Stereotaxic injection of C1ql2-expressing AAV into Bcl11b cKO DGN restores C1ql2 levels

a Experimental design to analyze the functions of C1ql2 in the MFS as a downstream target of Bcl11b. b AAV constructs injected in the DG of Bcl11b cKO and control littermates. c Western blot and d relative C1ql2 protein levels in mouse hippocampal homogenates. n=3. All data are presented as means; error bars indicate SEM. Two-way ANOVA and Tuckey’s PHC. Bcl11b cKO+EGFP-2A-C1ql2 vs. Bcl11b cKO+EGFP: *p=0.015; Bcl11b cKO+EGFP-2A-C1ql2 vs. Bcl11b cKO+EGFP-2A-C1ql3: *p=0.019; ns, not significant. e Immunohistochemistry of C1ql2 (cyan), GFP (green) and Bcl11b (red) on hippocampal sections. Scale bar: 200 μm. f Immunohistochemistry of C1ql2 (cyan), vGlut1 (magenta) and Homer1 (yellow) in the SL of CA3. White arrowheads indicate co-localizing puncta of all three proteins. Scale bar: 15 μm

C1ql2 reintroduction in Bcl11b cKO DGN rescues SV recruitment in MFS

a Electron microscope images of MFS and proximal SV. White bars mark synapse length from the postsynaptic side. Scale bar: 100 nm. b Average synapse score. Control+EGFP, n=3; Bcl11b cKO+EGFP, Bcl11b cKO+EGFP-2A-C1ql2, Bcl11b cKO+EGFP-2A-C1ql3, n=4. Two-way ANOVA and Tuckey’s PHC. Control+EGFP vs. Bcl11b cKO+EGFP: ***p=0.0002, and vs. Bcl11b cKO+EGFP-2A-C1ql3: ***p=0.0003; Bcl11b cKO+EGFP-2A-C1ql2 vs. Bcl11b cKO+EGFP and vs. Bcl11b cKO+EGFP-2A-C1ql3: ***p<0.0001; ns, not significant. c Relative frequency of synapse scores. d Number of docked vesicles per 100 nm AZ profile length. Control+EGFP, Bcl11b cKO+EGFP-2A-C1ql3, n=3; Bcl11b cKO+EGFP, Bcl11b cKO+EGFP-2A-C1ql2, n=4. All data are presented as means; error bars indicate SEM. Points represent the individual examined AZ and SV, respectively. Two-way ANOVA and Tuckey’s PHC. Control+EGFP vs. Bcl11b cKO+EGFP: *p=0.024, and vs. Bcl11b cKO+EGFP-2A-C1ql3: *p=0.045; Bcl11b cKO+EGFP-2A-C1ql2 vs. Bcl11b cKO+EGFP: *p=0.026, and vs. Bcl11b cKO+EGFP-2A-C1ql3: *p=0.049; ns, not significant. e Diameter of docked vesicles. Control+EGFP, Bcl11b cKO+EGFP-2A-C1ql3, n=3; Bcl11b cKO+EGFP, Bcl11b cKO+EGFP-2A-C1ql2, n=4; Two-way ANOVA. ns, not significant. f Cumulative and g relative frequency of the number of docked vesicles per 100 nm AZ profile length.

C1ql2 reintroduction in Bcl11b cKO DGN rescues mossy fiber LTP

a Representative fEPSP traces showing baselines before HFS (black), fEPSP changes 30-40 min after HFS (cyan) and following the application of 3 µM DCG-IV (red). b Time course of fEPSP slopes. The black arrow indicates HFS and the dashed line the baseline level. c Quantification of fEPSP facilitation at four different time intervals after HFS. Changes in fEPSP slope are shown as percentage of the mean baseline fEPSP. Control+EGFP, n=7; Bcl11b cKO+EGFP, Bcl11b cKO+EGFP-2A-C1ql3, n=8; Bcl11b cKO+EGFP-2A-C1ql2, n=6; All data are presented as means; error bars indicate SEM. One-way ANOVA followed by Bonferroni’s PHC for each time interval. 20-30 min: Control+EGFP vs. Bcl11b cKO+EGFP: **p=0.002, and vs. Bcl11b cKO+EGFP-2A-C1ql3: *p=0.011; Bcl11b cKO+EGFP-2A-C1ql2 vs. Bcl11b cKO+EGFP: *p=0.023; 30-40 min: Control+EGFP vs. Bcl11b cKO+EGFP: ***p<0.001, and vs. Bcl11b cKO+EGFP-2A-C1ql3: **p=0.002; Bcl11b cKO+EGFP-2A-C1ql2 vs. Bcl11b cKO+EGFP: *p=0.01 and vs. Bcl11b cKO+EGFP-2A-C1ql3: *p=0.0523; ns, not significant. d Representative fEPSP traces showing baselines before forskolin application (black), fEPSP changes 105-120 min after the start of application (cyan) and following the addition of 3 µM DCG-IV (red). e Time course of fEPSP slopes. The black solid line indicates forskolin perfusion and dashed line the baseline level. f Quantification of fEPSP facilitation at two different time intervals after the start of forskolin application. Changes in fEPSP slope are shown as percentage of the mean baseline fEPSP. n=8. All data are presented as means; error bars indicate SEM. Unpaired t-test for both time intervals. 15-30 min: **p=0.005; 105-120 min: **p=0.0025.

KD of C1ql2 in DGN of WT mice impairs SV recruitment and LTP

a Experimental design to analyze the MFS after AAV-mediated KD of C1ql2 in WT DGN. b Relative C1ql2 mRNA levels in DGN. n=4 (3). All data are presented as means; error bars indicate SEM. Unpaired t-test: ***p=0.0002. c Western blot of mouse hippocampal homogenates. d Immunohistochemistry of C1ql2 (red) and GFP (green) on hippocampal sections. Scale bar: 200 μm. e Average synapse score. n=4. Unpaired t-test. *p=0.025. f Number of docked vesicles per 100 nm AZ profile length. n=3. All data are presented as means; error bars indicate SEM. Points represent the individual examined AZ. Unpaired t-test. *p=0.018. g Representative fEPSP traces showing baselines before HFS (black), fEPSP changes 30-40 min after HFS (cyan) and following the application of 3 µM DCG-IV (red). h Time course of fEPSP slopes. The black arrow indicates HFS and the dashed line the baseline level. i Quantification of fEPSP facilitation at four different time intervals after HFS. Changes in fEPSP slope are shown as percentage of the mean baseline fEPSP. +shNS-EGFP, n=6; +shC1ql2-EGFP, n=7. All data are presented as means; error bars indicate SEM. Mann-Whitney U-test for each time interval. 10-20 min: **p=0.0012; 20-30 min: **p=0.0023; 30-40 min: **p=0.0023; ns, not significant.

C1ql2-Nrxn3 interaction recruits vGlut1 in vitro

a Immunocytochemistry of myc-tagged C1ql2, C1ql2.K262E or myc-tag (magenta) expressing HEK293 cells and GFP-Nrxn3α(25b+) (cyan) from contacting hippocampal neurons. Scale bar: 5 μm. b Nrxn3α(25b+) recruitment by differentially transfected HEK293 cells. n=3. All data are presented as means; error bars indicate SEM. One-way ANOVA and Tuckey’s PHC. myc-C1ql2 vs. myc-tag: *p=0.016, and vs. myc-K262E: *p=0.022; ns, not significant. c Immunocytochemistry of myc-tagged C1ql2, C1ql2.K262E or myc-tag (magenta) expressing HEK293 cells and vGlut1 (cyan) from contacting hippocampal neurons. Scale bar: 5 μm. d vGlut1 recruitment by differentially transfected HEK293 cells. n = 3. All data are presented as means; error bars indicate SEM. One-way ANOVA and Tuckey’s PHC. myc-C1ql2 vs. myc-tag: *p=0.04, and vs. myc-K262E: **p=0.007; ns: non-significant. e Immunocytochemistry of myc-tagged C1ql2 (magenta) expressing HEK293 cells and vGlut1 (cyan) from contacting control, Nrxn123 KO or Nrxn123 KO with Nrxn3α(25+) rescued hippocampal neurons. Scale bar: 5 μm. f vGlut1 recruitment by HEK293 cells in presence or absence of neuronal Nrxns. n = 3. All data are presented as means; error bars indicate SEM. One-way ANOVA and Tuckey’s PHC. inactive Cre vs. Cre: *p=0.023, and vs. Cre+ Nrxn3α(25+): p=0.21; ns: non-significant. g Trimeric structures of C1ql2 (PDB_ID: 4QPY, upper panels) and the variant C1ql2.K262E (lower panels). Residue 262 is the central residue (red, left & middle panels) of a larger area underneath the C1ql2-specific calcium and receptor binding loops (magenta, middle panel). The mutation K262E alters the charge of that surface area negative (yellow-circled area, right panels) and makes it potentially repulsive to bind Nrxn3(25b+).

C1ql2-Nrxn3(25b+) interaction is important for C1ql2 localization at the MFS and SV recruitment

a Experimental design to analyze the MFS after AAV-mediated expression of C1ql2.K262E in Bcl11b cKO DGN. b Western blot and c relative C1ql2.K262E protein levels in mouse hippocampal homogenates. n=3. All data are presented as means; error bars indicate SEM. Mann-Whitney U-test. ns: non-significant. d Immunohistochemistry of C1ql2 (red) and GFP (green) in hippocampal sections. Scale bar: 200 μm. Upper right corner of C1ql2 panels depicts close-ups from the SL of CA3. Scale bar: 15 μm. e Integrated density of C1ql2 fluorescence in the SL of CA3. n=3. All data are presented as means; error bars indicate SEM. Unpaired t-test. *p=0.008. f Electron microscope images of MFS and proximal SV. White bars mark synapse length from postsynaptic side. Scale bar: 100 nm. g Average synapse score. n=3. Unpaired t-test. **p=0.0012. h Number of docked vesicles per 100 nm AZ profile length. n=3. All data are presented as means; error bars indicate SEM. Points represent the individual examined AZ. Unpaired t-test. *p=0.018.

Nrxn KO perturbs C1ql2 localization at the MFS and SV

a Experimental design to analyze the MFS after AAV-mediated Nrxn KO. b Relative Nrxn3 mRNA levels. n=4. All data are presented as means; error bars indicate SEM. Unpaired t-test. **p=0.007. c Immunohistochemistry of C1ql2 (red) and GFP (green) in hippocampal sections. Scale bar: 200 μm. Upper right corner of C1ql2 panels depicts close-ups from the SL of CA3. Scale bar: 15 μm. d Integrated density of C1ql2 fluorescence in the SL of CA3. n=3. All data are presented as means; error bars indicate SEM. Unpaired t-test. *p=0.02. e Immunohistochemistry of ZnT3 (red) and GFP (green) in hippocampal sections. Scale bar: 200 μm. Upper right corner of ZnT3 panels depicts close-ups from the SL of CA3. Scale bar: 15 μm. f Electron microscope images of MFS and proximal SVs. White bars mark synapse length from postsynaptic side. Scale bar: 100 nm. g Average synapse score. n=3. Unpaired t-test. **p=0.009. h Number of docked vesicles per 100 nm AZ profile length. n=3. All data are presented as means; error bars indicate SEM. Points represent the individual examined AZ. Unpaired t-test. **p=0.007.

C1ql2 mRNA and protein are lost upon Bcl11b cKO in DGN

a mRNA in situ hybridization of C1ql2 on hippocampal sections. b Immunohistochemistry of C1ql2 (cyan) and Bcl11b (magenta) on hippocampal sections.

C1ql2 reintroduction in Bcl11b cKO DGN does not rescue MFS number and MFB complexity

a Immunohistochemistry of C1ql3 (red) and GFP (green) on hippocampal sections. Scale bar: 200 μm. b Relative C1ql3 mRNA levels in DGN. Control+EGFP, Bcl11b cKO+EGFP, Bcl11b cKO+EGFP-2A-C1ql2, n=4; Bcl11b cKO+EGFP-2A-C1ql3, n=3. All data are presented as means; error bars indicate SEM. Two-way ANOVA. ns, not significant. c Electron microscopy images of MFBs (purple) and contacting postsynaptic spines (yellow). Scale bar: 500 nm. d MFB perimeter-to-area ratio. n=5. All data are presented as means; error bars indicate SEM. Two-way ANOVA and Tuckey’s PHC. Control+EGFP vs. Bcl11b cKO+EGFP: *p=0.035, and vs. Bcl11b cKO+EGFP-2A-C1ql2: **p=0.0014; ns, not significant. e vGlut1 and Homer1 double positive puncta in selected CA3 SL ROIs. n=3. All data are presented as means; error bars indicate SEM. Two-way ANOVA and Tuckey’s PHC. Control+EGFP vs. Bcl11b cKO+EGFP: *p=0.047, and vs. Bcl11b cKO+EGFP-2A-C1ql2: *p=0.029; ns, not significant. f Representative fEPSP traces showing responses to a 20 V electrical stimulation (black, black arrowheads indicate stimulation). The signal is almost entirely blocked by the application of 3 µM DCG-IV (red). g Input-output curves generated by plotting fEPSP slope against fiber volley amplitude at increasing stimulation intensities. Control+EGFP, n = 35; Bcl11b cKO+EGFP, n = 32. The data are presented as means, error bars represent SEM.

KD of C1ql2 in DGN of WT mice impairs SV recruitment

a Immunohistochemistry of C1ql3 (red) and GFP (green) on hippocampal sections. Scale bar: 200μm. b Relative C1ql3 mRNA levels in DGN. n=4. All data are presented as means; error bars indicate SEM. Unpaired t-test. ns, not significant. c Relative frequency of synapse scores (refers to Figure 4e). d Cumulative and e relative frequency of the number of docked vesicles per 100 nm AZ profile length (refer to Figure 4f). f Diameter of the docked vesicles. n=3. All data are presented as means; error bars indicate SEM. Points represent the individual examined SV. Unpaired t-test. ns, not significant.

C1ql2-Nrxn3(25b+) interaction is important for SV recruitment at the MFS

a Immunohistochemistry for C1ql3 (red) and GFP (green) on hippocampal sections. Scale bar: 200 μm. b Relative frequency of synapse scores (refers to Figure 6g). c Cumulative and d relative frequency of the number of docked vesicles per 100 nm AZ profile length (refer to Figure 6h). e Diameter of the docked vesicles. n=3. All data are presented as means; error bars indicate SEM. Points represent the individual examined SV. Unpaired t-test. ns, not significant. f Representative fEPSP traces showing baselines before HFS (black), fEPSP changes 30-40 min after HFS (cyan) and following the application of 3 µM DCG-IV (red). g Time course of fEPSP slopes. The black arrow indicates HFS and the dashed line the baseline level. h Quantification of fEPSP facilitation at four different time intervals after HFS. Changes in the fEPSP slope are shown as percentage of the mean baseline fEPSP. Data for C1ql2.K262E from f-h in this figure are compared with control data from Figure 3a-c. Control+EGFP, n=7; Bcl11b cKO+EGFP-2A-K262E, n=5. All data are presented as means; error bars indicate SEM. Mann-Whitney U-test for each time interval. 0-10 min: **p=0.005; ns, not significant.

Nrxn KO perturbs C1ql2 localization and SV recruitment at the MFS

a Immunohistochemistry for C1ql3 (red) and GFP (green) on hippocampal sections. Scale bar: 200 μm. b Relative frequency of synapse scores (refers to Figure 7g). c Cumulative and d relative frequency of the number of docked vesicles per 100 nm AZ profile length (refer to Figure 7h). e Diameter of the docked vesicles. n=3. All data are presented as means; error bars indicate SEM. Points represent the individual examined SV. Unpaired t-test. ns, not significant.