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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 TrkC receptor downregulation on cortical cells apoptosis.A – triple-labeling (DAPI/GFP/casp3) of cortical progenitor cells cultured from P1 opossums in control conditions (transfected with control shRNA) and cells transfected with shRNA-TrkC3. Scale bars: 100 µm. B – graphs showing the percentage of apoptotic cells (transfected with control shRNA, shRNA-TrkC1, shRNA-TrkC2 and shRNA-TrkC3) in cultures obtained from opossums at P1 (P1-2DIV) and P7 (P7-2DIV).
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pone-0074346-g008: Effects of TrkC receptor downregulation on cortical cells apoptosis.A – triple-labeling (DAPI/GFP/casp3) of cortical progenitor cells cultured from P1 opossums in control conditions (transfected with control shRNA) and cells transfected with shRNA-TrkC3. Scale bars: 100 µm. B – graphs showing the percentage of apoptotic cells (transfected with control shRNA, shRNA-TrkC1, shRNA-TrkC2 and shRNA-TrkC3) in cultures obtained from opossums at P1 (P1-2DIV) and P7 (P7-2DIV).

Mentions: Cell survival was determined using an antibody recognizing cleaved caspase-3, which is expressed in dying cells undergoing apoptotic death (Figure 8). In control conditions, approximately 10% of GFP-positive cells (i.e., transfected with the control plasmid) were apoptotic, while transfections using the shRNA-TrkC plasmid increased the number of apoptotic cells (Figure 8 B). The highest rate of apoptosis was found in cell cultures transfected with shRNA-TrkC3 (25%) and shRNA-TrkC2 (20%) constructs. Similarly, in cell cultures obtained from P7 animals, the percentage of dying GFP-positive cells in control conditions was decreased (approximately 10%), while inhibition of TrkC receptor activity increased the number of dying cells by two-fold (Figure 8 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 TrkC receptor downregulation on cortical cells apoptosis.A – triple-labeling (DAPI/GFP/casp3) of cortical progenitor cells cultured from P1 opossums in control conditions (transfected with control shRNA) and cells transfected with shRNA-TrkC3. Scale bars: 100 µm. B – graphs showing the percentage of apoptotic cells (transfected with control shRNA, shRNA-TrkC1, shRNA-TrkC2 and shRNA-TrkC3) in cultures 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-g008: Effects of TrkC receptor downregulation on cortical cells apoptosis.A – triple-labeling (DAPI/GFP/casp3) of cortical progenitor cells cultured from P1 opossums in control conditions (transfected with control shRNA) and cells transfected with shRNA-TrkC3. Scale bars: 100 µm. B – graphs showing the percentage of apoptotic cells (transfected with control shRNA, shRNA-TrkC1, shRNA-TrkC2 and shRNA-TrkC3) in cultures obtained from opossums at P1 (P1-2DIV) and P7 (P7-2DIV).
Mentions: Cell survival was determined using an antibody recognizing cleaved caspase-3, which is expressed in dying cells undergoing apoptotic death (Figure 8). In control conditions, approximately 10% of GFP-positive cells (i.e., transfected with the control plasmid) were apoptotic, while transfections using the shRNA-TrkC plasmid increased the number of apoptotic cells (Figure 8 B). The highest rate of apoptosis was found in cell cultures transfected with shRNA-TrkC3 (25%) and shRNA-TrkC2 (20%) constructs. Similarly, in cell cultures obtained from P7 animals, the percentage of dying GFP-positive cells in control conditions was decreased (approximately 10%), while inhibition of TrkC receptor activity increased the number of dying cells by two-fold (Figure 8 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