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Expression of TrkC receptors in the developing brain of the Monodelphis opossum and its effect on the development of cortical cells.

Bartkowska K, Gajerska M, Turlejski K, Djavadian RL - PLoS ONE (2013)

Bottom Line: We found that in different neocortical areas located both at the rostral and caudal regions of the cortex, up to 98% of BrdU-labeled cells forming cortical layers (II-VI) had prominently expressed TrkC.The shRNAs were individually tested in transfected cortical progenitor cells grown on culture plates for 2 days.The effects of the shRNA-TrkC constructs were similar: blockade of TrkC receptors decreased the number of Ki67-positive and apoptotic cells, and it did not change the number of TUJ-positive neurons in vitro.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cellular Neurobiology, Nencki Institute of Experimental Biology, Warsaw, Poland.

ABSTRACT
In this study, we investigated the distribution, localization and several various functions of TrkC receptors during development of the Monodelphisopossum brain. Western blotting analysis showed that two different forms of the TrkC receptor, the full-length receptor and one of its truncated forms, are abundantly expressed in the opossum brain. The expression of TrkC receptors was barely detected in the brain of newborn opossums. At postnatal day (P) 3, the expression of full-length TrkC remained at low levels, while moderate expression of the TrkC truncated form was detected. The expression levels of both forms of this protein gradually increased throughout development, peaking at P35. We found that in different neocortical areas located both at the rostral and caudal regions of the cortex, up to 98% of BrdU-labeled cells forming cortical layers (II-VI) had prominently expressed TrkC. To assess which developmental processes of cortical cells are regulated by TrkC receptors, three different shRNAs were constructed. The shRNAs were individually tested in transfected cortical progenitor cells grown on culture plates for 2 days. The effects of the shRNA-TrkC constructs were similar: blockade of TrkC receptors decreased the number of Ki67-positive and apoptotic cells, and it did not change the number of TUJ-positive neurons in vitro. Thus, the lack of TrkC receptors in cultured progenitor cells provided insight on the potential role of these receptors in the regulation of proliferation and cell survival but not in the differentiation of cortical cells.

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Effect of TrkC receptor downregulation on cortical cell proliferation.A –triple-labeling (DAPI/GFP/Ki-67) of cortical progenitor cells cultured from P1 opossums in control conditions (transfected with control shRNA) and cells transfected with shRNA-TrkC1. Scale bars: 30 µm. B – graphs showing the percentage of dividing cortical cells in control (transfected with control shRNA) and transfected (shRNA-TrkC1, shRNA-TrkC2 and shRNA-TrkC3) cultures obtained from opossums at P1 (P1-2DIV) and P7 (P7-2DIV).
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pone-0074346-g007: Effect of TrkC receptor downregulation on cortical cell proliferation.A –triple-labeling (DAPI/GFP/Ki-67) of cortical progenitor cells cultured from P1 opossums in control conditions (transfected with control shRNA) and cells transfected with shRNA-TrkC1. Scale bars: 30 µm. B – graphs showing the percentage of dividing cortical cells in control (transfected with control shRNA) and transfected (shRNA-TrkC1, shRNA-TrkC2 and shRNA-TrkC3) cultures obtained from opossums at P1 (P1-2DIV) and P7 (P7-2DIV).

Mentions: The effect of TrkC receptor activity on the proliferation of cortical progenitors was examined using an antibody against the Ki-67 protein, which is present only in the nuclei of dividing cells (Figure 7 A). In control conditions, the percentage of dividing cells in cultures isolated at P1 was approximately 50% (Figure 7 B). Blockade of the endogenous TrkC receptor using shRNA-TrkC1, shRNA-TrkC2 or shRNA-TrkC3 decreased the rate of proliferation by approximately 30% (Figure 7 B). In cell cultures obtained from older animals (P7), the inhibition of TrkC receptors also decreased the rate of cell proliferation (Figure 7 B). One-way ANOVA showed a significant difference between groups (F3,11=10.24 P<0.004). All pairwise comparisons using the Holm-Sidak method showed significant differences in the number of Ki-67-labeled cells between cultures transfected with the control plasmid and those transfected with either the TrkC1, TrkC2 or TrkC3 plasmids.


Expression of TrkC receptors in the developing brain of the Monodelphis opossum and its effect on the development of cortical cells.

Bartkowska K, Gajerska M, Turlejski K, Djavadian RL - PLoS ONE (2013)

Effect of TrkC receptor downregulation on cortical cell proliferation.A –triple-labeling (DAPI/GFP/Ki-67) of cortical progenitor cells cultured from P1 opossums in control conditions (transfected with control shRNA) and cells transfected with shRNA-TrkC1. Scale bars: 30 µm. B – graphs showing the percentage of dividing cortical cells in control (transfected with control shRNA) and transfected (shRNA-TrkC1, shRNA-TrkC2 and shRNA-TrkC3) cultures obtained from opossums at P1 (P1-2DIV) and P7 (P7-2DIV).
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3760877&req=5

pone-0074346-g007: Effect of TrkC receptor downregulation on cortical cell proliferation.A –triple-labeling (DAPI/GFP/Ki-67) of cortical progenitor cells cultured from P1 opossums in control conditions (transfected with control shRNA) and cells transfected with shRNA-TrkC1. Scale bars: 30 µm. B – graphs showing the percentage of dividing cortical cells in control (transfected with control shRNA) and transfected (shRNA-TrkC1, shRNA-TrkC2 and shRNA-TrkC3) cultures obtained from opossums at P1 (P1-2DIV) and P7 (P7-2DIV).
Mentions: The effect of TrkC receptor activity on the proliferation of cortical progenitors was examined using an antibody against the Ki-67 protein, which is present only in the nuclei of dividing cells (Figure 7 A). In control conditions, the percentage of dividing cells in cultures isolated at P1 was approximately 50% (Figure 7 B). Blockade of the endogenous TrkC receptor using shRNA-TrkC1, shRNA-TrkC2 or shRNA-TrkC3 decreased the rate of proliferation by approximately 30% (Figure 7 B). In cell cultures obtained from older animals (P7), the inhibition of TrkC receptors also decreased the rate of cell proliferation (Figure 7 B). One-way ANOVA showed a significant difference between groups (F3,11=10.24 P<0.004). All pairwise comparisons using the Holm-Sidak method showed significant differences in the number of Ki-67-labeled cells between cultures transfected with the control plasmid and those transfected with either the TrkC1, TrkC2 or TrkC3 plasmids.

Bottom Line: We found that in different neocortical areas located both at the rostral and caudal regions of the cortex, up to 98% of BrdU-labeled cells forming cortical layers (II-VI) had prominently expressed TrkC.The shRNAs were individually tested in transfected cortical progenitor cells grown on culture plates for 2 days.The effects of the shRNA-TrkC constructs were similar: blockade of TrkC receptors decreased the number of Ki67-positive and apoptotic cells, and it did not change the number of TUJ-positive neurons in vitro.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cellular Neurobiology, Nencki Institute of Experimental Biology, Warsaw, Poland.

ABSTRACT
In this study, we investigated the distribution, localization and several various functions of TrkC receptors during development of the Monodelphisopossum brain. Western blotting analysis showed that two different forms of the TrkC receptor, the full-length receptor and one of its truncated forms, are abundantly expressed in the opossum brain. The expression of TrkC receptors was barely detected in the brain of newborn opossums. At postnatal day (P) 3, the expression of full-length TrkC remained at low levels, while moderate expression of the TrkC truncated form was detected. The expression levels of both forms of this protein gradually increased throughout development, peaking at P35. We found that in different neocortical areas located both at the rostral and caudal regions of the cortex, up to 98% of BrdU-labeled cells forming cortical layers (II-VI) had prominently expressed TrkC. To assess which developmental processes of cortical cells are regulated by TrkC receptors, three different shRNAs were constructed. The shRNAs were individually tested in transfected cortical progenitor cells grown on culture plates for 2 days. The effects of the shRNA-TrkC constructs were similar: blockade of TrkC receptors decreased the number of Ki67-positive and apoptotic cells, and it did not change the number of TUJ-positive neurons in vitro. Thus, the lack of TrkC receptors in cultured progenitor cells provided insight on the potential role of these receptors in the regulation of proliferation and cell survival but not in the differentiation of cortical cells.

Show MeSH
Related in: MedlinePlus