131], suggesting that a better understanding of germ cell biology will also contribute to the fields of regenerative and cancer biology. The zebrafish model system provides a tractable experimental system to gain mechanistic insights into these important topics relevant to human and animal health.
Acknowledgments
We thank current and former members of the Pelegri laboratory for useful discussions and participating on parts of the presented work. The research in our laboratory is funded by an National Institutes of Health (NIH) grant GM065303. C.E. has been additionally supported by NIH grant GM108449.
References
- 1.
Wolpert L. Principles of development. London/Oxford: Current Biology/University Press; 1998. - 2.
Swiers G, Chen Y-H, Johnson AD, Loose M. A conserved mechanism for vertebrate mesoderm specification in urodele amphibians and mammals. Dev. Biol. 2010;343:138-152. - 3.
Johnson AD, Bachvarova RF, Drum M. Expression of Axolotl DAZL RNA, a marker of germ plasm: widespread maternal RNA and onset of expression in germ cells approaching the gonad. Dev. Biol. 2001;234:402-415. - 4.
Smith LD. The role of a 'germinal plasm' in the formation of primordial germ cells in Rana pipiens . Dev. Biol. 1966;14:330-347. - 5.
Bachvarova RF, Crother BI, Manova K, Chatfield J, Shoemaker CM, Crews DP, Johnson AD. Expression of Dazl and Vasa in turtle embryos and ovaries: evidence for inductive specification of germ cells. Evol. Dev. 2009;11:525-534. - 6.
Tsunekawas N, Noito M, Sakai Y, Nishida T, Noce T. Isolation of chicken vasa homolog gene and tracing the origin of primordial germ cells. Development. 2000; 127: 2741-2750. - 7.
Evans TC, Wade CM, Chapman FA, Johnson AD, Loose M. Acquisition of germ plasm accelerates vertebrate evolution. Science. 2014;343:200-203. - 8.
Johnson AD, Richardson E, Bachvarova RF, Crother BI. Evolution of the germ line-soma relationship in vertebrate embryos. Reproduction. 2011;141:291-300. - 9.
Shibata N, Umesono Y, Orii H, Sajurai T, Watanabe K, Agata K. Expression of vasa (vas )-related genes in germline cells and totipotent somatic stem cells of planarians. Dev. Biol. 1999;206:73-87. - 10.
Anderson RA, Fulton N, Cowan G, Coutts S, Saunders PTK. Conserved and divergent patterns of expression of DAZL, VASA and OCT4 in the germ cells of the human fetal ovary and testis. BMC Dev. Biol. 2007;7:136. - 11.
Magnusdottir E, Surani MA. How to make a primordial germ cell. Electroenceph. Clin. Neurophys. 1987;66:529-538. - 12.
Boterenbrood EC, Nieuwkoop PD. The formation of the mesoderm in urodelean amphibians. V. Its regional induction by the endoderm. Wilhelm Roux' Arch. Dev. Biol. 1973;173:319-332. - 13.
Johnson AD, Crother B, White ME, Patient R, Bachvarova RF, Drum M, Masi T. Regulative germ cell specification in axolotl embryos: a primitive trait conserved in the mammalian lineage. Philos. Trans. R. Soc. Lond. B Biol. Sci. 2003;358:1371-1379. - 14.
Bachvarova RF, Masi T, Drum M, Parker N, Mason K, Patient R, Johnson AD. Gene expression in the axolotl germ line: Axdazl ,axvh ,axoct-4 , andAxkit . Dev. Dyn. 2004;231:871-880. - 15.
Lawson KA, Hage WJ. Clonal analysis of the origin of primordial germ cells in the mouse. Ciba Found. Symp. 1994;182:68-84. - 16.
Ying Y, Qi X, Zhao GQ. Induction of primordial germ cells from murine epiblasts by synergistic action of BMP4 and BMP8B signaling pathways. Proc. Natl. Acad. Sci. USA. 2001;98:7858-7862. - 17.
Ying Y, Zhao G-Q. Cooperation of endoderm-derived BMP2 and extraembryonic extoderm-derived BMP4 in primordial germ cell generation in the mouse. Dev. Biol. 2001;232:484-492. - 18.
Lawson KA, Dunn NR, Roelen BA, Zeinstra LM, Davis AM, Wright CV, Korving JP, Hogan BL. Bmp4 is required for the generation of primordial germ cells in the mouse embryo. Genes Dev. 1999;13:424-436. - 19.
Saga Y. Mouse germ cell development during embryogenesis. Curr. Opin. Genet. Dev. 2008;18:337-341. - 20.
Chen H-H, Welling M, Bloch DB, Muñoz J, Mientjes E, Chen X, Tramp C, Wu J, Yabuuchi A, Chou Y-F, Buecker C, Krainer A, Willemsen R, Heck AJ, Geijsen N. DAZL limits pluripotency, differentiation, and apoptosis in developing primordial germ cells. Stem Cell Rep. 2014;3:892-904. - 21.
Schrans-Stassen PT, Saunders PT, Cooke HJ, de Rooij DG. Nature of the spermatogenic arrest in Dazl-/- mice. Biol. Reprod. 2001;65:771-776. - 22.
Reijo R, Lee T-Y, Salo P, Alagappan R, Brown LG, Rosenberg M, Rozen S, Jaffe T, Strauss D, Hovatta O, de la Chapelle A, Silber S, Page DC. Diverse spermatogenic defects in humans caused by Y chromosome deletions encompassing a novel RNA binding protein gene. Nature Genetics. 1995;10:383-393. - 23.
Kee K, Angeles VT, Flores M, Nguyen HN, Reijo Pera RA. Human DAZL ,DAZ andBOULE genes modulate primordial germ-cell and haploid gamete formation. Nature. 2009;462:222-225. - 24.
Extavour CG, Akam M. Mechanisms of germ cell specification across the metazoans: epigenesis and preformation. Development. 2003;130:5869-5884. - 25.
Nakamura A, Seydoux G. Less is more: specification of the germline by transcriptional repression. Development. 2008;135:3817-3827. - 26.
Rangan P, DeGennaro M, Jaime-Bustamante K, Coux R-X, Martinho RG, Lehmann R. Temporal and spatial control of germ-plasm RNAs. Curr. Biol. 2009;19:72-77. - 27.
Huettner AF. The origin of the germ cells in Drosophila melanogaster . J. Morphol. 1923;37:385-423. - 28.
Mahowald AP. Assembly of the Drosophila germ plasm. Int. Rev. Cytol. 2001;203:187-213. - 29.
Geigy R. Action de l'ultra-violet sur le pole germinal dans l'oeuf de Drosophila melanogaster (Castration et mutabilite). Revue suisse Zool. 1931;38:187-288. - 30.
Okada M, Kleinman IA, and Schneiderman HA. Restoration of fertility in sterilized Drosophila eggs by transplantation of polar cytoplasm. Dev. Biol. 1974;37:43-54. - 31.
Mahowald AP. Fine structure of pole cells and polar granules in Drosophila melanogaster . J. Exp. Zool. 1962;151:201-216. - 32.
Lerit DA, Gavis ER. Transport of germ plasm on astral microtubules directs germ cell development in Drosophila . Curr. Biol. 2011;21:439-448. - 33.
Ephrussi A, Dickinson LK, and Lehmann R. oskar organizes the germ plasm and directs localization of the posterior determinant nanos. Cell. 1991;66:37-50. - 34.
Lynch JA, Özüak O, Khila A, Abouheif E, Desplan C, Roth S. The phylogenetic origin of oskar coincided with the origin of maternally provisioned germ plasm and pole cells at the base of the holometabola. PLoS Genet. 2011;7:e1002029. - 35.
Strome S, Wood WB. Generation of asymmetry and segregation of germ-line granules in early C. elegans embryos. Cell. 1983;35:15-25. - 36.
Boyd L, Guo S, Levitan D, Stinchcomb DT, Kemphues KJ. PAR-2 is asymmetrically distributed and promotes association of P granules and PAR-1 with the cortex in C. elegans embryos. Development. 1996;122:3075-3084. - 37.
Hird SN, White JG. Cortical and cytoplasmic flow polarity in early embryonic cells of Caenorhabditis elegans . J. Cell Biol. 1993;121:1343-1355. - 38.
Hird SN, Paulsen JE, Strome S. Segregation of germ granules in living Caenorhabditis elegans embryos: cell-type-specific mechanisms for cytoplasmic localisation. Development. 1996;122:1303-1312. - 39.
DeRenzo C, Reese KJ, Seydoux G. Exclusion of germ plasm proteins from somatic lineages by culin-dependent degradation. Nature. 2003;424:685-689. - 40.
Deppe U, Schierenberg E, Cole T, Krieg C, Schmitt D, Yoder B, von Ehrenstein G. Cell lineages of the embryo of the nematode Caenorhabditis elegans . Proc. Natl. Acad. Sci. USA. 1978;75:376-380. - 41.
Strome S, Wood WB. Immunofluorescence visualization of germ-line-specific cytoplasmic granules in embryos, larvae, and adults of Caenorhabditis elegans . Proc. Natl. Acad. Sci. USA. 1982;79:1558-1562. - 42.
Sulston JE, Schierenberg E, White JG, Thomson JN. The embryonic cell lineage of the nematode Caenorhabditis elegans . Dev. Biol. 1983;100:64-119. - 43.
Updike D, Strome S. P granule assembly and function in Caenorhabditis elegans germ cells. J. Androl. 2010;31:53-60. - 44.
Brown FD, Tiozzo S, Roux MM, Ishizuka K, Swalla BJ, De Tomaso AW. Early lineage specification of long-lived germline precursors in the colonial ascidian Botryllus schlosseri . Development. 2009;136:3485-3494. - 45.
Fujimura M, Takamura K. Characterization of an ascidian DEAD-box gene, Ci-DEAD: specific expression in the germ cells and its mRNA localization in the posterior-most blastomeres in early embryos. Dev. Genes Evol. 2000;210:64-72. - 46.
Shirae-Kurabayashi M, Nishikata T, Takamura K, Tanaka KJ, Nakamoto C, Nakamura A. Dynamic redistribution of vasa homolog and exclusion of somatic cell determinants during germ cell specification inCiona intestinalis . Development. 2006;133:2683-2693. - 47.
Brown FD, Swalla BJ. Vasa expression in a colonial ascidian, Botrylloides violaceus . Evol. Dev. 2007;9:165-177. - 48.
Takamura K, Fujimura M, Yamaguchi Y. Primordial germ cells originate from the endodermal strand cells in the ascidian Ciona intestinalis . Dev. Genes Evol. 2002;212:11-18. - 49.
Bontems F, Stein A, Marlow F, Lyautey J, Gupta T, Mullins MC, Dosch R. Bucky ball organizes germ plasm assembly in zebrafish. Curr. Biol. 2009;19:414-422. - 50.
Abrams EW, Mullins MC. Early zebrafish development: it's in the maternal genes. Curr. Opin. Genet. Dev. 2009;19:396-403. - 51.
Kosaka K, Kawakami K, Sakamoto H, Inoue K. Spatiotemporal localization of germ plasm RNAs during zebrafish oogenesis. Mech. Dev. 2007;124:279-289. - 52.
Ewen-Campen B, Schwager EE, Extavour CGM. The molecular machinery of germ line specification. Mol. Reprod. Dev. 2010;77:3-18. - 53.
Rocak S, Linder P. DEAD-box proteins: the driving forces behind RNA metabolism. Nature Rev. Mol. Cell Biol. 2004;5:232-241. - 54.
Lasko PF, Ashburner M. Posterior localization of vasa protein correlates with, but is not sufficient for, pole cell development. Genes Dev. 1990;4:905-921. - 55.
Gruidl ME, Smith PA, Kuznicki KA, McCrone JS, Kirchner J, Roussell DL, Strome S, Bennett KL. Multiple potential germ-line helicases are components of the germ-line-specific P granules of Caenorhabditis elegans . Proc. Natl. Acad. Sci. USA. 1996;93:13837-13842. - 56.
Sunanaga T, Watanabe A, Kawamura K. Involvement of vasa homolog in germline recruitment from coelomic stem cells in budding tunicates. Dev. Genes Evol. 2007;217:1-11. - 57.
Ohashi H, Umeda N, Hirazawa N, Ozaki Y, Miura C, Miura T. Expression of vasa (vas )-related genes in germ cells and specific interference with gene functions by double-stranded RNA in the monogenean,Neobenedenia girellae . Int. J. Parasitol. 2007;37:515-523. - 58.
Tanaka SS, Toyooka Y, Akasu R, Katoh-Fukui Y, Nakahara Y, Suzuki R, Yokoyama M, Noce T. The mouse homolog of Drosophila vasa is required for the development of male germ cells. Genes Dev. 2000;14:841-853. - 59.
Braat AK, van de Water S, Korving J, Zivkovic D. A zebrafish Vasa morphant abolishes Vasa protein but does not affect the establishment of the germline. Genesis. 2001;30:183-185. - 60.
Weidinger G, Wolke U, Köpprunner M, Klinger M, Raz E. Identification of tissues and patterning events required for distinct steps in early migration of zebrafish primordial germ cells. Development. 1999;126:5295-5307. - 61.
Kobayashi S, Yamada M, Asaoka M, Kitamura T. Essential role of the posterior morphogen nanos for germline development in Drosophila . Nature. 1996;380:708-711. - 62.
Forbes A, Lehmann R. Nanos and Pumilio have critical roles in the development and function of Drosophila germline stem cells. Development. 1998;125:679-690. - 63.
Deshpande G, Calhoun G, Yanowitz JL, Schedl PD. Novel functions of nanos in downregulating mitosis and transcription during the development of theDrosophila germline. Cell. 1999;99:271-281. - 64.
Köprunner M, Thisse C, Thisse B, Raz E. A zebrafish nanos-related gene is essential for the development of primordial germ cells. Genes Dev. 2001;15:2877-2885. - 65.
Kedde M, Strasser MJ, Boldajipour B, Oude Vrielink JA, Slanchev K, le Sage C, Nagel R, Voorhoeve PM, van Duijse J, Ørom UA, Lund AH, Perrakis A, Raz E, Agami R. RNA-binding protein Dnd1 inhibits microRNA access to target mRNA. Cell. 2007;131:1273-1286. - 66.
Mishima Y, Giraldez AJ, Takeda Y, Fujiwara T, Sakamoto H, Schier AF, Inoue K. Differential regulation of germline mRNAs in soma and germ cells by zebrafish miR-430. Curr. Biol. 2006;16:2135-2142. - 67.
Weidinger G, Stebler J, Slanchev K, Dumstrei K, Wise C, Lovell-Badge R, Thisse C, Thisse B, Raz E. dead end, a novel vertebrate germ plasm component, is required for zebrafish primordial germ cell migration and survival. Curr. Biol. 2003;13:1429-1434. - 68.
Blaser H, Eisenbeiss S, Neumann M, Reichman-Fried M, Thisse B, Thisse C, Raz E. Transition from non-motile behaviour to directed migration during early PGC development in zebrafish. J. Cell Sci. 2005;118:4027-4038. - 69.
Strasser MJ, Mackenzie NC, Dumstrei K, Nakkrasae L-I, Stebler J, Raz E. Control over the morphology and segregation of zebrafish germ cell granules during embryonic development. BMC Dev. Biol. 2008;8:58. - 70.
Arkov AL, Wang J-YS, Ramos A, Lehmann R. The role of Tudor domains in germline development and polar granule architecture. Development. 2006;133:4053-4062. - 71.
Lachke SA, Alkuraya FS, Kneeland SC, Ohn T, Aboukhalil A, Howell GR, Saadi I, Cavallesco R, Yue Y, Tsai AC-H, Nair KS, Cosma MJ, Smith RS, Hodges E, AlFadhli SM, Al-Hajeri A, Shamseldin HE, Bahbehani A, Hannon GJ, Bulyk ML, Drack AV, Anderson PJ, John SWM, Maas RL. Mutations in the RNA granule component TDRD7 cause cataract and glaucoma. Science. 2011;331:1571-1576. - 72.
Nair S, Marlow F, Abrams E, Kapp L, Mullins M, Pelegri F. The chromosomal passenger protein Birc5b organizes microfilaments and germ plasm in the zebrafish embryo. PLoS Genetics. 2013;9:e1003448. - 73.
Maegawa S, Yasuda K, Inoue K. Maternal mRNA localization of zebrafish DAZ-like gene. Mech. Dev. 1999;81:223-226. - 74.
Suzuki H, Maegawa S, Nishibu T, Sugiyama T, Yasuda K, Inoue K. Vegetal localization of the maternal mRNA encoding an EDEN-BP/Bruno-like protein in zebrafish. Mech. Dev. 2000;93:205-209. - 75.
Theusch EV, Brown KJ, Pelegri F. Separate pathways of RNA recruitment lead to the compartmentalization of the zebrafish germ plasm. Dev. Biol. 2006;292:129-141. - 76.
Takeda Y, Mishima Y, Fujiwara T, Sakamoto H, Inoue K. DAZL relieves miRNA-mediated repression of germline mRNAs by controlling Poly(A) tail length in zebrafish. PLoS ONE. 2009;4:e7513. - 77.
Hashimoto Y, Maegawa S, Nagai T, Yamaha E, Suzuki H, Yasuda K, Inoue K. Localized maternal factors are required for zebrafish germ cell formation. Dev. Biol. 2004;268:152-161. - 78.
Welch E, Pelegri F. Cortical depth and differential transport of vegetally localized dorsal and germ line determinants in the zebrafish embryo. Bioarchitecture 2015;5:13-26. - 79.
Yoon C, Kawakami K, Hopkins N. Zebrafish vasa homologue RNA is localized to the cleavage planes of 2- and 4-cell-stage embryos and is expressed in the primordial germ cells. Development. 1997;124:3157-3165. - 80.
Tran LD, Hino H, Quach H, Lim S, Shindo A, Mimori-Kiyosue Y, Mione M, Ueno N, Winkler C, Hibi M, Sampath K. Dynamic microtubules at the vegetal cortex predict the embryonic axis in zebrafish. Development. 2012;139:3644-3652. - 81.
Ge X, Grotjahn D, Welch E, Lyman-Gingerich J, Holguin C, Dimitrova E, Abrams EW, Gupta T, Marlow FL, Yabe T, Adler A, Mullins MC, Pelegri F. Hecate/Grip2a acts to reorganize the cytoskeleton in the symmetry-breaking event of embryonic axis induction. PLoS Genet. 2014;10:e1004422. - 82.
Campbell PD, Heim AE, Smith MZ, Marlow FL. Kinesin-1 interacts with Bucky ball to form germ cells and is required to pattern the zebrafish body axis. Development. 2015;142:2996-3008. - 83.
Robb DL, Heasman J, Raats J, Wylie C. A kinesin-like protein is required for germ plasm aggregation in Xenopus. Cell. 1996;87:823-831. - 84.
Tarbashevich K, Dzementsei A, Pieler T. A novel function for KIF13B in germ cell migration. Dev. Biol. 2011;349:169-178. - 85.
Pelegri F, Knaut H, Maischein H-M, Schulte-Merker S, Nüsslein-Volhard C. A mutation in the zebrafish maternal-effect gene nebel affects furrow formation andvasa RNA localization. Curr. Biol. 1999;9:1431-1440. - 86.
Eno C, Pelegri F. Gradual recruitment and selective clearing generate germ plasm aggregates in the zebrafish embryo. Bioarchitecture. 2013;3:125-132. - 87.
Kishimoto Y, Koshida S, Furutani-Seiki M, Kondoh H. Zebrafish maternal-effect mutations causing cytokinesis defects without affecting mitosis or equatorial vasa deposition. Mech. Dev. 2004;121:79-89. - 88.
Yabe T, Ge X, Lindeman R, Nair S, Runke G, Mullins M, Pelegri F. The maternal-effect gene cellular island encodes Aurora B kinase and is essential for furrow formation in the early zebrafish embryo. PLoS Genet. 2009;5:e1000518. - 89.
Danilchik MV, Funk WC, Brown E, Larkin K. Requirement for microtubules in new membrane formation during cytokinesis of Xenopus embryos. Dev. Biol. 1998;194:47-60. - 90.
Jesuthasan S. Furrow-associated microtubule arrays are required for the cohesion of zebrafish blastomeres following cytokinesis. J. Cell Sci. 1998;111:3695-3703. - 91.
Urven LE, Yabe T, Pelegri F. A role for non-muscle myosin II function in furrow maturation in the early zebrafish embryo. J. Cell Sci. 2006;119:4342-4352. - 92.
Knaut H, Pelegri F, Bohmann K, Schwarz H, Nüsslein-Volhard C. Zebrafish vasa RNA but not its protein is a component of the germ plasm and segregates asymmetrically prior to germ line specification. J. Cell Biol. 2000;149:875-888. - 93.
Pelegri F, Schulte-Merker S. A gynogenesis-based screen for maternal-effect genes in the zebrafish, Danio rerio . In: Detrich W, Zon LI, Westerfield M, editors. The Zebrafish: Genetics and Genomics. San Diego: Academic Press; 1999. p. 1-20. - 94.
Bashirullah A, Halsell SR, Cooperstock RL, Kloc M, Karaiskakis A, Fisher WW, Etkin LD, Lipshitz HD. Joint action of two RNA degradation pathways controls the timing of maternal transcript elimination at the midblastula transition in Drosophila melanogaster . The EMBO J. 1999;18:2610-2620. - 95.
Kashikawa M, Amikura R, Nakamura A, Kobayashi S. Mitochondrial small ribosomal RNA is present on polar granules in early cleavage embryos of Drosophila melanogaster . Dev. Growth Differ. 1999;41:495-502. - 96.
Kloc M, Dougherety MT, Bilinski S, Chan AP, Brey E, King ML, Patrick CW Jr, Etkin LD. Three-dimensional ultrastructural analysis of RNA distribution within germinal granules of Xenopus . Dev. Biol. 2002;241:79-93. - 97.
Savage R, Danilchik MV. Dynamics of germ plasm localization and its inhibition by ultraviolet irradiation in early cleavage Xenopus embryos. Dev. Biol. 1993;157:371-382. - 98.
Braat AK, Zandbergen T, van de Water S, Goos HJT, Zivkovic D. Characterization of zebrafish primordial germ cells: morphology and early distribution of vasa RNA. Dev. Dyn. 1999;216:153-167. - 99.
Whittington PM, Dixon KE. Quantitative studies of germ plasm and germ cells during early embryogenesis of Xenopus laevis . J. Embryol. Exp. Morph. 1975;33:57-74. - 100.
Lambert JD, Nagy LM. Asymmetric inheritance of centrosomally localized mRNAs during embryonic cleavages. Nature. 2002;420:682-686. - 101.
Kane DA, Kimmel CB. The zebrafish midblastula transition. Development. 1993;119:447-456. - 102.
Lu X, Li JM, Tavazole S, Wieschaus EF. Coupling of zygotic transcription to mitotic control at the Drosophila mid-blastula transition. Development. 2009;136:2101-2110. - 103.
Magnúsdóttir E, Surani MA. How to make a primordial germ cell. Development. 2014;141:245-252. - 104.
Houwing S, Kamminga LM, Berezikov E, Cronembold D, Girard A, van den Elst H, Filippov DV, Blaser H, Raz E, Moens CB, Plasterk RHA, Hannon GJ, Draper BW, Ketting RF. A role for Piwi and piRNAs in germ cell maintenance and transposon silencing in zebrafish. Cell. 2007;129:69-82. - 105.
Ku H-Y, Lin H. PIWI proteins and their interactors in piRNA biogenesis, germline development and gene expression. Natl. Sci. Rev. 2014; 1: 205-218. - 106.
Braat AK, van de Water S, Goos H, Bogerd J, Zivkovic D. Vasa protein expression and localization in the zebrafish. Mech. Dev. 2000;95:271-274. - 107.
Wolke U, Widinger G, Köprunner M, Raz E. Multiple levels of postranscriptional control lead to germ line-specific gene expression in the zebrafish. Curr. Biol. 2002;12:289-294. - 108.
Richardson BE, Lehmann R. Mechanisms guiding primordial germ cell migration: strategies from different organisms. Nature Rev. Mol. Cell Biol. 2010;11:37-49. - 109.
Paksa A, Raz E. Zebrafish germ cells: motility and guided migration. Curr. Opin. Cell Biol. 2015;36:80-85. - 110.
Hartwig J, Tarbashevich K, Seggewiß J, Stehling M, Bandemer J, Grimaldi C, Paksa A, Groß-Thebing T, Meyen D, Raz E. Temporal control over the initiation of cell motility by a regulator of G-protein signaling. Proc. Natl. Acad. Sci. USA. 2014;113:11389-11394. - 111.
Raz E. Guidance of primordial germ cell migration. Curr. Opin. Cell Biol. 2004;16:169-173. - 112.
Knaut H, Steinbeisser H, Schwarz H, Nüsslein-Volhard C. An evolutionary conserved region in the vasa 3'UTR targets RNA translation to the germ cells in the zebrafish. Curr. Biol. 2002;12:454-466. - 113.
Herpin A, Rohr S, Riedel D, Kluever N, Raz E, Schartl M. Specification of primordial germ cells in medaka ( Oryzias latipes ). BMC Dev. Biol. 2007;7:3. - 114.
Saito TL, Psenicka M, Goto R, Adachi S, Inoue K, Arai K, Yamaha E. The origin and migration of primordial germ cells in sturgeons. PLoS ONE. 2014;9:e86861. - 115.
Minakhina S, Steward R. Axes formation and RNA localization. Curr. Opin. Genet. Dev. 2005;15:416-421. - 116.
Zhou Y, King ML. Localization of Xcat2 RNA, a putative germ plasm component, to the mitochondrial cloud in Xenopus stage 1 oocyte. Development. 1996;122:2947-2953. - 117.
Kloc M, Etkin LD. Apparent continuity betwen the messenger transport organizer and late RNA pathways during oogenesis in Xenopus . Mech. Dev. 1988;73:95-106. - 118.
Kloc M, Larabell C, Etkin LD. Elaboration of the messenger transport organizer pathway for localization of RNA to the vegetal cortex of Xenopus oocytes. Dev. Biol. 1996;180:119-130. - 119.
Quaas J, Wylie C. Surface contraction waves (SCWs) in the Xenopus egg are required for the localization of the germ plasm and are dependent upon maternal stores of the kinesin-like protein Xklp1. Dev. Biol. 2002;243:272-280. - 120.
MacArthur H, Houston DW, Bubunenko M, Mosquera L, King ML. DEADSouth is a germ plasm specific DEAD-box RNA helicase in Xenopus related to eIF4A. Mech. Dev. 2000;95:291-295. - 121.
Whitingon PM, Dixon KE. Quantitative studies of germ plasm and germ cells during early embryogenesis of Xenopus laevis . J. Embryol. Exp Morphol. 1975;33:57-74. - 122.
Ikenishi K, Okuda T, Nakazato S. Differentiation of presumptive primordial germ cells (pPGC)-like cells in explants into PGCs in experimental tadpoles. Dev. Biol. 1984;103:258-262. - 123.
Wylie CC, Heasman J, Snape A, O'Driscoll M, Holwill S. Primordial germ cells of Xenopus laevis are not irreversibly determined early in development. Dev. Biol. 1985;112:66-72. - 124.
Technau G. A single cell approach to problems of cell lineage and commitment during embryogenesis of Drosophila melanogaster . Development. 1987;100:1-12. - 125.
Swift CH. Origin and early history of the primordial germ cells in the chick. Am. J. Anat. 1914;15:483-516. - 126.
Eyal-Giladi H, Ginsburg M, Farbarov A. Avian primordial germ cells are of epiblastic origin. J. Embryol. Exp. Morphol. 1981;165:139-147. - 127.
Karagenc L, Cinnamon Y, Ginsburg M, Petitte JN. Origin of primordial germ cells in the prestreak chick embryo. Dev. Genet. 1996;19:290-301. - 128.
Juliano CE, Swartz SZ, Wessel GM. A conserved germline multipotency program. Development. 2010;137:4113-4126. - 129.
Nagamatsu G, Kosaka T, Kawasumi M, Kinoshita T, Takubo K, Akiyama H, Sudo T, Kobayashi T, Oya M, Suda T. A germ cell-specific gene, Prmt5, works in somatic cell reprogramming. J. Biol. Chem. 2011;286:10641-10648. - 130.
Yohn CB, Pusateri L, Barbosa V, Lehmann R. l(3)malignant brain tumor and three novel genes are required for Drosophila germ-cell formation. Genetics. 2003;165:1889-1900. - 131.
Janiç A, Mendizabal L, Llamazares S, Rossell D, Gonzalez C. Ectopic expression of germline genes drives malignant brain tumor growth in Drosophila . Science. 2010;330:1824-1827.