To test whether these cells are actively producing Shh and might contact the subventricular zone, we pursued two strategies. click here First, we injected
Ad:CreStopLight (Ad:CSL) virus in the ventral SVZ of Shh-Cre animals. This virus contains a CMV promoter driving the expression of the gene encoding red fluorescent protein (and a STOP sequence) flanked by loxP sequences, followed by the gene encoding green fluorescent protein ( Yang and Hughes, 2001). After injecting Ad:CSL in the ventral region of the SVZ, we observed many RFP-positive cells at the injection site ( Figure S4F). We also observed a subset of GFP-positive cells located near the ventral SVZ in the bed nucleus of the stria terminalis, similar to the labeled cells identified in ShhCreER; R26YFP animals ( Figures S4G and S4H). We did not observe GFP-labeled cells after an equivalent injection
in the dorsal SVZ ( Figures S4I and S4J). These ventral cells were therefore actively producing Cre recombinase under the control of the Shh promoter. Previous studies in rodent brain Alectinib solubility dmso have suggested that Shh may be transported anterogradely along axons and secreted distally at axon terminals (Traiffort et al., 2001). To determine if more distant Shh-producing cells might also contact the SVZ, we used the retrograde tracer Fluorogold (hydroxystilbamidine). After treating P90 ShhCreER; R26YFP animals with tamoxifen, we waited two days to allow accumulation of YFP label in the absence of injury and injected Fluorogold into the lateral ventricle. At 2 days after tracer injection, we found significant Fluorogold labeling around the ventricles and in the bed nuclei of the stria terminalis, as well as many labeled cells in the medial septum
and preoptic nuclei. A small number of Fluorogold-labeled cells in the septum also expressed YFP ( Figures 3K–3M), STK38 suggesting that Shh produced in the septum could also reach the SVZ by transport along axonal extensions. We did not observe more distant Fluorogold-labeled cells in the midbrain or hindbrain. Finally, we performed immunohistochemical staining for Shh protein (rabbit mAb 95.9, kind gift from Genentech). We found that Shh protein was present in the septum in cell bodies associated with NeuN-positive nuclei and at low levels in the associated neuropil (Figure 3P). In the ventral SVZ, we also found Shh protein in the neuropil and in association with the apical and basal surfaces of SVZ cells (Figures 3O, 3R, and 4C). Infrequent, punctate staining for Shh protein was also observed in the dorsal SVZ (Figure 3Q), suggesting that a low level of ligand may be present in this region despite the absence of Shh-producing cells or their processes. Although Shh ligand production and pathway activity both occur in the ventral forebrain, this did not necessarily indicate a requirement for this signaling in the generation of particular olfactory interneurons.