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The chemokine SDF1 regulates migration of dentate granule cells

Anil Bagri1,2, Theresa Gurney2, Xiaoping He1, Yong-Rui Zou3,{dagger}, Dan R. Littman3, Marc Tessier-Lavigne2,*,{ddagger} and Samuel J. Pleasure1,{ddagger}

1 Neurodevelopmental Disorders Laboratory, Department of Neurology, Program in Neuroscience, University of California, San Francisco, CA 94143-0435, USA
2 Departments of Anatomy and of Biochemistry and Biophyics, Howard Hughes Medical Institute, University of California, San Francisco, CA 94143-0452, USA
3 Departments of Pathology and Microbiology, Skirball Institute of Biomolecular Medicine, Howard Hughes Medical Institute, New York University Medical Center, New York, NY 10016, USA
* Present address: Department of Biological Sciences, Howard Hughes Medical Institute, Stanford University, Stanford, CA 94305, USA
{dagger} Present address: Department of Microbiology, Columbia University College of Physicians and Surgeons, New York, NY 10032, USA



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  		Fig. 1. In utero intraventricular injections of nlslacZ retrovirus at various ages labeled different populations of cells. Coronal sections of hippocampus were processed for X-gal histochemistry to detect ß-gal activity and then counterstained with nuclear Fast Red. (A) The in utero intraventricular retroviral injection approach. Retrovirus was injected into the ventricle of embryonic rats in utero, allowing the virus to diffuse and infect cells in the ventricular zone (blue). During subsequent hippocampal development, the infected dentate granule cells and precursors (blue) migrated with uninfected cells (red) to the dentate gyrus. (B) Quantification of the percentage of cells positive for ß-gal in the dentate granule cell layer (blue), pyramidal cell layer (red) or other regions of the hippocampus (yellow) at P15 after in utero injections were performed at different developmental ages. The graph depicts an average of five different animals at each age. (C-E) The distribution of cells at P15 when nlslacZ retrovirus injections were made at E16 (C), E18 (D) or E20 (E). As ß-gal+ cells were difficult to resolve at this magnification and with the counterstain, black dots (indicating the position of each positive cell) were added to the images to facilitate their identification. Injections at E16 (C) result in the labeling of many dentate granule cells in addition to non-dentate granule cells. Injections at E18 (D) resulted in the labeling of a larger fraction of dentate granule cells, with a dramatic decrease in the labeling of pyramidal cells. Interestingly, injections at E20 (E) resulted in labeling of dentate granule cells primarily in the inferior blade of the granule cell layer. At this age, other cells comprised the largest cohort of labeled cells. (F-H) Injections of nlslacZ virus at E16 (F), E18 (G) and E20 (H) analyzed 72 hours after injection yielded a similar pattern of labeling in the dentate migratory stream, indicating that infected granule and precursor cells behaved in similar fashion, regardless of the age at which they were labeled. Scale bars: 250 µm in C-E; 500 µm in F-H.

 


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  		Fig. 2. Developmental time-course of migration from the ventricular zone to the dentate gyrus. Intraventricular injections of nlslacZ retrovirus were performed in E18 rats and the migratory pattern of labeled cells was analyzed after varying amounts of time. Coronal sections of hippocampus were processed for X-gal histochemistry to detect ß-gal activity and then counterstained with nuclear Fast Red. For reference, the fimbria is marked with a green asterisk. Each experiment was performed on embryos from at least three different litters. (A) At E19, 24 hours after retroviral injection, labeled cells (blue) can be seen in the ventricular zone (open arrowhead). Additionally, a few cells can be seen to have left the ventricular zone (arrowhead). (B) Forty-eight hours after retroviral injection (E20), labeled cells are still seen to populate the ventricular zone. Additionally, cells can be seen above the fimbria in the dentate migratory stream (arrowheads), but have not yet entered the dentate gyrus. (C) 72 hours after retroviral injection (E21), labeled cells have migrated into the dentate gyrus and can be found in the germinal center in the dentate hilus. In addition, the first labeled cells migrate into the granule cell layer (arrow). (D) 96 hours after injection (P1), fewer labeled cells can be seen in the migratory stream; however, many labeled cells are now seen in the dentate hilus and populating the granule cell layer (arrows). (E) At E21, the morphology of labeled cells within the ventricular zone can be seen as they migrate above the fimbria and the migrating cells extended from the VZ into the dentate gyrus. The pattern and extent of migrating cells agreed with the pattern seen in the nlslacZ experiments. (F-H) By P30, cells had migrated to their final position and developed morphologies characteristic of mature differentiated cells. In the dentate granule cell layer (G, boxed area in F), many granule cells were labeled (G), and their highly branched dendritic trees and axonal arbors extended into the molecular layer and hilus, respectively. Additionally, in the hilus (H, boxed area in F), glia were also seen, confirming that cells labeled at the ventricular zone that migrate to the dentate gyrus do not have a homogenous fate. Scale bars: in A, 200 µm for A-D; in E, 75 µm; in F, 50 µm for F; in H, 50 µm for G,H.

 


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  		Fig. 3. In vivo and in vitro dentate granule cell migration/gyrus development occurred in a comparable manner. (A,D,G) Prox1 immunostaining labeled granule cells in coronal sections of E15.5 (A), E17.5 (D) and P0 (G) wild-type mice, and showed the accumulation of these cells in the developing dentate gyrus (broken outline). In addition to increasing numbers of Prox1-positive cells in the dentate, more weakly positive Prox1 cells can also be seen in the migratory stream at the E17.5 and P0 time-points. Additional Prox1-positive cells are seen in the thalamus and more weakly staining ones transiently in the cortex. (B,E,H) Prox1 immunostaining of E15.5 hippocampal slice cultures showed a comparable rate and pattern of dentate granule cell accumulation. Cultures fixed at t=0 days in vitro (B) had similar low levels of Prox1 immunoreactivity in the dentate when compared with E15.5 tissue (A). Cultures analyzed after 2 days of culturing (E) had an increased number of Prox1-positive cells in the dentate and few weakly positive cells were also seen in the migratory stream, similar to E17.5 tissue (D). This was also seen when 4 day cultures (H) were compared with P0 tissue (G). (C,F,I) The schematic (C) shows that nlslacZ retrovirus injections into the ventricular zone adjacent to the fimbria resulted in the labeling of many cells. Cultures fixed after 1 day of culturing (F) and processed for X-gal histochemistry showed that labeled cells (blue) were restricted to a region close to the site of injection. However, by 4 days in vitro (I), many labeled cells were seen in the migratory stream and within the dentate gyrus. Scale bars: 200 µm.

 


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  		Fig. 4. The chemokine SDF1 and its receptor CXCR4 were expressed in complementary patterns in the developing hippocampus. Schematic representation of the migratory events ongoing at E14.5 and P0 (A,B). Red square shows enlarged area, migrating cells are in blue. Non-radioactive in situ hybridization for SDF1 (C,D) and CXCR4 (E,F) in E14.5 (C,E) and P0 (D,F) wild-type mouse tissue. (C,E) At E14.5, SDF1 (C) was expressed in the meninges, superficially in the cortex and within the developing dentate anlage (arrowhead). At the same age, CXCR-4 (E) was expressed in the dentate ventricular zone and in a continuum from the ventricular zone to the developing dentate (arrow). (D,F) At P0, SDF1 (D) was expressed primarily in the Cajal-Retzius cells that line the hippocampal fissure. CXCR4 (F) was expressed in the dentate migratory stream (arrow), starting above the fimbria and extending into the dentate gyrus. CXCR4 was also present in the forming granule cell layer. Scale bar in F: 250 µm for C-F.

 


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  		Fig. 5. Cxcr4 mutant mice had ectopic dentate granule cells. Prox1 immunohistochemistry in the E18.5 hippocampus of Cxcr4+/– and Cxcr4–/– mice. Scale bar: 250 µm. (A,B) In Cxcr4+/– mice, Prox-1 positive granule cells were in the dentate gyrus. In Cxcr4–/– mice many Prox1-positive granule cells were in the dentate gyrus, but appeared disorganized. Additionally, many Prox1-positive cells were outside the dentate gyrus, near the ventricular zone, above the fimbria and in the dentate migratory stream (arrows). (C,D) In Cxcr4+/– mice Phosphohistone-H3 positive precursor cells were distributed in the dentate subventricular zone, migratory stream and in the tertiary matrix. In addition, note the scattered positive cells throughout the ventricular zone of the hippocampus. In Cxcr4–/– mice the number of dividing precursor cells labeled with Phosphohistone-H3 was dramatically decreased in all the regions but is preserved outside the dentate gyrus in the ventricular zone. (E,F) Nestin antibody staining in Cxcr4+/– and Cxcr4–/– mice showing that the overall distribution of the radial glial network is intact.

 


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  		Fig. 6. Ectopic SDF1 expression disrupted migration of granule cells in vitro. GFP or SDF1 and GFP were electroporated into slice cultures and subsequently allowed to develop for 4 days. Cultures were then double immunostained for GFP and Prox1 to identify the extent of the electroporated tissue and location of granule cells, respectively, in the culture. (A) Schematic outlining the potential roles of SDF1 in dentate granule cell migration and the expected effect of ectopic expression (green shading) upon Prox1-positive cells (orange). (B) In order to express SDF1 ectopically in the hippocampus, we used focal electroporation. A schematic of the apparatus is shown. The slice culture on a membrane was placed on a 5% agarose section in contact with the positive electrode. Another agarose piece containing DNA was placed on the negative electrode and a current was applied. (C-F) Electroporation of GFP without SDF1 resulted in normal granule cell migration. Prox1 (red) is shown with GFP (green) staining to allow comparison of electroporation site with granule cell migration site. Additionally, to visualize clearly the location of all granule cells, Prox1 staining is shown alone. In both examples (C,E and D,F), granule cells migrate appropriately to the dentate gyrus despite GFP (green) expression throughout the hippocampus. (G-J) Electroporation of GFP and SDF1 resulted in disruption of granule cell migration. Again, GFP staining was used to identify the location of electroporated tissue. We show two distinct examples here, one with electroporation throughout the hippocampal formation, including the forming dentate gyrus (G,I) and the other throughout the hippocampus but not including the forming dentate gyrus (H,J). In both examples, Prox1-positive cells can be seen in the fimbrial region. Additionally, when ectopic expression included the middle of the developing dentate (G,I), many cells migrated to the position of the ectopically expressed SDF1 but failed to migrate any further into the dentate gyrus (arrows). When SDF1/GFP is electroporated into the hippocampus, excluding the dentate (H,J), very few cells leave the vicinity of the fimbria. For reference, the fimbria is labeled with an asterisk. Scale bar: 150 µm.

 

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