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Complementarity of medium-throughput in situ RNA hybridization and tissue-specific transcriptomics: case study of Arabidopsis seed development kinetics.

Francoz E, Ranocha P, Pernot C, Le Ru A, Pacquit V, Dunand C, Burlat V - Sci Rep (2016)

Bottom Line: Integration of results illustrates the complementarity of both datasets.Moreover, depending on the tissues and the developmental stages considered, one or the other technique appears more sensitive than the other.For each tissue/developmental stage, we finally determined tissue-specific transcriptomic threshold values compatible with the spatiotemporally-specific detection limits of ISH for lists of hundreds to tens-of-thousands of genes.

View Article: PubMed Central - PubMed

Affiliation: Laboratoire de Recherche en Sciences Végétales, Université de Toulouse, CNRS, UPS, 24 chemin de Borde Rouge, Auzeville, BP42617, 31326 Castanet Tolosan, France.

ABSTRACT
The rationale of this study is to compare and integrate two heterologous datasets intended to unravel the spatiotemporal specificities of gene expression in a rapidly growing and complex organ. We implemented medium-throughput RNA in situ hybridization (ISH) for 39 genes mainly corresponding to cell wall proteins for which we have particular interest, selected (i) on their sequence identity (24 class III peroxidase multigenic family members and 15 additional genes used as positive controls) and (ii) on their expression levels in a publicly available Arabidopsis thaliana seed tissue-specific transcriptomics study. The specificity of the hybridization signals was carefully studied, and ISH results obtained for the 39 selected genes were systematically compared with tissue-specific transcriptomics for 5 seed developmental stages. Integration of results illustrates the complementarity of both datasets. The tissue-specific transcriptomics provides high-throughput possibilities whereas ISH provides high spatial resolution. Moreover, depending on the tissues and the developmental stages considered, one or the other technique appears more sensitive than the other. For each tissue/developmental stage, we finally determined tissue-specific transcriptomic threshold values compatible with the spatiotemporally-specific detection limits of ISH for lists of hundreds to tens-of-thousands of genes.

No MeSH data available.


Related in: MedlinePlus

Illustration of the in situ hybridization results obtained for a selection of genes and developmental stages.Results for 10 AtPRXs (a–j) and for 10 non peroxidase genes (k–t) are displayed in the decreasing order of their maximal individual tissue-specific transcriptomic expression values7. For each gene and each developmental stage, a screen copy of the seed eFP browser tissue-specific transcriptomic map including the individual absolute heatmap scale (red-to-yellow colour codes correspond to high-to-low expression values) that is different for each gene12 is compared with the corresponding ISH results (for both the antisense and sense probes). The final corresponding new ISH map (re-coloured after original drawings from ref. 7 available at Seedgenenetwork http://estdb.biology.ucla.edu/seed/) gave increased cellular resolution with a unique colour code for all genes (red corresponding to visually detected strong signals, orange corresponding to moderate signals and white corresponding to the absence of detected ISH signal). Note that the naturally brown colour of seed coat does not correspond to ISH signal. The full ISH results corresponding to a selection of 5 developmental stages for all 39 genes can be found in Supplementary Figs. S4–S42. Scale bars: 100 μm. Abbreviations: AS, anti-sense; CZEN, chalazal endosperm; CZSC, chalazal seed coat; EP, embryo proper; MEN, micropylar endosperm; PEN, peripheral endosperm; S, sense; SC, seed coat; SUS, suspensor.
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f2: Illustration of the in situ hybridization results obtained for a selection of genes and developmental stages.Results for 10 AtPRXs (a–j) and for 10 non peroxidase genes (k–t) are displayed in the decreasing order of their maximal individual tissue-specific transcriptomic expression values7. For each gene and each developmental stage, a screen copy of the seed eFP browser tissue-specific transcriptomic map including the individual absolute heatmap scale (red-to-yellow colour codes correspond to high-to-low expression values) that is different for each gene12 is compared with the corresponding ISH results (for both the antisense and sense probes). The final corresponding new ISH map (re-coloured after original drawings from ref. 7 available at Seedgenenetwork http://estdb.biology.ucla.edu/seed/) gave increased cellular resolution with a unique colour code for all genes (red corresponding to visually detected strong signals, orange corresponding to moderate signals and white corresponding to the absence of detected ISH signal). Note that the naturally brown colour of seed coat does not correspond to ISH signal. The full ISH results corresponding to a selection of 5 developmental stages for all 39 genes can be found in Supplementary Figs. S4–S42. Scale bars: 100 μm. Abbreviations: AS, anti-sense; CZEN, chalazal endosperm; CZSC, chalazal seed coat; EP, embryo proper; MEN, micropylar endosperm; PEN, peripheral endosperm; S, sense; SC, seed coat; SUS, suspensor.

Mentions: The basis of this protocol comes from41 following several adaptations404243 and final simplification using a unique buffer for most of the steps and substituting the acetic anhydre/triethanolamine tedious charge equilibration step by a more simple diethylpyrocarbonate (DEPC)-mediated carbethoxylation step, inspired by16. 10 μm-serial sections of tissue microarrays were disposed on precoated microscopy slides. 40 slides, corresponding to 40 riboprobes were processed in one ISH experiment. Hybridization was performed overnight at 50 °C, and following stringent washing steps, hybridized probes were immunodetected using anti-Dig-alkaline phosphatase (AP) antibody (Roche) and colour development of AP reaction was performed overnight at room temperature. Step-by-step detailed protocol is provided with timing in Supplementary Methods. Slides were mounted in Eukitt and scanned at high-throughput using a nanozoomer HT slide scanner (Hamamatsu). We routinely scanned all slides at ×20 focus (=0.46 μm per pixel on a single z plan) and these scans were viewed and analysed using the NDP view freeware (Hamamatsu) and directly used to prepare Figures (Fig. 2; Supplementary Figs. S4–S45).


Complementarity of medium-throughput in situ RNA hybridization and tissue-specific transcriptomics: case study of Arabidopsis seed development kinetics.

Francoz E, Ranocha P, Pernot C, Le Ru A, Pacquit V, Dunand C, Burlat V - Sci Rep (2016)

Illustration of the in situ hybridization results obtained for a selection of genes and developmental stages.Results for 10 AtPRXs (a–j) and for 10 non peroxidase genes (k–t) are displayed in the decreasing order of their maximal individual tissue-specific transcriptomic expression values7. For each gene and each developmental stage, a screen copy of the seed eFP browser tissue-specific transcriptomic map including the individual absolute heatmap scale (red-to-yellow colour codes correspond to high-to-low expression values) that is different for each gene12 is compared with the corresponding ISH results (for both the antisense and sense probes). The final corresponding new ISH map (re-coloured after original drawings from ref. 7 available at Seedgenenetwork http://estdb.biology.ucla.edu/seed/) gave increased cellular resolution with a unique colour code for all genes (red corresponding to visually detected strong signals, orange corresponding to moderate signals and white corresponding to the absence of detected ISH signal). Note that the naturally brown colour of seed coat does not correspond to ISH signal. The full ISH results corresponding to a selection of 5 developmental stages for all 39 genes can be found in Supplementary Figs. S4–S42. Scale bars: 100 μm. Abbreviations: AS, anti-sense; CZEN, chalazal endosperm; CZSC, chalazal seed coat; EP, embryo proper; MEN, micropylar endosperm; PEN, peripheral endosperm; S, sense; SC, seed coat; SUS, suspensor.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4837347&req=5

f2: Illustration of the in situ hybridization results obtained for a selection of genes and developmental stages.Results for 10 AtPRXs (a–j) and for 10 non peroxidase genes (k–t) are displayed in the decreasing order of their maximal individual tissue-specific transcriptomic expression values7. For each gene and each developmental stage, a screen copy of the seed eFP browser tissue-specific transcriptomic map including the individual absolute heatmap scale (red-to-yellow colour codes correspond to high-to-low expression values) that is different for each gene12 is compared with the corresponding ISH results (for both the antisense and sense probes). The final corresponding new ISH map (re-coloured after original drawings from ref. 7 available at Seedgenenetwork http://estdb.biology.ucla.edu/seed/) gave increased cellular resolution with a unique colour code for all genes (red corresponding to visually detected strong signals, orange corresponding to moderate signals and white corresponding to the absence of detected ISH signal). Note that the naturally brown colour of seed coat does not correspond to ISH signal. The full ISH results corresponding to a selection of 5 developmental stages for all 39 genes can be found in Supplementary Figs. S4–S42. Scale bars: 100 μm. Abbreviations: AS, anti-sense; CZEN, chalazal endosperm; CZSC, chalazal seed coat; EP, embryo proper; MEN, micropylar endosperm; PEN, peripheral endosperm; S, sense; SC, seed coat; SUS, suspensor.
Mentions: The basis of this protocol comes from41 following several adaptations404243 and final simplification using a unique buffer for most of the steps and substituting the acetic anhydre/triethanolamine tedious charge equilibration step by a more simple diethylpyrocarbonate (DEPC)-mediated carbethoxylation step, inspired by16. 10 μm-serial sections of tissue microarrays were disposed on precoated microscopy slides. 40 slides, corresponding to 40 riboprobes were processed in one ISH experiment. Hybridization was performed overnight at 50 °C, and following stringent washing steps, hybridized probes were immunodetected using anti-Dig-alkaline phosphatase (AP) antibody (Roche) and colour development of AP reaction was performed overnight at room temperature. Step-by-step detailed protocol is provided with timing in Supplementary Methods. Slides were mounted in Eukitt and scanned at high-throughput using a nanozoomer HT slide scanner (Hamamatsu). We routinely scanned all slides at ×20 focus (=0.46 μm per pixel on a single z plan) and these scans were viewed and analysed using the NDP view freeware (Hamamatsu) and directly used to prepare Figures (Fig. 2; Supplementary Figs. S4–S45).

Bottom Line: Integration of results illustrates the complementarity of both datasets.Moreover, depending on the tissues and the developmental stages considered, one or the other technique appears more sensitive than the other.For each tissue/developmental stage, we finally determined tissue-specific transcriptomic threshold values compatible with the spatiotemporally-specific detection limits of ISH for lists of hundreds to tens-of-thousands of genes.

View Article: PubMed Central - PubMed

Affiliation: Laboratoire de Recherche en Sciences Végétales, Université de Toulouse, CNRS, UPS, 24 chemin de Borde Rouge, Auzeville, BP42617, 31326 Castanet Tolosan, France.

ABSTRACT
The rationale of this study is to compare and integrate two heterologous datasets intended to unravel the spatiotemporal specificities of gene expression in a rapidly growing and complex organ. We implemented medium-throughput RNA in situ hybridization (ISH) for 39 genes mainly corresponding to cell wall proteins for which we have particular interest, selected (i) on their sequence identity (24 class III peroxidase multigenic family members and 15 additional genes used as positive controls) and (ii) on their expression levels in a publicly available Arabidopsis thaliana seed tissue-specific transcriptomics study. The specificity of the hybridization signals was carefully studied, and ISH results obtained for the 39 selected genes were systematically compared with tissue-specific transcriptomics for 5 seed developmental stages. Integration of results illustrates the complementarity of both datasets. The tissue-specific transcriptomics provides high-throughput possibilities whereas ISH provides high spatial resolution. Moreover, depending on the tissues and the developmental stages considered, one or the other technique appears more sensitive than the other. For each tissue/developmental stage, we finally determined tissue-specific transcriptomic threshold values compatible with the spatiotemporally-specific detection limits of ISH for lists of hundreds to tens-of-thousands of genes.

No MeSH data available.


Related in: MedlinePlus