1 Supplementary Information A role for primary cilia in glutamatergic synaptic integration of adult-orn neurons Natsuko Kumamoto 1,4,5, Yan Gu 1,4, Jia Wang 1,4, Stephen Janoschka 1,2, Ken-Ichi Takemaru 3, Joel Levine 1 and Shaoyu Ge 1,* 1 Department of Neuroiology and Behavior, 2 Program in Neuroscience, 3 Department of Pharmacological Sciences, SUNY at Stony Brook, Stony Brook, NY 11794 4 These authors equally contriuted to the work 5 Current address: Department of Anatomy and Neuroscience, Osaka University Graduate School of Medicine, Suita, Japan Contents: Supplementary Figure 1-9 Supplementary Video 1 Nature Neuroscience: doi:1.138/nn.342
a 5 dtomato-f2a-ift88::egfp-3 a-1 dtomato dtomatoift88 dtomatoift88aciii Merged ACIII Merged 2 a-2 a-3 Neworn neurons with primary cilia (%) Ift88-EGFP * 1 5 5 14 21 Time (dpi) c 15 1 5 Centrin-2 expression 14dpi 14dpi Continual Induced Supplementary figure 1. Primary cilia assemly in developing adult-orn neurons. (a) A schematic diagram of the inducile retroviral vector for co-expression of dtomato and Ift88 fused with EGFP. Shown are representative confocal images (a-1, a-2 and a-3) of adult-orn neurons at 21 dpi expressing the dtomato and Ift88-EGFP, counterstained for DAPI and immunostained for ACIII. Arrows indicate primary cilia. Scale ar: 2.5 µm. () Quantification of primary cilia development in adult-orn neurons at 5, 14 and 21 dpi. (c) Ectopic expression of Centrin-2 has no significant effect on cilium assemly at 14 dpi. Shown is the percent of adult-orn DGCs with cilia after continuous expression of Centrin-2 for 14 days versus induced expression from12 dpi to 14 dpi y the administration of doxycycline. The group of neurons continuously expressing Centrin-2 is also presented in Fig. 1c. Values represent mean+sem (n=25-48 neurons; *: p<.1, ANOVA). Nature Neuroscience: doi:1.138/nn.342
3 a c Cilium length (µm) 6 3 3 6 12 24 GCL 14 21 28 (dpi).. θ SGZ 12 24 Primary cilia in the leading area (%) 1 5 12 24 14 21 Time (dpi) o ( ) Supplementary figure 2. Primary cilia consistently originate from the leading edge of migrating DGCs ut do not protrude in a fixed direction. (a) A sample image and schematic diagram of a developing neworn DGC, indicating the sugranular zone (SGZ), the granule cell layer (GCL), the stereotypic direction of migration (arrow), the leading edge of the nucleus (middle dashed line), centrosome (dots), primary cilium (solid line), and cilium angle (θ). () Percent of neworn DGCs at 14 or 21 dpi that assemle primary cilia in the leading side of the nucleus. Values represent mean+sem (n=32-48 neurons; *: p<.1, ANOVA). (c) Radial plot of cilia angle and length at 14, 21 and 28 dpi. Radial unit: 2 µm. Nature Neuroscience: doi:1.138/nn.342
Relative line Grey value LAMP-1dTomatoDAPI a Relative line Grey value LAMP-1dnKif3ADAPI -Cell 1 -Cell 2 -Cell 3 4 1 2 6 Line 1 4 2 6 Line 2 4 2 5 1 15 2 25 3 dnkif3a-cell 1 5 1 15 2 25 3 dnkif3a-cell 2 5 1 15 2 25 3 dnkif3a-cell 3 Line 1 6 4 2 Line 2 6 4 2 5 1 15 2 25 3 5 1 15 2 25 3 5 1 15 2 25 3 Supplementary figure 3. Asence of late endosome/lysosome clusters in adult-orn DGCs after ectopic expression of dnkif3a. (a-) Comparison of the intensity of LAMP-1 endosome immunostaining in wild-type versus experimental adult-orn DGCs at 21 dpi. Randomly selected cells from each group were analyzed along their major (center of nucleus to proximal apical dendrite) and minor axes, as indicated in the schematic overlay of the first panel. (a) At top, representative confocal images of three wild-type adult-orn neurons immunostained for LAMP-1 (green panels), as visualized with retroviral dtomato and DAPI (merged red-lue panels). At ottom, linear plots of LAMP-1 intensity along the axes. () LAMP-1 staining and measurement in three dnkif3a expressing DGCs. Scale ar: 5 µm. Nature Neuroscience: doi:1.138/nn.342
a dtomato Biocytin DAPI 5 dnkif3adnkif3a+ 8 Mean eepscs (pa) 6 4 2 dnkif3a Supplementary figure 4. Mature DGCs expressing dnkif3a have normal dendritic synaptic activity. (a) Mature DGCs infected y AAV-dnKif3a injected into the dentate gyrus. Shown are confocal images of two recorded DGCs filled with iocytin through the recording pipette. The left-most cell is negative for dnkif3a (left inset), and the right-most is positive for dnkif3a (right panels). The recording paradigm was the same as that in Fig. 3. Scale ar: 2 µm. () Synaptic responses as measured y evoked excitatory post-synaptic currents (eepscs) in mature dnkif3a- and dnkif3a+ DGCs. All values represent mean+sem (n=5-12 neurons; p<.1, ANOVA). Nature Neuroscience: doi:1.138/nn.342
6 a Total dendritic length (µm) 24 16 8 dnkif3a Total ranching numer 7.5 5. 2.5 dnkif3a Supplementary figure 5. Dendritic growth of control and dnkif3a+ adult-orn DGCs at 7 dpi. 7 dpi control neurons versus dnkif3a-induced (5 to 7 dpi) neurons were analyzed for (a) total dendritic length and () total dendritic ranching numer (n=25-26 cells; *: p<.1, ANOVA). All values represent mean±sem. Nature Neuroscience: doi:1.138/nn.342
a shrna -sh #1 #2 #3 #4 (kda) mift88-egfp 12 Normalized intensity againstt control (%) c Total dendritic length (µm) GAPDH 1..5. 9 6 3 -sh #1 * * #2 d #3 Total dendritic ranch numer 8 6 4 2 #4 38 5 U6-shRNA Ift88-EF1a-mCherry-3 Centrin-2 ACIII Merged -sh shift88#4 Cilia length (µm) 3 2 1 7 * -sh shift88#4 shift88#4 shift88#4 Supplementary figure 6. Primary cilia deletion via shrna knockdown in adult-orn neurons results in defective dendritic refinement in the entorhinal cortex projection field of the dentate gyrus. (a) Validation of the efficacy of various shrnas against mouse Ift88 in vitro. Retroviral constructs expressing control (randomly-generated sequence not complementary to any known gene) or different shrnas against mouse Ift88 were co-transfected together with an expression construct for mift88-egfp into HEK293 cells. The top panel shows a representative immunolot using anti-gfp and anti-gapdh antiodies. Shown elow is the densitometric quantification of the relative amounts of mift88-egfp. All values represent mean±sem (n=3; *: p<.1, ANOVA). () Disruption of primary cilia assemly upon Ift88 knock-down. At left, sample images of control and shift88#4+ DGCs at 28 dpi, immunostained for ACIII and counterstained with DAPI. At right, quantification of cilia length. Scale ar: 5 µm. (c) Dendritic lengths of 28 dpi control and shift88#4+ DGCs. (d) Dendritic ranch numer of 28 dpi control and shift88#4+. (For, c and d, n=3-35 cells, *: p<.1, ANOVA). All values represent mean±sem. Nature Neuroscience: doi:1.138/nn.342
a Centrin-2DAPIdTomato Distance from centrosome to nucleus (µm) 24 16 8 7dpi 14dpi 21dpi GL SGZ shift88#4 8 14 28 Time (dpi) 14 28 Time (dpi) Supplementary figure 7. Developing adult-orn DGCs with deleted primary cilia display normal centrosome migration. (a) Representative images of Centrin-2 laeled centrosomes in adult-orn DGCs. Shown are single plane images of the centrioles (arrows) at 7, 14 and 28 dpi, oriented with the SGZ at the ottom. Scale ar: 1µm. () Centrosome position within developing adult-orn DGCs. At left, the distance etween the centrosome and the center of nucleus in control adult-orn DGCs at 5, 14, 21 and 28 dpi, showing the transient migration of centrosomes away from the nucleus during development. At right, a summary of the distance etween centrosome and nucleus in control and shift88#4 expressing adult-orn DGCs at identical time points. Values in represent mean±sem (n=32-34 cells; control values in oth figures of were from the same group of cells; *: p<.5, ANOVA). Nature Neuroscience: doi:1.138/nn.342
9 LEnt MEnt MC GCL 14dpi 21dpi Wnts Wnts β-catenin β-catenin Dendritic elongation Synaptogenesis Centrosome Primary cilium Glutamatergic Synapses Supplementary figure 8. A model of the effects of primary cilia assemly on dendritic refinement and synapse formation with entorhinal cortical projections in adult-orn DGCs. At 14 dpi, cilia have not yet assemled, Wnt/β-catenin signaling is active and dendrites are immature. At 21dpi, newly formed cilia suppress Wnt/β-catenin signaling allowing for further growth of dendrites. GCL, granule cell layer. MC, mossy cell projecting layer. Nature Neuroscience: doi:1.138/nn.342
1 dnkif3a 2 eepscs amplitude (pa) 1..2.4 Stimulating Intensity (ma) Supplementary figure 9. eepscs amplitude in control and dnkif3a+ adult-orn neurons. Increasing the stimulus intensity did not evoke eepscs in cilia-deleted neurons without synaptic response. Nature Neuroscience: doi:1.138/nn.342