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1.
Figure 3

Figure 3. YFP+ cells induced by TM administration at E8.5 are reelin-positive CR cells.. From: Inducible Genetic Lineage Tracing of Cortical Hem Derived Cajal-Retzius Cells Reveals Novel Properties.

A–C: X-gal staining on E18.5 Fzd10 CreER™/R26R-LacZ brain sections. B shows β-gal+ CR cells settled at the marginal zone of the dentate gyrus. C: Arrows showing the β-gal+ CR cells at the marginal zone of the cortex. D–F: Double immunostaining shows that induced YFP-positive cells are reelin-positive at E14.5 when TM was administered at E8.5. DG, dentate gyrus; Cx, cortex. Scale bars: A, 2 mm; B–C: 200 µm; others: 300 µm.

Xiaochun Gu, et al. PLoS One. 2011;6(12):e28653.
2.
Figure 2

Figure 2. The progenitor zone that will later generate CR cells is specified as early as E6.5.. From: Inducible Genetic Lineage Tracing of Cortical Hem Derived Cajal-Retzius Cells Reveals Novel Properties.

A–B: Whole-mount X-gal staining of E18.5 Fzd10 CreER™/R26R-LacZ brains when TM induction was given at E6.5. Arrows show CR cells migrating out of the cortical hem. C–H: Double immunostaining shows that induced YFP-positive cells are reelin positive at E16.5 (C–E, arrows) and E18.5 (F–H, arrows) in the neocortical MZ. I–N: Double immunostaining shows that induced YFP-positive cells are also P73 positive. O–Q: YFP and P73 are co-localized at the marginal zone of the hippocampal dentate gyrus (arrows). DG, dentate gyrus; MZ, marginal zone. Scale bars:A, B: 1 mm; C–E: 225 µm; others: 200 µm.

Xiaochun Gu, et al. PLoS One. 2011;6(12):e28653.
3.
Figure 7

Figure 7. Hem-derived CR cells survive to postnatal stages.. From: Inducible Genetic Lineage Tracing of Cortical Hem Derived Cajal-Retzius Cells Reveals Novel Properties.

A–B: Whole-mount X-gal staining of Fzd10 CreER™/R26R-LacZ brains when TM induction was performed at P2 and examined at P4. C–E: X-gal staining on P8 brain coronal sections showing hem-derived β-gal+ cells located at the MZ of the dentate gyrus (D) and neocortex (E) when TM was administered at P0. F–H: YFP+ cells induced postnatally were reelin positive. I–K: YFP+ cells were also P73 positive, indicating that these cells are CR cells. L–O: Z-stack magnification views show that the YFP-positive cells express the CR cell markers P73 and reelin. P2–P4, TM was administered at P2, and brains were stained at P4, the same as P0–P8. DG, dentate gyrus; Cx, cortex. Scale bars: A–B, 2 mm; others: 200 µm.

Xiaochun Gu, et al. PLoS One. 2011;6(12):e28653.
4.
Figure 1

Figure 1. Cortical hem-derived CR cells originating from progenitors expressing Fzd10.. From: Inducible Genetic Lineage Tracing of Cortical Hem Derived Cajal-Retzius Cells Reveals Novel Properties.

A–B: RNA in situ hybridization showing that Cre expression mimics endogenous Fzd10 in the cortical hem in the Fzd10-CreER™ mouse. C–D: Whole-mount X-gal staining of E18.5 Fzd10 CreER™/R26R-LacZ brains when TM was administered at E11.5, showing the output of hem-derived CR cells. C: Lateral view of the hemisphere. D: Middle view. E–J: YFP reporter-positive cells are both reelin+ and P73+, indicating that these cells are CR cells originating from the cortical hem. E–G: When TM was injected at E11.5 and double immunostaining was performed at E16.5, YFP+ cells expressed reelin. H–J: YFP+ cells were also P73 positive. E11.5–E18.5, TM was administered at E11.5 and brains were stained at E18.5. Scale bars: A, B: 100 µm; C–D: 2 mm; others: 200 µm.

Xiaochun Gu, et al. PLoS One. 2011;6(12):e28653.
5.
Figure 6

Figure 6. The fimbrial radial glial scaffold derived from cortical hem is necessary for CR cell migration.. From: Inducible Genetic Lineage Tracing of Cortical Hem Derived Cajal-Retzius Cells Reveals Novel Properties.

Reelin-positive CR cells cannot migrate out and ectopically settled at the fimbrial area after the radial glial scaffold is disrupted by DTA toxin expression. A shows that the BLBP-positive radial glial scaffold is disrupted in Fzd10 CreER™/ROSA26DTA mice compared to control mice in E. B shows that reelin-positive CR cells (arrows) were arrested at the fimbria after the disruption of the fimbrial radial glial scaffold by DTA expression, but reelin-positive cells could not be detected in the control fimbria in F. D The number of Reelin+ cells (arrows) in Fzd10 CreER™/ROSA26DTA hippocampal MZ was dramatically decreased compared to control. Fi, fimbria; MZ, marginal zone; DG, dentate gyrus. Scale bars: D, H: 100 µm; others: 50 µm.

Xiaochun Gu, et al. PLoS One. 2011;6(12):e28653.
6.
Figure 5

Figure 5. The hem-derived CR cells migrate to the hippocampal marginal zone along the fimbrial radial glial scaffold.. From: Inducible Genetic Lineage Tracing of Cortical Hem Derived Cajal-Retzius Cells Reveals Novel Properties.

A–D: Triple immunostaining for YFP/reelin/Tbr2 in E14.5 brains when TM was injected at E10.5. A: Cortical hem-derived YFP-positive cells consisting of two types of cells, radial glial scaffold (arrow) and migrating cells (arrowheads). B: Anti-Tbr2 staining shows two Tbr2+ migrating streams, one from the dentate neuroepithelium to produce dentate granule cells indicated by yellow arrows, the other from the cortical hem (arrowheads). They meet at the area of the future dentate. C: Hem-derived reelin-positive CR cells. D: Yellow arrows indicate double-labeled YFP+/Tbr2+ cells migrating out of the cortical hem, and white arrows indicate YFP+/reelin+ cells settled at the MZ zone of the hippocampus. E: Some scaffold-like YFP-positive cells are derived from the cortical hem and distribute to the dentate gyrus from the hem (arrows). F–L: The YFP+ scaffold is positive for the radial glial marker BLBP. F–H: YFP+/BLBP+ radial glial scaffold extend to the DG from the fimbria (arrows). I–L: YFP+/BLBP+ radial glial scaffold in the fimbria. Arrowheads indicate the processes of the radial glia, and the arrow shows its cell body. J–L show high-magnification views of the area boxed in I. M–P: CR cell progenitors, Tbr2+ cells migrate out of the hem along the YFG-positive radial glial scaffold also derived from the cortical hem. Arrows in N–P show migrating Tbr2+ CR progenitors. N–P: High-magnification view of the boxed area in M. dnp, dentate neuroepithelium; CH, cortical hem; Fi, fimbria; MZ, marginal zone; DG, dentate gyrus. Scale bars: A–E: 200 µm; others: 50 µm.

Xiaochun Gu, et al. PLoS One. 2011;6(12):e28653.
7.
Figure 4

Figure 4. Hem-derived CR cells preferentially settled at the hippocampal marginal zone.. From: Inducible Genetic Lineage Tracing of Cortical Hem Derived Cajal-Retzius Cells Reveals Novel Properties.

A–H: X-gal staining of E18.5 hemispheres when TM injection was given at E10.5 (A–C), E11.5 (D) and E13.5 (E–H). A, E, D, H: The hem-derived CR cells preferentially populated the dorsal-caudal cortical surface. I-L:β-gal+ cells can be detected at P4 (I, J) and P16 (K, L) at the hippocampal and neocortical marginal zone. I, K: X-gal staining showing β-gal+ cells in P4 and P16 neocortical marginal zones, respectively. J, L: X-gal staining showing β-gal+ cells in P4 and P16 hippocampal marginal zones, respectively. M–P: Double staining shows that the YFP-positive cells that settled at the hippocampal marginal zone are both reelin (M, N) and P73 positive (O, P), indicating that these cells are CR cells. Q: Schematic drawing of the cortical and hippocampal areas for CR cell counting, the surface of the cortex was divided into sub-regions of H (hippocampus), D (dorsal cortex),V (ventral cortex),I (intermediate cortex) and C (cingulate cortex) sub-regions. R: Statistical analysis of the distribution of CR cells in sub-regions. S. More β-gal+ CR cells populated the hippocampal MZ than other cortical regions (*p<0.05, **P<0.01, ***P<0.001). S Statistical analysis of the total number of CR cells settled at hippocampal and cortical MZ at different developmental stages of E10.5, E11.5, E12.5 and E13.5 respectively (*P<0.05) CR cells preferentially migrated to the hippocampal MZ at most developmental stages. At the stage of E11.5, when the production peak of CR cells appears, the total number of CR cells in the whole cortical MZ seems larger than that of in the hippocampal MZ. However the number of CR cells in the hippocampal MZ (H) is more than any other sub-regions (C, D, I, V) in the cortex as shown in R. T: schematic drawing of the distribution of CR cells derived from the cortical hem. Black dots represent CR cells. H, hippocampus; C, cingulate cortex; D, dorsal cortex; I, intermediate cortex; V, ventral cortex. DG, Dentate gyrus; CX, Cortex. rs, rostral cortex; cd, caudal cortex; ds, dorsal cortex; MZ, marginal zone; r, rostral telencephalon ; c, caudal telencephalon,. Scale bars: A, E, D, H: 2 mm; others: 400 µm.

Xiaochun Gu, et al. PLoS One. 2011;6(12):e28653.

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