Article Figures & Data
Figures
- Figure 1.
Temporal properties of in utero electroporation. BrdU was administered immediately after IUE with pCAG-RFP to label cells in S-phase at the moment of IUE (0 h) (Ai). Using this paradigm, the majority of cells transfected by IUE (RFP+) were colabeled with BrdU (70.87 ± 6.60%) (D). Administering BrdU 2 h before IUE labeled cells throughout S- to M-phase at the moment of IUE (−2 h) (Aii). Nearly the entire RFP+ population also expressed BrdU using this protocol (95.97 ± 4.00%) (B, B′, D). The arrow in B and B′ denotes an RFP+ cell that was not BrdU+. These data demonstrate that IUE preferentially transfects cells that are in S- and M-phases at the time of electroporation. C, C′, Only a small percentage (23.29 ± 4.16%) of cells were colabeled with RFP and BrdU when BrdU was injected 6 h after IUE; the arrows point to the minority of cells that were colabeled. D, The percentage of colabeled BrdU+RFP+ cells decreased as the time between IUE and BrdU administration increased, indicating that plasmid viability is limited. Together, these data indicate that the cohort of electroporated cells is temporally limited to those in S- through M-phase at the moment of IUE, with the leading edge of the transfected population in M-phase, and that the electroporated plasmid is only viable for 6–8 h after IUE. Error bars represent SE.
- Figure 2.
Short neural precursors have distinct cell cycle kinetics. A, Labeling schemes of IUE/BrdU and IUE/M–M-phase methods for estimating TG1 and TC, respectively. TG1 represents the time it takes transfected cells (red) to transit through G1-phase and reenter S-phase, determined by administering BrdU at 2 h intervals after IUE until cells become colabeled with BrdU (yellow). TC represents the cell cycle duration, determined by identifying the time necessary to find mitotic electroporated cells (green) at the ventricular surface. B, For proof of principle, IUE/BrdU method was first performed with CAG-RFP plasmid to label all cells within the VZ; cells reentered S-phase 12–14 h after IUE (arrow). B′, To measure TG1 for individual precursor populations, IUE was performed with pTα1-hGFP or pGLAST-eGFPf; SNPs entered into the next S-phase 4 h later than RGCs (TG1 of 16 vs 12 h, marked with arrows; n ≥ 3 for all time points; ANOVA, *p < 0.0001). C, To determine M-to-M-phase duration, RFP+ cells with condensed chromatin at the ventricular surface were counted after staining with SYTO 24. Transfected RFP+ cells entered into the next M-phase 20–22 h after IUE (arrow). C′, M-phase reentry after IUE for SNPs was delayed by 4 h compared with RGCs (TC of 24–26 vs 20–22 h, marked with arrows; n ≥ 3 for all time points; median regression, *p < 0.009). In all graphs, error bars represent SE.
- Figure 3.
Laminar allocation differences in VZ-derived cells at the end of neurogenesis. A, Genetic fate mapping experiments were performed by coelectroporation of cell type-specific Cre plasmids together with a floxed stop GFP reporter plasmid. B, Embryos electroporated at E13.5 were harvested at E17.5 for analysis of the distribution of GFP+ progeny across the thickness of the neocortical wall. C, In collapsed confocal Z-stacks, the thickness of the cortical wall was divided into five bins, yielding regions corresponding to the VZ, SVZ, IZ, and lower and upper CP, and GFP+ cells were counted across a 300 μm width in each region. Most GFP+ progeny of SNPs were found in the lower CP (second box from top on all bars) by the end of neurogenesis, whereas more RGC progeny were found in the upper CP (top box on all bars). pNestin+ NSCs generated progeny with an intermediate distribution between that of RGCs and SNPs. Graph shows percentage of total GFP+ cells (per 300 μm column) in each region. Error bars indicate SE. n = 3 for pNestin and pTα1; n = 4 for pGLAST. Scale bar, 100 μm.
- Figure 4.
RGCs and SNPs labeled on the same embryonic day generate neuronal progeny specified to different cortical laminae. After IUE with Cre and GFP reporter plasmids at E14.5, brains were harvested at P10. Composite, low-magnification images of GFP+ neuronal progeny and callosal axons are shown for each precursor population in A–C. On examination at higher magnification (A′–C″), variations in cell location became apparent. RGC progeny were grouped tightly in the deep part of layer II/III (labeled with Brn1 staining) (B″). In contrast, SNP progeny formed a thin band deep to layer II/III, in layer IV (C″). D, The average distance (in micrometers) of all GFP+ neuronal soma from the top of layer II/III was significantly greater for cells generated from SNPs (pTα1) compared with neurons produced by RGCs (pGLAST) or NSCs (pNestin). E, GFP+ pyramidal neurons showed similar distribution patterns compared with the entire neuronal populations of NSC (pNestin), RGC (pGLAST), and SNP (pTα1) progeny. Error bars show SE. n = 3 for pNestin and pTα1; n = 2 for pGLAST. *p < 0.01, **p < 0.001, Student's t test. Scale bars: A–C, 900 μm; A′–C″, 100 μm.
- Figure 5.
RGC progeny are amplified by proliferative IPCs whereas SNPs generate neurons directly from the VZ. Immunostaining for Tbr2, Ki67, TUJ1 (βIII-tubulin), and Pax6 was performed on tissue electroporated at E14.5 with pTα1-hGFP or pGLAST-eGFPf and fixed 24 h later (A–C′). The arrows in A–C point to GFP+ RGCs double positive for Tbr2, TUJ1, or Pax6, respectively. Orthogonal views of single Z-sections in A′–C′ show double labeling of GFP+ SNPs (outlined in white boxes). D, Nearly twice as many RGCs coexpressed Tbr2 compared with SNPs. Threefold more SNPs coexpressed TUJ1 compared with RGCs. Differences between RGCs and SNPs in coexpression of Ki67 or Pax6 were not statistically significant. Error bars represent SE. n = 4 per group. *p < 0.01, Student's t test. Scale bar, 20 μm.
- Figure 6.
Differential neuronal production from VZ precursor subtypes. The experiments in this study were conducted at mid-neurogenesis (E13.5–E14.5), the stage during which stellate and pyramidal neurons begin to be generated from precursors in the VZ and SVZ. Our data demonstrate that pGLAST+ RGCs and pTα1+ SNPs exhibit markedly different cell cycle kinetics and that SNPs persist in the VZ for at least two cell divisions (Fig. 2). The Cre/lox fate-mapping studies show that SNPs and RGCs present in the VZ at the same time produce different types of neurons, as defined by their laminar position (Fig. 4). Because of the inside-out nature of cortical layer formation, layer IV neurons are born before neurons residing in layer II/III. Furthermore, these experiments demonstrate that RGCs divide in the VZ to directly generate a small number of layer IV neurons and that they also indirectly generate layer II/III neurons via IPC divisions in the SVZ. These IPC divisions take additional time; thus, progeny of the RGC antecedents are spread radially over a large laminar area. In contrast, SNPs within the E14.5 VZ do not generate appreciable numbers of INPs but rather generate neuronal progeny directly from the VZ, which are allocated more quickly and specifically to layer IV. Thus, RGCs contribute more substantially to laminar expansion by producing neurons for multiple layers over a longer period of time, whereas SNPs generate discrete neuronal populations over a short time window.
Tables
Percentage of GFP+ cells colabeled with Ki67 Pax6 Tbr2 TUJ1 RGCs 11.73 ± 2.41 15.66 ± 5.64 7.98 ± 1.40 2.96 ± 0.84 SNPs 7.75 ± 0.83 17.60 ± 0.69 4.78 ± 0.63 9.65 ± 1.39 -
Immunostaining was performed on tissue harvested 24 h after electroporation at E14.5 with either pTα1-hGFP or pGLAST-eGFPf. Cell counts were performed on 40× collapsed Z-stacks, across the entire width of the image (225 μm) and up to 200 μm from the ventricular surface. Values represent the percentage of GFP+ cells coexpressing each protein or transcription factor ± SEM; n = 4 per group.
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