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Spatiotemporal analyses of neural lineages after embryonic and postnatal progenitor targeting combining different reporters.

Figueres-Oñate M, García-Marqués J, Pedraza M, De Carlos JA, López-Mascaraque L - Front Neurosci (2015)

Bottom Line: To address this issue, we performed postnatal and in utero co-electroporations of different fluorescent reporters to label, in both cerebral cortex and olfactory bulb, the progeny of subventricular zone neural progenitors.Further, while neuronal lineages arise from many progenitors in proliferative zones after few divisions, glial lineages come from fewer progenitors that accomplish many cell divisions.Together, these data provide a useful guide to select a strategy to track the cell fate of a specific cell population and to address whether a different proliferative origin might be correlated with functional heterogeneity.

View Article: PubMed Central - PubMed

Affiliation: Instituto Cajal-Consejo Superior de Investigaciones Científicas, Department of Molecular, Cellular and Developmental Neurobiology Madrid, Spain.

ABSTRACT
Genetic lineage tracing with electroporation is one of the most powerful techniques to target neural progenitor cells and their progeny. However, the spatiotemporal relationship between neural progenitors and their final phenotype remain poorly understood. One critical factor to analyze the cell fate of progeny is reporter integration into the genome of transfected cells. To address this issue, we performed postnatal and in utero co-electroporations of different fluorescent reporters to label, in both cerebral cortex and olfactory bulb, the progeny of subventricular zone neural progenitors. By comparing fluorescent reporter expression in the adult cell progeny, we show a differential expression pattern within the same cell lineage, depending on electroporation stage and cell identity. Further, while neuronal lineages arise from many progenitors in proliferative zones after few divisions, glial lineages come from fewer progenitors that accomplish many cell divisions. Together, these data provide a useful guide to select a strategy to track the cell fate of a specific cell population and to address whether a different proliferative origin might be correlated with functional heterogeneity.

No MeSH data available.


Olfactory bulb interneurons after postnatal electroporation (P1). (A–D) P6 transfected cells expressed the integrated and non-integrated constructs interchangeably. (B) In the SVZ, transfected red and green cells showed glial morphology. (C) In the rostral migratory stream large cells labeled were directed to the olfactory bulb. (D) OB displayed a huge number of neuroblasts in the ependymal zone. (E–H) Adult transfected cells. (F) In the SVZ glial-like cells co-expressed both plasmids. (G) In the RMS migrating neuroblast mostly expressed just the EGFP plasmid (integrated). (H) Transfected OB cells co-expressed both constructs. Sagittal sections. (I–K) Quantitative analyses after postnatal electroporation (P1). (I) Short-term analyses after postnatal electroporation (P1 to P6) showed that OB interneurons mostly expressed both constructs: yellow cells, 68.5 ± 1.5%; green cells, 19.6 ± 4.3% and red cells, 11.9 ± 5%. (J) Long term survival times after postnatal electroporation (P1 to P30) showed the following distribution: yellow cells, 42.1 ± 4.6%; green cells, 45.9 ± 3.3% and red cells, 12 ± 3%. (K) Increment of red cells in short term analyses, One-Way ANOVA showed a high statistical significance lower that P < 0.001 Ctx, cortex; OB, Olfactory bulb; RMS, rostral migratory stream; SVZ, subventricular Zone.
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Figure 3: Olfactory bulb interneurons after postnatal electroporation (P1). (A–D) P6 transfected cells expressed the integrated and non-integrated constructs interchangeably. (B) In the SVZ, transfected red and green cells showed glial morphology. (C) In the rostral migratory stream large cells labeled were directed to the olfactory bulb. (D) OB displayed a huge number of neuroblasts in the ependymal zone. (E–H) Adult transfected cells. (F) In the SVZ glial-like cells co-expressed both plasmids. (G) In the RMS migrating neuroblast mostly expressed just the EGFP plasmid (integrated). (H) Transfected OB cells co-expressed both constructs. Sagittal sections. (I–K) Quantitative analyses after postnatal electroporation (P1). (I) Short-term analyses after postnatal electroporation (P1 to P6) showed that OB interneurons mostly expressed both constructs: yellow cells, 68.5 ± 1.5%; green cells, 19.6 ± 4.3% and red cells, 11.9 ± 5%. (J) Long term survival times after postnatal electroporation (P1 to P30) showed the following distribution: yellow cells, 42.1 ± 4.6%; green cells, 45.9 ± 3.3% and red cells, 12 ± 3%. (K) Increment of red cells in short term analyses, One-Way ANOVA showed a high statistical significance lower that P < 0.001 Ctx, cortex; OB, Olfactory bulb; RMS, rostral migratory stream; SVZ, subventricular Zone.

Mentions: To further individually explore the postnatal progeny of precursor cells, co-electroporation of the plasmid mixture was performed into the neonatal SVZ (Figure 3). Short-term expression analysis (P6, Figures 3A–D,I) revealed an equivalent expression of both integrable and non-integrable reporters. Labeled cells corresponding to either progenitor cells remained within the SVZ (Figure 3B), neuroblasts along the rostral migratory stream (RMS, Figure 3C) or incoming neuroblasts and immature interneurons within the OB (Figure 3D). Those interneurons mostly expressed both constructs after short-term survival times (yellow cells: 68.5 ± 1.5%). Equivalent expressions of both integrable (green cells: 19.6 ± 4.3%) and non-integrable (red cells: 11.9 ± 5%) reporters were determined after quantification analysis (Figure 3I). There were not significant differences between the percentage of labeled green and red cells after One-Way ANOVA analyses. Different expression pattern was observed at long-term survival analysis (P30, Figures 3E–H,J). In the SVZ, glial-like cells strongly co-expressed both plasmids (Figure 3F), suggesting that they did not undergo enough division rounds to lose the non-integrable plasmids. Cell expression of non-integrable (red) plasmids decreased along the RMS cells (Figure 3G) that mostly expressed the integrable EGFP plasmid. Besides, OB interneurons co-expressed both plasmids (Figures 3H,J; yellow cells: 42.1 ± 4.6%), but since in the RMS the neuroblasts were green labeled, those red positive cells in the OB probably corresponded to the first incoming OB interneurons. This was corroborated after the One-Way ANOVA analysis, showing a significant increment (P < 0.001) in the percentage of green OB interneurons in long-term experiments (Figure 3K). By contrast there were not significant differences in the percentage of red cells comparing short- and long-term experiments. Thus, those data provided insights into cell fate determination analyses of SVZ progenitor cells.


Spatiotemporal analyses of neural lineages after embryonic and postnatal progenitor targeting combining different reporters.

Figueres-Oñate M, García-Marqués J, Pedraza M, De Carlos JA, López-Mascaraque L - Front Neurosci (2015)

Olfactory bulb interneurons after postnatal electroporation (P1). (A–D) P6 transfected cells expressed the integrated and non-integrated constructs interchangeably. (B) In the SVZ, transfected red and green cells showed glial morphology. (C) In the rostral migratory stream large cells labeled were directed to the olfactory bulb. (D) OB displayed a huge number of neuroblasts in the ependymal zone. (E–H) Adult transfected cells. (F) In the SVZ glial-like cells co-expressed both plasmids. (G) In the RMS migrating neuroblast mostly expressed just the EGFP plasmid (integrated). (H) Transfected OB cells co-expressed both constructs. Sagittal sections. (I–K) Quantitative analyses after postnatal electroporation (P1). (I) Short-term analyses after postnatal electroporation (P1 to P6) showed that OB interneurons mostly expressed both constructs: yellow cells, 68.5 ± 1.5%; green cells, 19.6 ± 4.3% and red cells, 11.9 ± 5%. (J) Long term survival times after postnatal electroporation (P1 to P30) showed the following distribution: yellow cells, 42.1 ± 4.6%; green cells, 45.9 ± 3.3% and red cells, 12 ± 3%. (K) Increment of red cells in short term analyses, One-Way ANOVA showed a high statistical significance lower that P < 0.001 Ctx, cortex; OB, Olfactory bulb; RMS, rostral migratory stream; SVZ, subventricular Zone.
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Figure 3: Olfactory bulb interneurons after postnatal electroporation (P1). (A–D) P6 transfected cells expressed the integrated and non-integrated constructs interchangeably. (B) In the SVZ, transfected red and green cells showed glial morphology. (C) In the rostral migratory stream large cells labeled were directed to the olfactory bulb. (D) OB displayed a huge number of neuroblasts in the ependymal zone. (E–H) Adult transfected cells. (F) In the SVZ glial-like cells co-expressed both plasmids. (G) In the RMS migrating neuroblast mostly expressed just the EGFP plasmid (integrated). (H) Transfected OB cells co-expressed both constructs. Sagittal sections. (I–K) Quantitative analyses after postnatal electroporation (P1). (I) Short-term analyses after postnatal electroporation (P1 to P6) showed that OB interneurons mostly expressed both constructs: yellow cells, 68.5 ± 1.5%; green cells, 19.6 ± 4.3% and red cells, 11.9 ± 5%. (J) Long term survival times after postnatal electroporation (P1 to P30) showed the following distribution: yellow cells, 42.1 ± 4.6%; green cells, 45.9 ± 3.3% and red cells, 12 ± 3%. (K) Increment of red cells in short term analyses, One-Way ANOVA showed a high statistical significance lower that P < 0.001 Ctx, cortex; OB, Olfactory bulb; RMS, rostral migratory stream; SVZ, subventricular Zone.
Mentions: To further individually explore the postnatal progeny of precursor cells, co-electroporation of the plasmid mixture was performed into the neonatal SVZ (Figure 3). Short-term expression analysis (P6, Figures 3A–D,I) revealed an equivalent expression of both integrable and non-integrable reporters. Labeled cells corresponding to either progenitor cells remained within the SVZ (Figure 3B), neuroblasts along the rostral migratory stream (RMS, Figure 3C) or incoming neuroblasts and immature interneurons within the OB (Figure 3D). Those interneurons mostly expressed both constructs after short-term survival times (yellow cells: 68.5 ± 1.5%). Equivalent expressions of both integrable (green cells: 19.6 ± 4.3%) and non-integrable (red cells: 11.9 ± 5%) reporters were determined after quantification analysis (Figure 3I). There were not significant differences between the percentage of labeled green and red cells after One-Way ANOVA analyses. Different expression pattern was observed at long-term survival analysis (P30, Figures 3E–H,J). In the SVZ, glial-like cells strongly co-expressed both plasmids (Figure 3F), suggesting that they did not undergo enough division rounds to lose the non-integrable plasmids. Cell expression of non-integrable (red) plasmids decreased along the RMS cells (Figure 3G) that mostly expressed the integrable EGFP plasmid. Besides, OB interneurons co-expressed both plasmids (Figures 3H,J; yellow cells: 42.1 ± 4.6%), but since in the RMS the neuroblasts were green labeled, those red positive cells in the OB probably corresponded to the first incoming OB interneurons. This was corroborated after the One-Way ANOVA analysis, showing a significant increment (P < 0.001) in the percentage of green OB interneurons in long-term experiments (Figure 3K). By contrast there were not significant differences in the percentage of red cells comparing short- and long-term experiments. Thus, those data provided insights into cell fate determination analyses of SVZ progenitor cells.

Bottom Line: To address this issue, we performed postnatal and in utero co-electroporations of different fluorescent reporters to label, in both cerebral cortex and olfactory bulb, the progeny of subventricular zone neural progenitors.Further, while neuronal lineages arise from many progenitors in proliferative zones after few divisions, glial lineages come from fewer progenitors that accomplish many cell divisions.Together, these data provide a useful guide to select a strategy to track the cell fate of a specific cell population and to address whether a different proliferative origin might be correlated with functional heterogeneity.

View Article: PubMed Central - PubMed

Affiliation: Instituto Cajal-Consejo Superior de Investigaciones Científicas, Department of Molecular, Cellular and Developmental Neurobiology Madrid, Spain.

ABSTRACT
Genetic lineage tracing with electroporation is one of the most powerful techniques to target neural progenitor cells and their progeny. However, the spatiotemporal relationship between neural progenitors and their final phenotype remain poorly understood. One critical factor to analyze the cell fate of progeny is reporter integration into the genome of transfected cells. To address this issue, we performed postnatal and in utero co-electroporations of different fluorescent reporters to label, in both cerebral cortex and olfactory bulb, the progeny of subventricular zone neural progenitors. By comparing fluorescent reporter expression in the adult cell progeny, we show a differential expression pattern within the same cell lineage, depending on electroporation stage and cell identity. Further, while neuronal lineages arise from many progenitors in proliferative zones after few divisions, glial lineages come from fewer progenitors that accomplish many cell divisions. Together, these data provide a useful guide to select a strategy to track the cell fate of a specific cell population and to address whether a different proliferative origin might be correlated with functional heterogeneity.

No MeSH data available.