Supplementary Figure 6: Dentate gyrus gene expression and connectivity.

(a) Gene expression patterns within the DG revealed three gene expression domains within the granule layer and two distinct cell- groups within the polymorph layer. Images show DG mapped gene expression subdomains from HGEA level 89 aligned with example gene expression patterns (arrowheads mark the boundaries between regions in all images). Btg1 is highly expressed in all parts of the DG granule cell layer, whereas Slc26a10 and Trhr are restricted to dorsal and ventral parts of the granule cell layer respectively. The DGi is defined as the area of overlap between these two unique expression patterns. In the polymorph layer, we found two expression patterns that suggest dorsal (ex. Bid) and ventral (ex. Etv1) cell groups which are clearly segregated at rostral levels but more mixed at caudal levels (see Supplementary Figure 4). (b) DGpo was revealed to contain at least 2 distinct gene expression populations, the DGpod and DGpov. Tracer coinjection into the DGpod near the DGi produced bilateral anterogradely-labeled fibers in the inner molecular layer of the DGd and DGi and bilateral retrogradely-labeled neurons within the DGpov (SW110914-04A). In contrast, retrograde tracer injection in the DGd only labels neurons within the DGpod (SW110914-02A). Anterograde tracer injection within the DGpov produces bilateral fiber labeling within the DGv and DGi only (SW160909-01A). Taken together, the DGpo neuron populations provide a primarily unidirectional ventral-to-dorsal hippocampus pathway. (c) The three DG granule layer subregions (DGd, DGi, and DGv) are differentially connected to the CA3 regions as shown by multiple cases with DG anterograde tracer injections. All DGd granule cell mossy fibers display a hairpin connectivity motif, starting locally onto the DGpo, then travelling rostrally for up to 1mm before turning caudally (innervating multiple levels of CA3dd, CA3d, or CA3id along the way depending on DG granule cell location) to eventually terminate at a topographic location within the CA2 (yellow arrows follow the mossy fiber path across rostrocaudal sections). The geometric length of the mossy fiber pathway is limited by the rostral pole of the hippocampus. (d) In contrast to the hairpin connectivity motif of the DGd granule cells, granule cells within the DGi and DGv project rostrally in a linear orientation through the stratum lucidum of the CA3ic and CA3v/CA3vv, respectively. DGi and DGv mossy fiber pathways do not innervate the CA2. (e) Retrograde injection into the caudal ventral CA2 shows that the CA2 receives convergent input from DG granule cells across multiple rostrocaudal levels. Note the location of retrogradely-labeled DG neurons within the granule layer changes such that DG neurons at rostral levels (right) are close to CA3 and labeled neurons at caudal levels (left) are near the tip of the granule cell layer. (f) Retrograde injections within the CA3v and CA3vv retrogradely labels separate neuron populations within the DGv. For the number of tracer experiments and cross-validated results, see the Supplementary Methods.