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Files in this Data Supplement:
Fig. S1. Relationship of Nestin-GFP, Tbr2 and Prox1 expression in the perinatal dentate gyrus. (A) At birth, subpial Nestin-GFP+ cells were proliferative as shown by active BrdU incorporation (A′) and mostly nonoverlapped with Prox1 (A′′). (B) By P1, subpial Tbr2+ cells were proliferating as shown by active BrdU incorporation. Boxed area was shown as inset at higher power. (C) By P1, subpial Tbr2+ cells were distinct from the reelin+ Cajal-Retzius cells (arrows in inset). Boxed area was shown as and inset at higher power. (D-F) By P5, Prox1 were weakly expressed in the remaining Tbr2+ cells in the MZ (E-E′′), whereas in the newly formed SGZ, the weak Tbr2+ cells were also positive for Prox1 (arrows in F-F′′). Boxed areas in D were shown in E-E′′ and F-F′′ at higher power. GCL, granule cell layer; HF, hippocampal fissure; ML, molecular layer; MZ, marginal zone; SGZ, subgranular zone.
Fig. S2. Embryonic expression of Cxcl12 and Cxcr4 by in situ hybridization during the migration of the dentate precursors. (A,C) In situ hybridization showed that Cxcl12 was exclusively expressed in the meninges at E13.5 and E16.5, respectively. (B) At E13.5, Cxcr4 was expressed in the dentate primordium (arrow) and marginal Cajal-Retzius cells (arrowheads). (D) At E16.5, Cxcr4 was expressed in the migratory stream (arrow) of the dentate gyrus. Scale bar: 100μm in A,B; 200μm in C,D.
Fig. S3. Emx1-Cxcr4 cKO phenocopied the Cxcr4 null in the developing dentate gyrus. At E17.5, the migratory stream showed by the transit amplifying cell marker Tbr2 was very compact in the controls (A,E), whereas it became widely distributed in both Emx1-Cxcr4 cKO (B) and Cxcr4-null (F). Similarly, compared with the controls (arrows in C,G), both Emx1-Cxcr4 cKO and Cxcr4-null animals failed to form a distinct upper blade as labeled by the granule cell marker Prox1 (arrows in D,H).
Fig. S4. Postnatal organization of the subgranular niche in Emx1-Cxcr4 cKOs. (A-H) Abnormal organization of the subgranular zone at early postnatal ages in Emx1-Cxcr4 cKO. By P5, most Nestin-GFP+ cells were organized at the SGZ and focally concentrated in the growing tip of the lower blade in the control (A), whereas they were scattered in the hilus with decreased number and lost focal collection in the growing tip of the lower blade in the Emx1-Cxcr4 cKOs (B). The neurogenic Tbr2+ cells were mainly localized in the molecular layer in the control (C), but most of them were dispersed in the hilus in the Emx1-Cxcr4 cKO (D). There were many more Ki67+ proliferating cells in the hilus in the Emx1-Cxcr4 cKO (F) compared with the control (E). Compared with the control (G), the Prox1+ granule cells also assumed a nearly normal layer organization except for the consistently slightly thinner upper blade and somewhat thicker lower blade in the Emx1-Cxcr4 cKO (H). (I-P) Subgranular zone has recovered by the second postnatal week in Emx1-Cxcr4 cKO. By P14, BLBP staining showed normal transgranular radial glial scaffolding in the Emx1-Cxcr4 cKOs (J) compared with the control (I). Very similar to the control (K,M), the SGZ also showed largely normal organization in the Emx1-Cxcr4 cKO as revealed by Tbr2 (L) and BrdU (N) staining, except that there were some ectopic Tbr2+ cells in the granule cell layer and ectopic BrdU+ cells in the hilus (arrows in L,N). Boxed area in L is shown as inset for ectopic Tbr2+ cells. There was no discernible difference in the organization of granule cell layer labeled by Prox1 between controls (O) and cKOs (P). Scale bar: 100 μm in A-H; 200 μm in I-P. H, hilus; SGZ, subgranular zone.
Fig. S5. Recovered subgranular niche in the adult Emx1-Cxcr4 cKOs. At P45, the organization of the subgranular zone in the Emx1-Cxcr4 cKOs as shown by BLBP, Tbr2 and Ki67 (A′-C′) remained essentially similar to the controls (A-C). The granule cell layer in cKOs (D′) also showed normal organization compared to the controls (D), except that small collections of ectopic granule cells could be detected occasionally in cKO (arrow in D′′). The whole dentate pole of the cKO is shown at lower power as an inset in D′. Scale bar: 200 μm in all pictures.
Fig. S6. Emx1ires-cre showed complete penetrance in the dentate gyrus. (A,B) Recombination mediated by Emx1ires-cre completely covered the whole medial wall, including hippocampal field (Hip), primordium for dentate gyrus (DG), fimbria (F) and some of the choroids plexus (CP). (C) Recombination penetrance mediated by Emx1ires-cre was domonstrated by the presence of the Cre reporter Z/EG in the dentate gyrus of the Emx1-Cxcr4 cKO animals at P14. Boxed area in C is showed at higher power in D-F. (D-F) All the Prox1-labeled granule cells (red) were co-stained with the recombination marker GFP (green). Nuclei were labeled by DAPI in C-F. Outer granule cells showed stronger GFP staining than inner granule cells as they were born earlier. Scale bar in D applies to E and F. H, hilus; GCLinner, inner granule cell layer; GCLouter, outer granule cell layer; ML, molecular layer.
Fig. S7. Abnormal development of the dentate gyrus in the Emx1-PTX animals at birth. (A,B) Most Nestin-GFP+ precursors were restricted at subpial region of the forming dentate pole in the control (A), whereas they were aberrantly scattered in the hilus in the Emx1-PTX animals (arrow in B). (C,D) Consistently, Tbr2+ transit amplifying precursors, which were organized into a thin layer subpially in the control, instead spread into the incipient granule cell layer and hilus (arrow in D) and formed a broad stream (arrowhead in D) in the Emx1-PTX animals. (E,F) In contrast to the well-defined upper blade as shown by Prox1 staining in the control (arrow in E), the tip of the upper blade was quite obscured in the Emx1-PTX animals (arrow in F). Moreover, extra Prox1+ granule cells were retained at the fimbriodentate junction (arrowhead in F). F, fimbria; H, hilus. Scale bar in F applies to A-F.
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