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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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Expression of TrkC receptors in the developing opossum brain.A - Western blotting analysis was performed in opossum brain homogenates at different ages (P1, P3, P7, P12, P20, P35, P60 and adult). GAPDH was used as a loading control. The arrows indicate the positions of the full-length (TrkC-F) and truncated (TrkC-T) TrkC receptors. B - graph illustrates the mean amount of TrkC protein from three different animals at each age for both full-length (black bars) and truncated forms (gray bars) of TrkC receptors. Error bars indicate ± S.E.M. Note that the highest level of TrkC expression was observed at P35 and P60. * indicates P<0.05; ** indicate P<0.01; *** indicate P<0.001 for all figures.
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pone-0074346-g001: Expression of TrkC receptors in the developing opossum brain.A - Western blotting analysis was performed in opossum brain homogenates at different ages (P1, P3, P7, P12, P20, P35, P60 and adult). GAPDH was used as a loading control. The arrows indicate the positions of the full-length (TrkC-F) and truncated (TrkC-T) TrkC receptors. B - graph illustrates the mean amount of TrkC protein from three different animals at each age for both full-length (black bars) and truncated forms (gray bars) of TrkC receptors. Error bars indicate ± S.E.M. Note that the highest level of TrkC expression was observed at P35 and P60. * indicates P<0.05; ** indicate P<0.01; *** indicate P<0.001 for all figures.

Mentions: To detect TrkC protein in the opossum brain, three different antibodies were used. However, two of the antibodies were not specific to the opossum brain. Western blotting analysis was performed using whole brain lysates. Although we detected several labeled bands on the immunoblots using TrkC (H-300) and TrkC (798) antibodies, none of these proteins recognized the 145-kDa neurotrophic TrkC receptor protein. However, the TrkC (C44H5) antibody revealed the expression of one of the truncated forms and the full-length form of the TrkC receptor in whole brain lysates (Figure 1 A). The full-length form of the TrkC receptor (TrkC-F, fully glycosylated protein) exhibits a molecular mass of approx. 145 kDa. Full-length TrkC was slightly expressed in newborn animals and its expression significantly increased by the end of the first week of life. Its levels continued to increase until day 20 and remained unchanged thereafter (Figure 1 B). One-way ANOVA showed that there was a statistically significant difference between groups (F7,23 = 9.89 p<0.001). Holm-Sidak multiple comparisons also showed that there were significant differences between P1 and P12; P1 and P20, P35, P60 and adult; P3 and P35; and P7 and P35 animal groups. The same antibody detected another band with a molecular weight of 95 kDa, which corresponded to one of the truncated forms of the TrkC (TrkC-T) receptor. Its low-level expression was detected in newborn opossum brains and then gradually increased until P35. The highest expression level of this truncated protein was observed at P35, the stage when the opossum starts to open its eyes. The truncated protein expression decreased slightly in later development and remained at a stable level in adult animals (Figure 1 A, B). Statistically significant differences were also observed between groups (F7,23 = 32.4 p<0.001).


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)

Expression of TrkC receptors in the developing opossum brain.A - Western blotting analysis was performed in opossum brain homogenates at different ages (P1, P3, P7, P12, P20, P35, P60 and adult). GAPDH was used as a loading control. The arrows indicate the positions of the full-length (TrkC-F) and truncated (TrkC-T) TrkC receptors. B - graph illustrates the mean amount of TrkC protein from three different animals at each age for both full-length (black bars) and truncated forms (gray bars) of TrkC receptors. Error bars indicate ± S.E.M. Note that the highest level of TrkC expression was observed at P35 and P60. * indicates P<0.05; ** indicate P<0.01; *** indicate P<0.001 for all figures.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3760877&req=5

pone-0074346-g001: Expression of TrkC receptors in the developing opossum brain.A - Western blotting analysis was performed in opossum brain homogenates at different ages (P1, P3, P7, P12, P20, P35, P60 and adult). GAPDH was used as a loading control. The arrows indicate the positions of the full-length (TrkC-F) and truncated (TrkC-T) TrkC receptors. B - graph illustrates the mean amount of TrkC protein from three different animals at each age for both full-length (black bars) and truncated forms (gray bars) of TrkC receptors. Error bars indicate ± S.E.M. Note that the highest level of TrkC expression was observed at P35 and P60. * indicates P<0.05; ** indicate P<0.01; *** indicate P<0.001 for all figures.
Mentions: To detect TrkC protein in the opossum brain, three different antibodies were used. However, two of the antibodies were not specific to the opossum brain. Western blotting analysis was performed using whole brain lysates. Although we detected several labeled bands on the immunoblots using TrkC (H-300) and TrkC (798) antibodies, none of these proteins recognized the 145-kDa neurotrophic TrkC receptor protein. However, the TrkC (C44H5) antibody revealed the expression of one of the truncated forms and the full-length form of the TrkC receptor in whole brain lysates (Figure 1 A). The full-length form of the TrkC receptor (TrkC-F, fully glycosylated protein) exhibits a molecular mass of approx. 145 kDa. Full-length TrkC was slightly expressed in newborn animals and its expression significantly increased by the end of the first week of life. Its levels continued to increase until day 20 and remained unchanged thereafter (Figure 1 B). One-way ANOVA showed that there was a statistically significant difference between groups (F7,23 = 9.89 p<0.001). Holm-Sidak multiple comparisons also showed that there were significant differences between P1 and P12; P1 and P20, P35, P60 and adult; P3 and P35; and P7 and P35 animal groups. The same antibody detected another band with a molecular weight of 95 kDa, which corresponded to one of the truncated forms of the TrkC (TrkC-T) receptor. Its low-level expression was detected in newborn opossum brains and then gradually increased until P35. The highest expression level of this truncated protein was observed at P35, the stage when the opossum starts to open its eyes. The truncated protein expression decreased slightly in later development and remained at a stable level in adult animals (Figure 1 A, B). Statistically significant differences were also observed between groups (F7,23 = 32.4 p<0.001).

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