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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.


Neuronal lineage after embryonic electroporation. (A) Cortical P0 cells transfected at E14. Neurons in layers II and IV expressed interchangeably red or green plasmids (upper box in a,b). Cells located close to subventricular zone lost the mCherry non-integrable vector. This proliferative zone mostly expressed the integrated plasmid (EGFP), because the mCherry-plasmid dilution through cell division rounds (lower box in a,b). (B) Adult (P30) cortical cells labeled after IUE at E13 expressed either green (arrow) or red (arrowhead) reporters. Yellow cells (asterisk) co-expressed both constructs. At adult stages non-integrable constructs maintained a stable cell expression. (C,D) IUE performed at E13 and analyzed at adult stages (P30). Tbr1- (Ca) and NeuN-positive cells (Da) revealed the neuronal fate of cortical neurons expressed both the integrated (green, b) and non-integrated (red, c) constructs. (E) Percentage of adult cortical neurons (IUE at E14, analysis P30, n = 4 animals, 4 sections per animal) expressing green- (9.6 ± 1.5%), red- (8.1 ± 1.5%) or both constructs (82.3 ± 1.6%). (F) Interneurons in the olfactory bulb after IUE at E13. Olfactory bulb transfected interneurons expressed only the green integrated construct (a). Separate confocal channels demonstrate the lack of mCherry signal (b). IUE: In utero electroporation.
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Figure 2: Neuronal lineage after embryonic electroporation. (A) Cortical P0 cells transfected at E14. Neurons in layers II and IV expressed interchangeably red or green plasmids (upper box in a,b). Cells located close to subventricular zone lost the mCherry non-integrable vector. This proliferative zone mostly expressed the integrated plasmid (EGFP), because the mCherry-plasmid dilution through cell division rounds (lower box in a,b). (B) Adult (P30) cortical cells labeled after IUE at E13 expressed either green (arrow) or red (arrowhead) reporters. Yellow cells (asterisk) co-expressed both constructs. At adult stages non-integrable constructs maintained a stable cell expression. (C,D) IUE performed at E13 and analyzed at adult stages (P30). Tbr1- (Ca) and NeuN-positive cells (Da) revealed the neuronal fate of cortical neurons expressed both the integrated (green, b) and non-integrated (red, c) constructs. (E) Percentage of adult cortical neurons (IUE at E14, analysis P30, n = 4 animals, 4 sections per animal) expressing green- (9.6 ± 1.5%), red- (8.1 ± 1.5%) or both constructs (82.3 ± 1.6%). (F) Interneurons in the olfactory bulb after IUE at E13. Olfactory bulb transfected interneurons expressed only the green integrated construct (a). Separate confocal channels demonstrate the lack of mCherry signal (b). IUE: In utero electroporation.

Mentions: IUE was performed as previously described (García-Marqués et al., 2014). Briefly, E13-E14 pregnant mice were anesthetized with isofluorane (Isova vet, Centauro), and placed in a thermic plate. The skin and the abdominal cavity were cut, opened, and the uterine horns exposed. The DNA mixture solution consisted of an equal amount of pPB-UbC-EGFP and UbC-mCherry plasmids, plus half the amount of mPBase and 0.1% Fast Green. The final concentration was 1 μg/μL, and 1 μl of the solution was injected into the lateral ventricle of each embryo by a pulled glass micropipette. After that, the head of each embryo was placed between 3 mm tweezer-type electrodes (Sonidel) and 5 electric pulses of 50 ms length were passed after 950 ms intervals using an electroporator. In all cases, the electroporated region was the ventricular zone in the dorso-lateral area (Figure 2B). The voltage varied depending on the embryonic day from 33 V at E13 to 35 V at E14. After electroporation, the uterus was repositioned and the abdominal cavity was sutured. Pregnant mice received a subcutaneous injection of both 5 mg/kg of the antibiotic enrofloxacine (Baytril; Bayer, Kiel, DE) and 300 μg/kg of the anti-inflammatory/analgesic meloxicam (Metacam; Boehringer Ingelheim) and embryos were allowed to continue developing until desired.


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)

Neuronal lineage after embryonic electroporation. (A) Cortical P0 cells transfected at E14. Neurons in layers II and IV expressed interchangeably red or green plasmids (upper box in a,b). Cells located close to subventricular zone lost the mCherry non-integrable vector. This proliferative zone mostly expressed the integrated plasmid (EGFP), because the mCherry-plasmid dilution through cell division rounds (lower box in a,b). (B) Adult (P30) cortical cells labeled after IUE at E13 expressed either green (arrow) or red (arrowhead) reporters. Yellow cells (asterisk) co-expressed both constructs. At adult stages non-integrable constructs maintained a stable cell expression. (C,D) IUE performed at E13 and analyzed at adult stages (P30). Tbr1- (Ca) and NeuN-positive cells (Da) revealed the neuronal fate of cortical neurons expressed both the integrated (green, b) and non-integrated (red, c) constructs. (E) Percentage of adult cortical neurons (IUE at E14, analysis P30, n = 4 animals, 4 sections per animal) expressing green- (9.6 ± 1.5%), red- (8.1 ± 1.5%) or both constructs (82.3 ± 1.6%). (F) Interneurons in the olfactory bulb after IUE at E13. Olfactory bulb transfected interneurons expressed only the green integrated construct (a). Separate confocal channels demonstrate the lack of mCherry signal (b). IUE: In utero electroporation.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4362314&req=5

Figure 2: Neuronal lineage after embryonic electroporation. (A) Cortical P0 cells transfected at E14. Neurons in layers II and IV expressed interchangeably red or green plasmids (upper box in a,b). Cells located close to subventricular zone lost the mCherry non-integrable vector. This proliferative zone mostly expressed the integrated plasmid (EGFP), because the mCherry-plasmid dilution through cell division rounds (lower box in a,b). (B) Adult (P30) cortical cells labeled after IUE at E13 expressed either green (arrow) or red (arrowhead) reporters. Yellow cells (asterisk) co-expressed both constructs. At adult stages non-integrable constructs maintained a stable cell expression. (C,D) IUE performed at E13 and analyzed at adult stages (P30). Tbr1- (Ca) and NeuN-positive cells (Da) revealed the neuronal fate of cortical neurons expressed both the integrated (green, b) and non-integrated (red, c) constructs. (E) Percentage of adult cortical neurons (IUE at E14, analysis P30, n = 4 animals, 4 sections per animal) expressing green- (9.6 ± 1.5%), red- (8.1 ± 1.5%) or both constructs (82.3 ± 1.6%). (F) Interneurons in the olfactory bulb after IUE at E13. Olfactory bulb transfected interneurons expressed only the green integrated construct (a). Separate confocal channels demonstrate the lack of mCherry signal (b). IUE: In utero electroporation.
Mentions: IUE was performed as previously described (García-Marqués et al., 2014). Briefly, E13-E14 pregnant mice were anesthetized with isofluorane (Isova vet, Centauro), and placed in a thermic plate. The skin and the abdominal cavity were cut, opened, and the uterine horns exposed. The DNA mixture solution consisted of an equal amount of pPB-UbC-EGFP and UbC-mCherry plasmids, plus half the amount of mPBase and 0.1% Fast Green. The final concentration was 1 μg/μL, and 1 μl of the solution was injected into the lateral ventricle of each embryo by a pulled glass micropipette. After that, the head of each embryo was placed between 3 mm tweezer-type electrodes (Sonidel) and 5 electric pulses of 50 ms length were passed after 950 ms intervals using an electroporator. In all cases, the electroporated region was the ventricular zone in the dorso-lateral area (Figure 2B). The voltage varied depending on the embryonic day from 33 V at E13 to 35 V at E14. After electroporation, the uterus was repositioned and the abdominal cavity was sutured. Pregnant mice received a subcutaneous injection of both 5 mg/kg of the antibiotic enrofloxacine (Baytril; Bayer, Kiel, DE) and 300 μg/kg of the anti-inflammatory/analgesic meloxicam (Metacam; Boehringer Ingelheim) and embryos were allowed to continue developing until desired.

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.