Molecular markers to characterize the hermaphroditic reproductive system of the planarian Schmidtea mediterranea
© Chong et al; licensee BioMed Central Ltd. 2011
Received: 20 August 2011
Accepted: 10 November 2011
Published: 10 November 2011
The freshwater planarian Schmidtea mediterranea exhibits two distinct reproductive modes. Individuals of the sexual strain are cross-fertilizing hermaphrodites with reproductive organs that develop post-embryonically. By contrast, individuals of the asexual strain reproduce exclusively by transverse fission and fail to develop reproductive organs. These different reproductive strains are associated with distinct karyotypes, making S. mediterranea a useful model for studying germline development and sexual differentiation.
To identify genes expressed differentially between these strains, we performed microarray analyses and identified >800 genes that were upregulated in the sexual planarian. From these, we characterized 24 genes by fluorescent in situ hybridization (FISH), revealing their expression in male germ cells or accessory reproductive organs. To identify additional markers of the planarian reproductive system, we also used immuno- and fluorescent lectin staining, identifying several antibodies and lectins that labeled structures associated with reproductive organs.
Collectively, these cell-type specific markers will enable future efforts to characterize genes that are important for reproductive development in the planarian.
While the planarian has re-emerged as an animal model for studying regeneration and stem cell biology [1–5], recent studies of Schmidtea mediterranea have also contributed to understanding germ cell biology and sexual development [6–9]. Two modes of specifying germ cells have evolved among animals, involving either localized determinants or inductive signaling [10–12]. Sexual planarians have a reproductive system that can be regenerated de novo from stem cells after amputation [9, 13], suggesting that they specify their germline inductively like mammals and many basal metazoans [10, 14, 15].
In contrast to the sexual strain, the asexual strain of S. mediterranea does not develop reproductive organs, and reproduces exclusively by transverse fission. While asexual planarians do not elaborate well-developed gonads, they do possess primordial germ cells that fail to differentiate further [8, 9, 20, 21]. Although the asexual strain is distinguished by a chromosomal translocation, the exact mechanisms that account for the two divergent modes of reproduction are unknown [22, 23]. However, neuropeptide signaling was recently shown to be required for the development of reproductive organs in sexual planarians. RNA interference (RNAi) knockdown of a single neuropeptide gene, npy-8 [GenBank: BK007010], resulted in animals that failed to develop or maintain reproductive organs. Interestingly, npy-8 mRNA was not detected in asexual planarians .
The existence of two divergent modes of reproduction in a single species presents a unique opportunity to identify conserved and species-specific genes that are important for germ cell development and reproductive maturation. Thus, we used two approaches to characterize differences between these strains: (i) microarray analysis to identify genes that are expressed differentially between sexual and asexual planarians, followed by in situ hybridization to identify the cell types in which these genes are expressed; and (ii) morphological analysis using confocal microscopy to identify cell type-specific markers that label components of the sexual reproductive system. We show that several genes identified through this transcriptional comparison serve as useful markers for somatic and germ cells of the planarian reproductive system. We also introduce several antibodies and fluorescent lectin-conjugates that will be useful for visualizing components of the planarian reproductive system. These studies provide complementary approaches for studying the genetic and morphological differences between sexual and asexual modes of reproduction in planarians.
Results and Discussion
Identification of genes with significantly higher expression in sexual planarians
Genes that are upregulated in sexual planarians are expressed in the planarian reproductive system
To validate our microarray results, a primary whole-mount in situ hybridization (WISH) screen involving 122 genes that showed significant differential expression in the sexual planarians (upregulated ≥ 2 standard deviations from the mean) was performed on mature sexual animals. We focused on genes from four different COG functional categories: signal transduction, transcription, cytoskeleton, and genes of unknown function. Of the 122 genes examined, 100 (82%) were expressed in the reproductive system: 96 genes were expressed in the testes, and 4 genes were expressed in accessory reproductive organs (data not shown). To characterize further the cellular distribution of several of the transcripts, 24 that were expressed either in accessory reproductive organs or during different stages of spermatogenesis were analyzed using fluorescence in situ hybridization (FISH) (Additional file 3, Figure S1 and Additional file 4, Table S3).
Genes identified through the sexual/asexual array are expressed in accessory reproductive organs or enriched in different stages of germ cell development in the testes.
Accessory reproductive organs
Sperm duct/seminal vesicles
Glands around copulatory apparatus
Mechanisms regulating different stages of germ cell differentiation are vital for the production of gametes. Based on the assigned putative functions of the genes examined and the enrichment of their mRNA transcripts in different domains of the testes, these genes may be involved in regulating the differentiation of male germ cells. Several studies have shown that germ cells rely upon post-transcriptional regulation to maintain their genomic plasticity [12, 28]. A recent functional genomic screen in S. mediterranea showed that RNA-binding proteins act as critical regulators of germ cell development in planarians , similar to germ cell regulation across metazoans, in which RNA-binding proteins regulate germ cell proliferation, stem cell maintenance, and sex determination [29–32]. For future work on genes upregulated in sexual planarians, it will be important to functionally characterize genes from other COG categories, such as RNA processing and modification, as well as translation, ribosomal structure and biogenesis, with emphasis on genes encoding RNA-binding proteins.
The aforementioned screen to identify genes required for germ cell development in planarians  used microarrays to investigate changes in gene expression in animals lacking germ cells after nanos [GenBank: EF035555] RNAi. These experiments were designed to identify differences in the "early" germ cell populations found in asexual planarians and juvenile sexual planarians (i.e., well before reproductive maturity); however, they would not detect genes expressed in the latest stages of reproductive maturation. By contrast, the experiments reported here to compare asexual and fully mature sexual planarians should detect genes expressed in the accessory reproductive organs and/or during the latest stages of reproductive system development. The nanos knockdown experiments identified 103 genes that were downregulated after nanos (RNAi) in asexual planarians and 275 genes that were downregulated after nanos (RNAi) in juvenile sexual planarians ; whereas, we found >800 genes that were dramatically upregulated in mature sexual planarians relative to asexuals. Given the large number of reproductive organs (gonads and the many accessory organs) that are present in mature sexual animals, it is not surprising that there are many more genes expressed differentially and at much greater differential levels than were observed in the nanos RNAi experiments.
Comparing these data sets revealed that 237/275 genes (86%) that were downregulated in juvenile sexual nanos (RNAi) animals (i.e., lacking germ cells) were upregulated (>2 s.d.) in sexual planarians relative to asexuals. Similarly, 88/103 genes (85%) that were downregulated in asexual nanos (RNAi) animals were upregulated (>2 s.d.) in sexual planarians. Of the 13 genes shown to be required for different stages of germ cell development  nine were upregulated >2 s.d. and two were upregulated >1 s.d. (>3.12-fold) in sexual planarians. Thus, there was excellent agreement between these different data sets. Because asexual planarians also possess early nanos-expresssing germ cells, it is possible that some of the genes that do not show differential expression between asexual and sexual planarians are expressed in early germ cells. Among the genes characterized here by in situ hybridization, 15/24, including those expressed in the somatic accessory structures were not expressed differentially in nanos (RNAi) versus control sexuals, validating this complementary approach to identify genes involved in planarian sexual development.
Our in situ hybridization analyses did not detect genes whose transcripts were expressed exclusively in the ovaries. The difficulty of isolating ovary-specific genes is most likely based upon the paucity of ovarian tissue relative to the animal as a whole. The two ovaries reside at the base of the cephalic ganglia, whereas the testes are distributed throughout the dorso-lateral margin, along most of the length of the animal. Several other accessory reproductive organs also occupy a much greater portion of the planarian body than do the ovaries. Because these experiments have used RNAs isolated from whole planarians, ovarian RNAs are likely to be under-represented relative to testis-enriched RNAs. Thus, isolation of additional ovary-specific transcripts will require other experimental approaches.
Immunofluorescent and fluorescent lectin-conjugate labeling of the planarian reproductive system
Antibodies and lectins that label accessory reproductive organs and gonads in S. mediterranea.
Accessory reproductive organs
Glands around the copulatory apparatus
Membranes in/around testes
Muscle (pre-immune serum)^
Peanut agglutinin (PNA)*
Ricinus communis agglutinin (RCA)*
Erythrina cristagalli lectin (ECL)*
Lens culinaris agglutinin (LCA)*
The male accessory reproductive organs were also labeled by a variety of antibodies and lectins. The ciliated sperm ducts were labeled by anti-tubulin δ2 (Figure 6A, dashed box) while the seminal vesicles, tubular structures suspended in a network of muscle fibers, were visualized with anti-muscle (Figure 6G) and anti-phosphotryosine immunostaining (Additional file 5, Figure S2B). The penis papilla was labeled by anti-phosphotyrosine (Figure 6H) and ECL (Additional file 5, Figure S2C). Finally, the common gonopore could be visualized by staining with muscle antibody (Additional file 5, Figure S2D).
The markers described thus far allow the labeling of male and female reproductive structures of the planarian. An interesting question in simultaneous hermaphrodites is whether male and female reproductive structures are co-dependent on each other. While some genes function in both male and female developmental pathways, there are two potentially distinct pathways that are responsible for either male or female sexual development. This idea is supported by the fact that it is possible to use RNAi to generate animals devoid of male germ cells . The presence of sex-specific pathways in planarians like S. mediterranea could allow the evolution of simultaneous hermaphrodites into distinct male or female sexes, as seen in more derived flatworm species like the parasitic schistosomes. Interestingly, glands that are labeled with lectins in planarians are also labeled by similar lectins in Schistosoma mansoni females, suggesting a common evolutionary origin for these organs . The ability to visualize both male and female components of the planarian reproductive system with antibodies, lectins, and FISH as shown here will prove useful as we proceed with functional studies to identify genes involved in sexual development to gain insight into the mechanisms responsible for sex-specific development.
Using microarray analyses, we have identified >800 genes that are upregulated in the sexual vs. asexual planarian. A subset of these genes, validated through whole-mount in situ hybridization, provides markers of the planarian reproductive system. Their differential expression in differentiating germ cells will be useful for dissecting the spatial and temporal development of planarian reproductive organs. These transcriptomic data, combined with the ability to simultaneously label both male and female components of the planarian reproductive system through the markers identified in this study, will enable functional studies to dissect pathways and mechanisms that are involved in inductive germ cell specification, as well as sexual differentiation and development.
Sexual and asexual S. mediterranea were maintained as previously described [9, 37], and starved at least 1 week before use. For all in situ hydridization, immuno and lectin experiments, sexually mature animals were used.
Sequences for oligonucleotide arrays
Sequences from three S. mediterranea EST libraries (Alvarado et al. 2002, Zayas et al. 2005, unpublished ESTs in NCBI) were obtained from NCBI and assembled into 17,568 unique sequences using a variety of approaches (e.g. BLAST, Sequencher and CAP3, and hand curation). These sequence data were used to generate an oligonucleotide array that was submitted to Roche Nimblegen for probe creation. Of the 17,568 sequences submitted, 628 had no suitable regions for probe creation; 129 shared all their probes with 1 other sequence; 12 shared all their probes with 2+ other sequences; and 357 had suitable sequence available for less than 10 probes. After these considerations, the oligonucleotide array had probes representing 16,786 ESTs. Each gene was represented by 10 probe pairs with a mean length of 60 nucleotides.
RNA extraction and purification
Total RNA was extracted from equivalent weights of asexual and sexual animals using a modified Trizol protocol that included an optional spin after homogenization and isopropanol/high salt solution precipitation (Invitrogen). RNA was treated with RNase-free DNAse (Promega) using standard protocols, purified using an RNeasy Mini Spin kit (Qiagen), and quantified with both a Nanodrop ND-1000 spectrophotometer (Thermo Scientific) and an Agilent 2100 Bioanalyzer (Agilent Technologies). The purified RNA samples were sent to Roche Nimblegen for labeling and hybridization to custom oligonucleotide arrays.
Oligonucleotide array data analysis
Expression data were received from Roche Nimblegen Inc. and EST calls were generated using the Robust Multichip Average (RMA) algorithm [39, 40]. Z-scores were calculated to assess differential expression. Microarray data have been deposited in the Gene Expression Omnibus (Accession number GSE32450).
Riboprobe synthesis and whole-mount in situ hybridization
To generate riboprobes for in situ hybridization, clones from the hermaphroditic S. mediterranea EST Database  were used as templates. In vitro transcription reactions with T3 RNA polymerase were performed using standard approaches allowing the incorporation of digoxigenin-12-UTP (Roche), fluorescein-12-UTP (Roche), or dinitrophenol-11-UTP (PerkinElmer). Whole-mount in situ hybridization of mature sexual planarians was performed as previously described [8, 42]. In situ hybridizations were conducted either manually or with the Insitu ProVS (Intavis AG Bioanalytical Instruments). For FISH, following post-hybridization washes and blocking, animals were incubated in anti-digoxigenin peroxidase, (1:1000 [Roche]), anti-FITC peroxidase, (1:1000 [Roche]) and/or anti-dinitrophenol peroxidase, (1:100 [PerkinElmer]) overnight at 4°C. Samples were washed at least 6 times (20 min each), and developed with FITC- or Cy3-Tyramide (PerkinElmer) using the manufacturer's protocol. For double FISH, the peroxidase after the first tyramide development was quenched with 2% H2O2 in PBTX (1× PBS + 0.1% Triton X-100) for 1 hour before subsequent antibody incubation. Samples were mounted in Vectashield (Vector Laboratories) for imaging.
Whole-mount immunofluorescence and Fluorescent Lectin-Conjugate Staining
Planarians were killed with 2% HCl for 5 minutes on ice and fixed for 2 hours with either methacarn (methanol: chloroform: acetic acid [6:3:1]), or 4% formaldehyde in 1× PBS. Following formaldehyde fixation, animals were bleached in 6% H2O2 in 1× PBS for one or two nights depending on size/pigmentation. For methacarn fixation, animals were incubated in methanol (MeOH) for 1-2 hours before bleaching in 6% H2O2 in MeOH and then rehydrated in a 75, 50, 25% MeOH/PBTX (1× PBS + 0.3% Triton X-100) gradient. For both methacarn and formaldehyde fixed samples, animals were washed twice (five minutes each) with PBTX, and incubated in blocking buffer (0.6% IgG free BSA, 0.45% fish gelatin in PBTX) for 2-4 hours. Primary antibody incubation was performed overnight at 4°C at the following concentrations (dilutions in blocking buffer): carbonic anhydrase II human erythrocytes rabbit polyclonal antibody (1:100 [Chemicon International, AB1828]), phospho-tyrosine mouse monoclonal antibody (1:500 [Cell Signaling Technology, 9411]), anti-tubulin δ2 rabbit polyclonal antibody (1:200 [Millipore, AB3203]), rabbit pre-immune serum/anti-muscle (1:500 [generated by Francesc Cebrià and Tingxia Guo, ]). After at least six one-hour washes in PBTX, animals were incubated in secondary antibody (goat anti-rabbit Alexa 568, 1:1000 [Molecular Probes, Invitrogen, A11036]; goat anti-mouse Alexa 488, 1:400 [Molecular Probes, Invitrogen, A11029]) overnight at 4°C. For staining with lectins, samples were incubated overnight at 4°C with FITC- or rhodamine-conjugated lectins (1:500 [Vector Laboratories]) diluted from 2 mg/ml stocks in blocking buffer. All immuno and lectin samples were counterstained with DAPI. Animals were washed several times before they were mounted in Vectashield and flattened for imaging.
Samples were imaged with a Leica M205A stereomicroscope and DFC420 camera, a Zeiss Stereo Lumar V12 and/or a LSM 710 confocal microscope. Confocal microscopy was performed as previously described . Images were processed using either Zen 2008/9 (Carl Zeiss) or Adobe Photoshop CS4 (Adobe).
We thank James Sikes and Jim Collins for insightful discussion and helpful comments on the manuscript. We also thank Nina Hosmane for the initial work on carbonic anhydrase immunostaining. This work was supported by grants from NIH (R01 HD043403) and NSF (IOS-0744689) to PAN. PAN is an investigator of the Howard Hughes Medical Institute.
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