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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