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Digital gene expression approach over multiple RNA-Seq data sets to detect neoblast transcriptional changes in Schmidtea mediterranea.

Rodríguez-Esteban G, González-Sastre A, Rojo-Laguna JI, Saló E, Abril JF - BMC Genomics (2015)

Bottom Line: These results are accessible via web for the community of researchers.DGE is a valuable tool for gene discovery, quantification and annotation.The application of DGE in S. mediterranea confirms the planarian stem cells or neoblasts as a complex population of pluripotent and multipotent cells regulated by a mixture of transcription factors and cancer-related genes.

View Article: PubMed Central - PubMed

Affiliation: Departament de Genètica, Facultat de Biologia, Universitat de Barcelona (UB), and Institut de Biomedicina de la Universitat de Barcelona (IBUB), Av. Diagonal 643, Barcelona, 08028, Catalonia, Spain. gresteban@scientist.com.

ABSTRACT

Background: The freshwater planarian Schmidtea mediterranea is recognised as a valuable model for research into adult stem cells and regeneration. With the advent of the high-throughput sequencing technologies, it has become feasible to undertake detailed transcriptional analysis of its unique stem cell population, the neoblasts. Nonetheless, a reliable reference for this type of studies is still lacking.

Results: Taking advantage of digital gene expression (DGE) sequencing technology we compare all the available transcriptomes for S. mediterranea and improve their annotation. These results are accessible via web for the community of researchers. Using the quantitative nature of DGE, we describe the transcriptional profile of neoblasts and present 42 new neoblast genes, including several cancer-related genes and transcription factors. Furthermore, we describe in detail the Smed-meis-like gene and the three Nuclear Factor Y subunits Smed-nf-YA, Smed-nf-YB-2 and Smed-nf-YC.

Conclusions: DGE is a valuable tool for gene discovery, quantification and annotation. The application of DGE in S. mediterranea confirms the planarian stem cells or neoblasts as a complex population of pluripotent and multipotent cells regulated by a mixture of transcription factors and cancer-related genes.

No MeSH data available.


Related in: MedlinePlus

Smed-meis-like is essential for anterior regeneration.A - WISH reveals that Smed-meis-like is expressed in the cephalic ganglia, the pharynx, the tip of the head (arrowhead) and the parenchyma, from where it is downregulated three days after irradiation. B - Double FISH of Smed-meis-like together with the neoblast marker Smed-h2b, shows that Smed-meis-like is expressed in neoblasts (arrowheads) as well as in differentiated cells (asterisk). DAPI labels the cell nuclei. See Additional file 11A for the separate channels of fluorescence. C - Smed-meis-like(RNAi) produce defects in anterior regeneration, which range from an squared head with elongated eyes, cyclops, to complete loss of anterior regeneration. The marker of brain branches Smed-gpas also shows this different penetrance. D - Double FISH with Smed-opsin and Smed-tph shows aberrant eyes in the less severe phenotype. E - The anterior markers Smed-notum, Smed-sfrp1, Smed-cintillo, and the eye progenitor marker Smed-ovo disappear after Smed-meis-like(RNAi), while the posterior marker Smed-wnt-1 remains. F - Quantification of mitotic cells by α-H3P immunohistochemistry in the whole animal (p < 0.001, t-test). All the experiments are done on bipolar regenerating trunks, at 11 days of regeneration after three rounds of injection.
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Fig6: Smed-meis-like is essential for anterior regeneration.A - WISH reveals that Smed-meis-like is expressed in the cephalic ganglia, the pharynx, the tip of the head (arrowhead) and the parenchyma, from where it is downregulated three days after irradiation. B - Double FISH of Smed-meis-like together with the neoblast marker Smed-h2b, shows that Smed-meis-like is expressed in neoblasts (arrowheads) as well as in differentiated cells (asterisk). DAPI labels the cell nuclei. See Additional file 11A for the separate channels of fluorescence. C - Smed-meis-like(RNAi) produce defects in anterior regeneration, which range from an squared head with elongated eyes, cyclops, to complete loss of anterior regeneration. The marker of brain branches Smed-gpas also shows this different penetrance. D - Double FISH with Smed-opsin and Smed-tph shows aberrant eyes in the less severe phenotype. E - The anterior markers Smed-notum, Smed-sfrp1, Smed-cintillo, and the eye progenitor marker Smed-ovo disappear after Smed-meis-like(RNAi), while the posterior marker Smed-wnt-1 remains. F - Quantification of mitotic cells by α-H3P immunohistochemistry in the whole animal (p < 0.001, t-test). All the experiments are done on bipolar regenerating trunks, at 11 days of regeneration after three rounds of injection.

Mentions: WISH on intact animals shows that it is expressed in the cephalic ganglia, the pharynx, the tip of the head, and the parenchyma (Figure 6A). The downregulation observed three days after irradiation suggests that the parenchyma-associated expression is related to neoblasts and early postmitotic cells. To corroborate this, a double fluorescence in situ hybridization (FISH) together with the neoblast marker Smed-h2b [59] has been carried out (Figure 6B and Additional file 11A). Confocal microscopy shows colocalization of both genes in some cells, which confirms the expression of Smed-meis-like in neoblasts and, thus, the DGE results. Nevertheless, not all Smed-meis-like positive cells are expressing Smed-h2b, reinforcing the idea that Smed-meis-like is not exclusive of neoblasts.Figure 6


Digital gene expression approach over multiple RNA-Seq data sets to detect neoblast transcriptional changes in Schmidtea mediterranea.

Rodríguez-Esteban G, González-Sastre A, Rojo-Laguna JI, Saló E, Abril JF - BMC Genomics (2015)

Smed-meis-like is essential for anterior regeneration.A - WISH reveals that Smed-meis-like is expressed in the cephalic ganglia, the pharynx, the tip of the head (arrowhead) and the parenchyma, from where it is downregulated three days after irradiation. B - Double FISH of Smed-meis-like together with the neoblast marker Smed-h2b, shows that Smed-meis-like is expressed in neoblasts (arrowheads) as well as in differentiated cells (asterisk). DAPI labels the cell nuclei. See Additional file 11A for the separate channels of fluorescence. C - Smed-meis-like(RNAi) produce defects in anterior regeneration, which range from an squared head with elongated eyes, cyclops, to complete loss of anterior regeneration. The marker of brain branches Smed-gpas also shows this different penetrance. D - Double FISH with Smed-opsin and Smed-tph shows aberrant eyes in the less severe phenotype. E - The anterior markers Smed-notum, Smed-sfrp1, Smed-cintillo, and the eye progenitor marker Smed-ovo disappear after Smed-meis-like(RNAi), while the posterior marker Smed-wnt-1 remains. F - Quantification of mitotic cells by α-H3P immunohistochemistry in the whole animal (p < 0.001, t-test). All the experiments are done on bipolar regenerating trunks, at 11 days of regeneration after three rounds of injection.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4494696&req=5

Fig6: Smed-meis-like is essential for anterior regeneration.A - WISH reveals that Smed-meis-like is expressed in the cephalic ganglia, the pharynx, the tip of the head (arrowhead) and the parenchyma, from where it is downregulated three days after irradiation. B - Double FISH of Smed-meis-like together with the neoblast marker Smed-h2b, shows that Smed-meis-like is expressed in neoblasts (arrowheads) as well as in differentiated cells (asterisk). DAPI labels the cell nuclei. See Additional file 11A for the separate channels of fluorescence. C - Smed-meis-like(RNAi) produce defects in anterior regeneration, which range from an squared head with elongated eyes, cyclops, to complete loss of anterior regeneration. The marker of brain branches Smed-gpas also shows this different penetrance. D - Double FISH with Smed-opsin and Smed-tph shows aberrant eyes in the less severe phenotype. E - The anterior markers Smed-notum, Smed-sfrp1, Smed-cintillo, and the eye progenitor marker Smed-ovo disappear after Smed-meis-like(RNAi), while the posterior marker Smed-wnt-1 remains. F - Quantification of mitotic cells by α-H3P immunohistochemistry in the whole animal (p < 0.001, t-test). All the experiments are done on bipolar regenerating trunks, at 11 days of regeneration after three rounds of injection.
Mentions: WISH on intact animals shows that it is expressed in the cephalic ganglia, the pharynx, the tip of the head, and the parenchyma (Figure 6A). The downregulation observed three days after irradiation suggests that the parenchyma-associated expression is related to neoblasts and early postmitotic cells. To corroborate this, a double fluorescence in situ hybridization (FISH) together with the neoblast marker Smed-h2b [59] has been carried out (Figure 6B and Additional file 11A). Confocal microscopy shows colocalization of both genes in some cells, which confirms the expression of Smed-meis-like in neoblasts and, thus, the DGE results. Nevertheless, not all Smed-meis-like positive cells are expressing Smed-h2b, reinforcing the idea that Smed-meis-like is not exclusive of neoblasts.Figure 6

Bottom Line: These results are accessible via web for the community of researchers.DGE is a valuable tool for gene discovery, quantification and annotation.The application of DGE in S. mediterranea confirms the planarian stem cells or neoblasts as a complex population of pluripotent and multipotent cells regulated by a mixture of transcription factors and cancer-related genes.

View Article: PubMed Central - PubMed

Affiliation: Departament de Genètica, Facultat de Biologia, Universitat de Barcelona (UB), and Institut de Biomedicina de la Universitat de Barcelona (IBUB), Av. Diagonal 643, Barcelona, 08028, Catalonia, Spain. gresteban@scientist.com.

ABSTRACT

Background: The freshwater planarian Schmidtea mediterranea is recognised as a valuable model for research into adult stem cells and regeneration. With the advent of the high-throughput sequencing technologies, it has become feasible to undertake detailed transcriptional analysis of its unique stem cell population, the neoblasts. Nonetheless, a reliable reference for this type of studies is still lacking.

Results: Taking advantage of digital gene expression (DGE) sequencing technology we compare all the available transcriptomes for S. mediterranea and improve their annotation. These results are accessible via web for the community of researchers. Using the quantitative nature of DGE, we describe the transcriptional profile of neoblasts and present 42 new neoblast genes, including several cancer-related genes and transcription factors. Furthermore, we describe in detail the Smed-meis-like gene and the three Nuclear Factor Y subunits Smed-nf-YA, Smed-nf-YB-2 and Smed-nf-YC.

Conclusions: DGE is a valuable tool for gene discovery, quantification and annotation. The application of DGE in S. mediterranea confirms the planarian stem cells or neoblasts as a complex population of pluripotent and multipotent cells regulated by a mixture of transcription factors and cancer-related genes.

No MeSH data available.


Related in: MedlinePlus