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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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Effects of reduced activity of TrkC on the neuronal differentiation of cortical cells.A - triple-labeling (DAPI/GFP/TUJ) of control (transfected with the control plasmid) and shRNA-TrkC3 transfected cortical progenitor cells cultured from opossums at age P7. Scale bars: 25 µm. B – graphs showing the percentage of neurons (cells stained with neuron-specific β3-tubulin) in cultured cells transfected with control shRNA, shRNA-TrkC1, shRNA-TrkC2 and shRNA-TrkC3 obtained from opossums at P1 (P1-2DIV) and P7 (P7-2DIV).
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pone-0074346-g009: Effects of reduced activity of TrkC on the neuronal differentiation of cortical cells.A - triple-labeling (DAPI/GFP/TUJ) of control (transfected with the control plasmid) and shRNA-TrkC3 transfected cortical progenitor cells cultured from opossums at age P7. Scale bars: 25 µm. B – graphs showing the percentage of neurons (cells stained with neuron-specific β3-tubulin) in cultured cells transfected with control shRNA, shRNA-TrkC1, shRNA-TrkC2 and shRNA-TrkC3 obtained from opossums at P1 (P1-2DIV) and P7 (P7-2DIV).

Mentions: To examine the effect of reduced TrkC receptor activity on the differentiation of isolated cortical cells, immunofluorescent staining using a neuron-specific marker, β3-tubulin (TUJ), was performed (Figure 9). The number of TUJ-positive neurons was approximately 30% in control cell cultures in P1 opossums, whereas this number decreased for cells transfected with shRNA-TrkC1 (Figure 9 B). In contrast, there was no effect on cell differentiation in cell cultures derived from P7 opossums (Figure 9 B).


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)

Effects of reduced activity of TrkC on the neuronal differentiation of cortical cells.A - triple-labeling (DAPI/GFP/TUJ) of control (transfected with the control plasmid) and shRNA-TrkC3 transfected cortical progenitor cells cultured from opossums at age P7. Scale bars: 25 µm. B – graphs showing the percentage of neurons (cells stained with neuron-specific β3-tubulin) in cultured cells transfected with control shRNA, shRNA-TrkC1, shRNA-TrkC2 and shRNA-TrkC3 obtained from opossums at P1 (P1-2DIV) and P7 (P7-2DIV).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0074346-g009: Effects of reduced activity of TrkC on the neuronal differentiation of cortical cells.A - triple-labeling (DAPI/GFP/TUJ) of control (transfected with the control plasmid) and shRNA-TrkC3 transfected cortical progenitor cells cultured from opossums at age P7. Scale bars: 25 µm. B – graphs showing the percentage of neurons (cells stained with neuron-specific β3-tubulin) in cultured cells transfected with control shRNA, shRNA-TrkC1, shRNA-TrkC2 and shRNA-TrkC3 obtained from opossums at P1 (P1-2DIV) and P7 (P7-2DIV).
Mentions: To examine the effect of reduced TrkC receptor activity on the differentiation of isolated cortical cells, immunofluorescent staining using a neuron-specific marker, β3-tubulin (TUJ), was performed (Figure 9). The number of TUJ-positive neurons was approximately 30% in control cell cultures in P1 opossums, whereas this number decreased for cells transfected with shRNA-TrkC1 (Figure 9 B). In contrast, there was no effect on cell differentiation in cell cultures derived from P7 opossums (Figure 9 B).

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