Generating Germ Cells in Schmidtea mediterranea

By PamelaSara Head

Methods of Generation

Germ cells are the generators of gametes and are essential for the propagation of sexually reproducing organisms. In metazoans, there are two main methods for specification of germ cells during development. One method is preformation or the determination of germ cells by maternally inherited determinants. This process occurs early on in development and it has been seen in such organisms as C. elegans, and D. melanogaster.

The other method is epigenesis in which germ cells are specified by inductive signals between cells/surrounding tissues. Specification by this method occurs much later on in development. This is the more common method seen in basal metazoans as well as higher organisms such as mice. This finding indicates that it is the ancestral method of germ cell specification while preformation is more novel. For more on the differences between these methods feel free to read the following article at this site.

Modes of germ cell specification across the Metazoa

Extavour, Cassandra G, and Michael Akam 2003 Figure 3 :Red boxes indicate they use epigenesis in germ cell specification, Blue boxes indicate preformation, boxes half blue and half red have species that show one or the other, and white boxes indicate the mechanism used is unknown. “Asterisks indicate phyla in which epigenesis has been claimed, but recent data suggest preformation.” Note here that basal metazoans use epigenesis and that there are more metazoans that use epigenesis rather than preformation.

Production and Maintenance of Germ cells and their Descendants

Despite the differences in how germ cells can be specified there are key steps common to both pathways:

First: pPGCs or presumptive primordial germ cells divide mitotically generating one PGC and one somatic cell.

Second: PGCs or primordial germ cells give rise to germ stem cells by clonal mitotic divisions.

Third: Once germ cells become specified and differentiated they enter the first stage of gametogenosis and become known as (spremato-/oo-) gonia.

Fourth: The gonia transform into oocytes and spermatocytes through the processes of oogenesis and spermatogenesis respectively.

Fifth: They then mature completely into ova and spermatozoa. So the question becomes, how does Schmidtea mediterrane specify, maintain, and regenerate its germ cells?

Modes of germ cell specification: preformation and epigenesis

Preformation:

Preformation:

Extavour, Cassandra G, and Michael Akam 2003 Box 1. During oogenesis in Drosophila melanogaster, RNAs and proteins are synthesised by the nurse cells.These products (blue) are transported through cytoplasmic bridges (blue arrows) to the oocyte. They become localised to the posterior of the ooplasm both by molecular anchoring at the posterior of the oocyte, and by posterior-specific translational and transcriptional regulation. This posterior ooplasm is the germ plasm, or germ line determinant. During early embryogenesis, cells which inherit the germ plasm become the primordial germ cells (PGCs; red).

Epigenesis:

Extavour, Cassandra G, and Michael Akam 2003 Box 1. No maternally deposited germ plasm has been observed in the oocytes. of the mouse Mus musculus. Instead, PGC determination takes place after the segregation of embryonic and extraembryonic tissues. A subpopulation of the pluripotent epiblast cells express `germline competence genes' (striped). These cells are able to interpret the inductive signals that arrive from neighbouring tissues and differentiate into PGCs (red). The inductive signals come from the extraembryonic ectoderm (blue) and endoderm (yellow).

Specification of Germ Cells in Schmidtea mediterranea

As previously stated, epigenesis is more likely the ancestral mechanism of germ cell specification and has been observed in basal metazoans. It therefore should come as no surprise that findings indicate Schmidtea mediterranea also specify their germ cells via epigenesis. This also means that germ cells are specified post-embryonically and in fact the sex organs don’t develop until after the organism hatches. Due to their mechanism of germ cell specification, Schmidtea mediterranea are good model organisms for studying the processes for other higher metazoans such as humans. Here we shall focus on the function of a particular inductive signal in this mechanism that has been of interest recently. This signal molecule is known as Smed-nanos (also known as Smednos) shall be discussed.

In Schmidtea mediterranea, the reproductive organs are generated in a specific order. First the ovaries are formed behind cephalic ganglia, then the testes are formed dorsolaterally, followed by the oviducts and vitelline glands. The copulatory apparatus and the gonads don’t develop until a certain time of year (mating season). For more on their general anatomy and other fun facts feel free to visit the geochembio website, the wikipedia page, and/or the Sànchez lab home page.

Figure 4: Anatomy and physiology in the common planarian.“Planarian.png (PNG Image, 550 × 402 Pixels) – Scaled (0%).” http://www.geochembio.com/IMG/planarian.png. 23 Mar. 2012.

Step1: Identifying the Nanos homologue in Schmidtea mediterranea

Smed-nanos is the homologue of the well known and well conserved protein Nanos. Nanos is an RNA binding protein (usually targets the 3’ untranslated regions of specific mRNAs) that was first identified in fruit fly development and its homologues have been found in mice, zebra fish, and amphibians. It is necessary in the prevention of premature mitotic divisons of PGCs. If they enter mitosis too early they will be unable to migrate to the correct positions of the organisms and therefore will undergo apoptosis (programmed cell death) and become unable to retain stem cell identity in the organism’s adulthood. In the paper “nanos function is essential for development and regeneration of planarian germ cells” by Wang et al, Smed-nanos was shown to be crucial in Schmidtea mediterranea.

Hashimoto, Hiroshi et al. 2010 Fig. 1: Nanos as found in zebrafish. The two conserved zinc finger domains are found at the C-terminus and in the amino acid code one can see the Cys-Cys-His-Cys pattern highlighted in pink.

In order to identify and track Smed-nanos in germ cells, investigators looked for sequences encoding the zinc finger motifs conserved in nanos and its homologues (composed of the code for the amino acids Cys-Cys-His-Cys in that order in the C-terminal ends). To accomplish this, researchers used shot-gun sequencing of the hermophoditic genome and then isolated full-length cDNAs of Smed-nanos from a cDNA library. They then preformed a Northern blot and found the Smed-nanos transcript (it was 0.8 kilobase pairs long and it encoded a 233 amino acid long protein with two zinc finger motifs).

Step 2: Evidence that Smed-nanos acts in germ cell specification later on in development:

As can be seen in Figure 1, in situ hybridization of Smed-nanos indicated the time and location of its expression. Here experimenters also preformed an in situ hybridization of the control molecules germinal Histone H4 and neural marker anosmin-1. Germinal H4 is a transcript found in pPGCs and somatic neoblasts. It was not until the third and seventh days after the flatworms had hatched that Smed-nanos was observed dorsolaterally (where pPGCs of the testes can be found as indicated by the arrows in Figure 1.).

In juvenile (individuals that had yet to develop fully mature reproductive structures) testes and ovaries as well as adult flatworm testes and ovaries, Smed-nanos RNA was detected. Germinal H4 was also detected in the clusters of dorsolateral clusters in day four old animals but not earlier of Smed-nanos positive cells further indicating the identity of those cells.

Wang, Y. et al. 2007 Fig. 1: A. O is shorthand for ovaries, t is for testes, od is for the oviducts, ca is for copulatory apparatus. B. the top most organism is a hatchling and no testes have formed (or germ cells). As one proceeds downward the organism is growing from a juvenile to a mature adult. The arrows are pointing to the forming testes. C. This shows the same thing but here the ovaries are being highlighted. This demonstrates how germ cells come later on in development.

In this protion of the experiment, experimenters also indicated that neoblasts were required for Smed-nanos function by using 30 Gy (“The gray is the SI derived unit of absorbed radiation dose of ionizing radiation, and is defined as the absorption of one joule of ionizing radiation by one kilogram of matter”- wikipedia) to eliminate the neoblasts. Without the neoblasts, Smed-nanos positive cells were not detected in dorsolateral clusters as in control groups three days after hatching. In conclusion, Smed-nanos expression is concurrent with specification of germ cell precursors and its expression requires neoblasts (or cell divison).

Step 3: Regeneration of Germ Cells in Schmidtea mediterranea evidence of epigenetic control:

Here the experimenters examined the expression of Smed-nanos in amputated head regions of Schmidtea mediterranea that lacked any and all reproductive tissues. If its expression could be generated without reproductive tissues (by somatic tissues) and after the event already occurred once, then germ cells are specified post-embryonically by non-maternal determinants (epigenetic mechanism). Using FISH and confocal microscopy, experimenters detected Smed-nanos postitive cell clusters in regenerated portions of the severed head regions. Fourteen days after amputation, 100% of flatworms expressed the protein and positive cell clusters were larger and greater in number twenty-one days post amputation.

Wang, Y. et al. 2007 Fig. 2: amputated head fragments during regeneration. Detection of Smed-nanos mRNA at 7, 14, and 21 days after amputation.

Concurrent in situ hybridization of T-plastin mRNA (T-plastin is expressed in spermatocytes and sprematids) gave further evidence that germ cells had regenerated. Its expression was absent until 15 days after amputation (after Smed-nanos began to be expressed once more). The experimenters went on to perform RNAi (gene knockdown) of Smed-nanos by feeding the flatworms with bacteria expressing double-stranded DNA.

First, they were given two feedings (once weekly) and then their head regions were amputated with their ovaries intact but not their testes. Feeding then continued two weeks later to allow for growth and maturation (once every week for 2 to 3 months). A control group was feed bacteria containing and empty vector and they regenerated their reproductive structures normally. Those fed dsRNA (for Smed-nanos) did not form reproductive structures until three months after amputation (after the feedings were stopped). Using DAPI staining, the state of reforming testes structures (dorsolateral Smed-nanos positive cell clusters) were monitored.

Wang, Y. et al. 2007 Fig. 3: Regeneration of the testes. A. detection of T-plastin in whole and regenerating fragments. B. detection of Smed-nanos in whole and regenerating fragments. C. FISH analysis of Smed-nanos mRNA in whole and regenerating fragments.

In Smed-nanos RNAi individuals these clusters were absent. Control individuals showed normal testes and were examined using anti-tubulin immunoflorsecence (microtubules are highly used in components of spermatozoa). Spermatozoa were not seen in RNAi individuals. T-plastin was seen in control specimens but not in specimens that underwent Smed-nanos RNAi. Examination of expression of germinal H4 (found in ovaries, testes, and neoblasts) showed that ovaries were also absent in RNAi individuals. These experiments indicated that Smed-nanos is necessary for regeneration of the gonads in flatworms.

In individuals starved after amputation for two weeks, FISH was used to detect germinal H4 mRNA, and it was only found in neoblast cells in RNAi treated individuals. The indicated the Smed-nano “RNAi phenotype during regeneration is the result of failure to form or maintain testes primordial during early stages of testes regeneration rather than loss of mature testes after normal regeneration.”

Step 4: Maintenance of Germ Cells in Schmidtea mediterranea:

Adult individuals that were fed Smed-nanos dsRNA every 4 to 5 days for one month ended up losing their gonads. This gives evidence that Smed-nanos is also required for germ cell maintenance.

Step 5: Post-embryonic development of the Planarian reproductive system:

Instead of using fragmented adults, experimenters preformed Smed-nanos RNAi (feed them every 4 to 5 days for 2 to 3 months) on one day old hatchlings (2 months are required for flatworms to reach maturity). They grew up normally in every physical aspect except they did not have ovaries, testes or gonopores. Thus, Smed-nanos is required for the post-embryonic development of the flatworm’s reproductive system.

Wang, Y. et al. 2007 Fig. 4: A and B. There is no regeneration of ovaries or testes after Smed-nanos RNAi. C and D is the immunofluorescent image of the same areas seen in A and B. Anti-tubulin immunofluorescence is seen inside the testes (arrows) in the control (C) a.k.a. the testes are present. The testes are not present in D when there is Smed-nanos RNAi. E and F: In the testes of planaria, when spermatogonia divide they do so incompletely and end in cysts composed of 8 spermatocytes which become 32 spermatids after meiosis. The cells dividing within the cysts can be labled with H3-Sp10P antibodies. There are H3-S10P positive clusters in the testes in the control (E) but not in the Smed-nanos RNAi individual (F). G is an overlay of B and C while H is an overlay of D and F. I through N is an in situ hybridization of gonad markers. In I (control) there was expression of T-plastin but not in the Smed-nanos RNAi individual (J). K and L looks at the expression of germinal H4 which is present in the control’s testes (K) but not in the individual who underwent RNAi. M: germinal H4 is expressed in the ovaries (arrows) in controls but not in the RNAi individual (N).

Interesting findings in the asexual strain of Schmidtea mediterranea:

In the asexual strain, there is no formation of reproductive organs but when experimenters preformed a Northern blot for Smed-nanos mRNA, they discovered it was expressed at comparable levels to that in the sexual strain.

In addition, an in situ hybridization of Smed-nanos indicated its mRNA was found in cells distributed in a similar way to how presumptive testes primordial of sexual worms is distributed. It was also found that germinal H4 was also expressed in the same way pattern wise. So why doesn’t the asexual strain produce sexual structures if Smed-nanos is present?

Wang, Y. et al. 2007 Fig. 6: Smed-nanos expression in the asexual strain and the effect of Smed-nanos RNAi on it. A: whole mount in situ hybridization to Smed-nanos in clusters of dorsolateral cell clusters where the testes would be. B. three days after 30 Gy, the cells from A are no longer there. C. germinal H4 is expressed in neoblasts and the cell clusters. D. 30 Gy eliminates expression seen in C. E is a control after a month past amputation. These individuals were feed control untreated bacteria and had germinal H4 expression like that seen in untreated individuals. F was fed bacteria with Smed-nanos dsRNA (RNAi). The lost expression of germinal H4 expression from the cell clusters, while its expression in neoblasts was unaffected.

It is stipulated that the genes encoding these mRNAs are expressed solely in a small subset of the proliferating cell population, neoblasts give rise to short-lived germ cells, or downstream inhibitors of these mRNAs/proteins expressed in the asexual strain. Further experiments are required to uncover the actual mechanism.

Another hypothesis suggests that even though Smed-nanos is produced it is not functional. So Smed-nanos RNAi was performed on the asexual strain followed by amputation seven days following the second feeding of dsRNA. One week later they continued the feeding and examined the organisms one month later.

They lacked the dorsolateral population of cells expressing germinal H4. This result was repeated in unamputated organisms that underwent Smed-nanos RNAi. Although the experiment showed that Smed-nanos is required for germinal H4 expression in pPGCs in whole and regenerating asexual flatworms, it failed to determine whether or not Smed-nanos was funtionaly in all aspects of generating germ cells other than its role in activating production of germinal H4.

Last comments:

The only suggestion I could give for improving this paper is for the researchers to have preformed more in depth studies on how Smed-nanos works in planaria to activate germ cell specification or how it activates germinal H4, or T-plastin. There also needs to be more research/experiments to both “clarify the basis of the radiation sensitivity to the presumptive germ cells,” and to identify other genes required for epigenetic germ cell specification in this model organism.

Bibliography:

  1. Handberg-Thorsager, Mette, and Emili Saló. “The Planarian Nanos-like Gene Smednos Is Expressed in Germline and Eye Precursor Cells During Development and Regeneration.” Development Genes and Evolution 217.5 (2007): 403–411. Web. 24 Mar. 2012.
  2. Hashimoto, Hiroshi et al. “Crystal Structure of Zinc-finger Domain of Nanos and Its Functional Implications.” EMBO reports 11.11 (2010): 848–853. Web. 18 Mar. 2012.
  3. Extavour, Cassandra G, and Michael Akam. “Mechanisms of Germ Cell Specification Across the Metazoans: Epigenesis and Preformation.” Development 130.24 (2003): 5869–5884. Web. 23 Mar. 2012.
  4. Mitinori Saitou, Germ cell specification in mice, Current Opinion in Genetics & Development, Volume 19, Issue 4, August 2009, Pages 386-395, ISSN 0959-437X, 10.1016/j.gde.2009.06.003.
  5. “Planarian.png (PNG Image, 550 × 402 Pixels) – Scaled (0%).” http://www.geochembio.com/IMG/planarian.png. 23 Mar. 2012.
  6. Wang, Y. et al. “Nanos Function Is Essential for Development and Regeneration of Planarian Germ Cells.” Proceedings of the National Academy of Sciences 104.14 (2007): 5901–5906. Web. 9 Mar. 2012.
  7. Zayas, Ricardo M et al. “The Planarian Schmidtea Mediterranea as a Model for Epigenetic Germ Cell Specification: Analysis of ESTs from the Hermaphroditic Strain.” Proceedings of the National Academy of Sciences of the United States of America 102.51 (2005): 18491–18496. Web. 24 Mar. 2012.

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