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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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Phenotype of cortical cells expressing TrkC receptors.Low-magnification (A–D) and high-magnification (E–P) confocal images showing triple immunofluorescence staining of cortical sections. Immunostaining was performed with DAPI (blue) to label the cell nucleus, TrkC receptors with an antibody against TrkC (red) and neurons (G, H) with NeuN antibody (green), or oligodendrocytes (K, L) with Olig2 (green), or astrocytes (O, P) using an antibody against GFAP (green).
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pone-0074346-g005: Phenotype of cortical cells expressing TrkC receptors.Low-magnification (A–D) and high-magnification (E–P) confocal images showing triple immunofluorescence staining of cortical sections. Immunostaining was performed with DAPI (blue) to label the cell nucleus, TrkC receptors with an antibody against TrkC (red) and neurons (G, H) with NeuN antibody (green), or oligodendrocytes (K, L) with Olig2 (green), or astrocytes (O, P) using an antibody against GFAP (green).

Mentions: To determine the phenotype of cortical cells expressing TrkC receptors, triple immunofluorescence staining was performed and high-magnification confocal images were obtained (Figure 5). We analyzed TrkC-expressing cells in the opossum cerebral cortex that were injected with BrdU at P3, P7 and P12. A total of 250-270 TrkC-expressing cells per animal were quantified in two specific regions (the anterior and caudal part of the cerebral cortex). In the neocortex, nearly all (98%) of the TrkC-labeled cells prominently expressed NeuN and demonstrated a neuronal phenotype (Figure 5 E–H). Moreover, glial cells also expressed TrkC receptors. In addition, the number of double-labeled cells was very low in the cerebral cortex. Approximately 1% of cells (for example, 2 cells from 250 TrkC cells in one case) were co-labeled with anti-GFAP, a marker for astrocytes, TrkC receptors (Figure 5 M–P). Cells labeled using the oligodendrocyte marker Olig2 were often located very close to the soma of the TrkC neurons but were never colocalized in the same cell (Figure 5 I–L).


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)

Phenotype of cortical cells expressing TrkC receptors.Low-magnification (A–D) and high-magnification (E–P) confocal images showing triple immunofluorescence staining of cortical sections. Immunostaining was performed with DAPI (blue) to label the cell nucleus, TrkC receptors with an antibody against TrkC (red) and neurons (G, H) with NeuN antibody (green), or oligodendrocytes (K, L) with Olig2 (green), or astrocytes (O, P) using an antibody against GFAP (green).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0074346-g005: Phenotype of cortical cells expressing TrkC receptors.Low-magnification (A–D) and high-magnification (E–P) confocal images showing triple immunofluorescence staining of cortical sections. Immunostaining was performed with DAPI (blue) to label the cell nucleus, TrkC receptors with an antibody against TrkC (red) and neurons (G, H) with NeuN antibody (green), or oligodendrocytes (K, L) with Olig2 (green), or astrocytes (O, P) using an antibody against GFAP (green).
Mentions: To determine the phenotype of cortical cells expressing TrkC receptors, triple immunofluorescence staining was performed and high-magnification confocal images were obtained (Figure 5). We analyzed TrkC-expressing cells in the opossum cerebral cortex that were injected with BrdU at P3, P7 and P12. A total of 250-270 TrkC-expressing cells per animal were quantified in two specific regions (the anterior and caudal part of the cerebral cortex). In the neocortex, nearly all (98%) of the TrkC-labeled cells prominently expressed NeuN and demonstrated a neuronal phenotype (Figure 5 E–H). Moreover, glial cells also expressed TrkC receptors. In addition, the number of double-labeled cells was very low in the cerebral cortex. Approximately 1% of cells (for example, 2 cells from 250 TrkC cells in one case) were co-labeled with anti-GFAP, a marker for astrocytes, TrkC receptors (Figure 5 M–P). Cells labeled using the oligodendrocyte marker Olig2 were often located very close to the soma of the TrkC neurons but were never colocalized in the same cell (Figure 5 I–L).

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