Journal: bioRxiv

Article Title: Extracellular matrix proteolysis maintains synapse plasticity during brain development

doi: 10.1101/2025.02.27.640672

Figure Lengend Snippet: a) Schematic of the perisynaptic ECM which consists of a hyaluronan sugar matrix connected by proteoglycans including brevican, linking proteins like tenascins, and others. b) Representative images of zebrafish hindbrain at 14 and 60 days post fertilization (dpf) showing brevican (antibody staining) and hyaluronan labeled with a genetically encoded sensor ubi:ssncan-GFP . Scales: 100 µm. c) Quantification of fluorescence intensity of brevican and hyaluronan ( ubi:ssncan-GFP ) over development from 7-90 dpf. Mean fluorescence intensity for brevican and hyaluronan were normalized to the maximum intensity measured for each (90 dpf and 28 dpf, respectively). Brevican quantification, number of fish: 7 dpf, n=9; 14 dpf, n=8; 21 dpf, n=8; 28 dpf, n=7; 60 dpf, n=7; 90 dpf, n=6. For hyaluronan, number of fish: 7 dpf, n=11; 14 dpf, n=10; 21 dpf, n=6; 28 dpf, n=10; 60 dpf, n=7; 90 dpf, n=3. d) Schematic of zebrafish larval hindbrain. Gray dots indicate cell bodies and the synaptic region is shown by pink. A cholinergic neuron with a cell body and dendrites is shown by black. e) Dorsal view of zebrafish hindbrain at 10 dpf shows sparsely labeled cholinergic neurons expressing a TdTomato (TdT) tagged FingR construct that binds to the excitatory postsynaptic marker PSD-95. Tg(chata:gal4);Tg(zcUAS:PSD95.FingR-TdT-CCR5TC-KRAB(A)), hereafter abbreviated Chat-PSD95 FingR . Scale: 20 µm. f) Strategy for quantification of synapses using Chat-PSD95 FingR . Insets of region in e shows (i) several sparsely labeled neurons and (ii) a dendritic segment from one cholinergic neuron with synapses indicated by asterisks. Scales: 20 µm. g) Immunostaining for brevican protein and Chat-PSD95 FingR at 14 dpf. Synaptic region is indicated in the image. Scale: 5 µm. h) Schematic of 24 hours time lapse imaging assay to quantify changes of synapse density. i) Representative images show a single Chat-PSD95 FingR dendrite imaged at 7 dpf (t=0) and 8 dpf (t=24). Pink arrowheads: synapses present at t=0 and absent at t=24 “lost synapses”. Green arrowheads: synapses that appear at t=24 “new synapses”. White circles: present at both time points. Scale: 5 µm. j) Quantification of total excitatory synapse density and dynamics over the live imaging window of hindbrain development (5-14 dpf), based on Chat-PSD95 FingR-TdT puncta normalized to µm of dendrite length. Black line indicates static synapse density per day (p=0.3437, One-way ANOVA). Pink bars indicate lost synapses (F (4,78) p=0.0040**, One-way ANOVA). Green bars indicate new synapses (F (4,78) p=0.018*, One-way ANOVA). Asterisks in the figure represent results of Tukey’s multiple comparisons with respect to the 5-6 dpf. Number of fish: 5-6 dpf, n=15; 7-8 dpf, n=18; 9-10 dpf, n=18; 11-12 dpf, n=15; 13-14 dpf, n=17. Results from individual fish in . k) Schematic of time lapse imaging to determine the fate of individual synapses at the indicated timepoints. Synapses at t=0 were defined as “stable” synapses. Synapses born between t=0 and t=6 were defined as “new” synapses and subsequently followed with the 6 hour timepoint set as t=0 (lower timecourse). Experiments were performed at 10-12 dpf. l) Representative image of a single excitatory synapse imaged at t=0, 6, 12, and 24 shows a “new synapse” born between t=0 and t=6, which disappeared between t=12 and t=24. Left shows a low power image of a Chat-PSD95 FingR cholinergic neuron. Dashed square indicates inset. Inset shows raw fluorescence (top) and fluorescence overlaid with 3D reconstruction of synapses (bottom). Arrowheads: newborn synapse. Circle: site of newborn synapse. Scale: 5 µm in low power image and 2 µm in inset. m) Kaplan-Meier plot of survival of individual synapses over time (Data from n=19 fish, n=427 stable synapses and n=25 new synapses). n) Quantification of the distance from the nearest synapse for stable and new synapses at t=6 (Stable, n=116 inter-synapse intervals from n=14 fish; New, n=25 inter-synapse intervals from n=14 fish; p<0.0001****, Welch’s t-test, performed for synapses). The inter-synapse intervals were normalized by the mean of stable synapses for each cell. Values were plotted as mean ±SEM. ****: p<0.0001; **: p<0.01; *: p<0.05; ns: not significant.

Article Snippet: The following primary antibodies were used: anti-GFP chicken polyclonal antibody (Aves Labs GFP-1020, 1:1000), anti-brevican mouse monoclonal antibody (1:100) , Living Colors DsRed polyclonal antibody (Clontech 632496, 1:1000), anti-SV2 mouse monoclonal antibody (DSHB, 1:500), anti-4C4 mouse monoclonal antibody (Gift from Hitchcock lab, 1:200), anti-HA rabbit monoclonal antibody (Cell Signaling Technology 3724T, 1:500).

Techniques: Staining, Labeling, Fluorescence, Expressing, Construct, Marker, Immunostaining, Imaging

Journal: bioRxiv

Article Title: Extracellular matrix proteolysis maintains synapse plasticity during brain development

doi: 10.1101/2025.02.27.640672

Figure Lengend Snippet: a) Representative images of brevican and hyaluronan ( ubi:ssncan-GFP ) in the hindbrain at 7, 14, 28, and 60 dpf. Scales: 100 µm. b) Representative image of PNN (perineuronal net)-like brevican staining at 60 dpf. Square indicates the region of inset on the right. Arrowheads indicate PNN-like brevican signals. Scale: 100 µm.

Article Snippet: The following primary antibodies were used: anti-GFP chicken polyclonal antibody (Aves Labs GFP-1020, 1:1000), anti-brevican mouse monoclonal antibody (1:100) , Living Colors DsRed polyclonal antibody (Clontech 632496, 1:1000), anti-SV2 mouse monoclonal antibody (DSHB, 1:500), anti-4C4 mouse monoclonal antibody (Gift from Hitchcock lab, 1:200), anti-HA rabbit monoclonal antibody (Cell Signaling Technology 3724T, 1:500).

Techniques: Staining

Journal: bioRxiv

Article Title: Extracellular matrix proteolysis maintains synapse plasticity during brain development

doi: 10.1101/2025.02.27.640672

Figure Lengend Snippet: a) Schematic of strategy for ECM digestion by hyaluronidase injection into the hindbrain ventricle. Tissues fixed or imaged 6 hours after injection with hyaluronidase or vehicle (PBS). b) Representative images of Chat-PSD95 FingR dendrites at 14 dpf from fixed section stained for ΤdT after vehicle or hyaluronidase injection. Scale: 5 µm. c) Quantification of synapse density at 14 dpf (Chat-PSD95 FingR puncta) per µm of dendrite length. Dots represent means per fish, with at least 1 dendrite quantified per fish (vehicle, n=14 fish; hyaluronidase, n=17 fish; p=0.020*, Welch’s t-test). d) Representative merged images of Chat-PSD95 FingR collected before injection (t=0, pink) and 6 hours after (t=6, green) hyaluronidase or vehicle injection. Overlap of pink and green appears white. Pink arrowheads: lost synapses, green arrowheads: newly observed synapses. Experiment performed at 10-12 dpf. Non-merged images in . Scale: 5 µm. e) Quantification of newly observed, lost, or stable synapses between t=0 and t=6 after hyaluronidase vs. vehicle injection (vehicle, n=17 fish; Hyal, n=16 fish; p=0.017* for newly observed, p=0.117 for lost, p=0.722 for stable, Welch’s t-test). f) Schematic of generation of brevican knock-out ( bcan -/- ) fish by CRISPR genome editing. Guide RNAs targeting exon 3 and exon 14 were injected to delete 19 kbp of the bcan gene. The truncation resulted in loss of bcan mRNA and brevican protein (see Extended Data Fig 3e,f). g) Representative images of Chat-PSD95 FingR dendrites at 14 dpf from fixed section stained for ΤdT from bcan +/+ vs. bcan -/- fish. Scale: 5 µm. h) Quantification of synapse density at 14 dpf (Chat-PSD95 FingR puncta) per µm of dendrite length. Dots represent means per fish, with at least 1 dendrite quantified per fish ( bcan +/+ , n=16 fish; bcan -/- , n=14 fish; p=0.031*, Welch’s t-test). i) Representative merged images of Chat-PSD95 FingR collected from bcan +/+ and bcan -/- at t=0 (pink) and t=24 (green). Pink arrowheads: lost synapses, green arrowheads: new synapses. Experiment performed at 10-12 dpf. Non-merged images in . Scale: 5 µm. j) Quantification of newly observed, lost and stable synapses between t=0 and t=24 bcan +/+ vs. bcan -/- fish ( bcan +/+ , n=13 fish; bcan -/- , n=15 fish; p=0.010* for newly observed, p=0.288 for lost, p=0.085 for stable, Welch’s t-test). Values were plotted as mean ±SEM. *: p<0.05; ns: not significant.

Article Snippet: The following primary antibodies were used: anti-GFP chicken polyclonal antibody (Aves Labs GFP-1020, 1:1000), anti-brevican mouse monoclonal antibody (1:100) , Living Colors DsRed polyclonal antibody (Clontech 632496, 1:1000), anti-SV2 mouse monoclonal antibody (DSHB, 1:500), anti-4C4 mouse monoclonal antibody (Gift from Hitchcock lab, 1:200), anti-HA rabbit monoclonal antibody (Cell Signaling Technology 3724T, 1:500).

Techniques: Injection, Staining, Knock-Out, CRISPR

Journal: bioRxiv

Article Title: Extracellular matrix proteolysis maintains synapse plasticity during brain development

doi: 10.1101/2025.02.27.640672

Figure Lengend Snippet: a) Representative images and quantification of hyaluronan depletion after hyaluronidase injection at 14 dpf, measured as mean fluorescence intensity of GFP ( ubi:ssncan-GFP ) at 6 hours post injection (hpi) (vehicle, n=7 fish; Hyal, n=8 fish; p=0.0023**, Welch’s t-test) and 24 hpi (vehicle, n=5 fish; Hyal, n=4 fish; p=0.392, Welch’s t-test). GFP intensity normalized to mean of vehicle control. Dashed lines indicate hindbrain regions. Scale: 100 µm. b) Representative images and quantification of hyaluronan depletion at 6 hpi at 60 dpf, measured as mean fluorescence intensity of GFP ( ubi:ssncan-GFP ) nomalized to vehicle control (vehicle, n=3 fish; Hyal, n=3 fish; p=0.049*, Welch’s t-test). Scale: 100 µm. c) Representative images and quantification of brevican depletion at 6 hours after hyaluronidase injection at 14 dpf (vehicle, n=19 fish; Hyal, n=18 fish; p=0.0025**, Welch’s t-test). Brevican intensity normalized to mean of vehicle control. Dashed lines indicate hindbrain regions. Scale: 20 µm. d) Representative images and quantification of SV2 presynapse marker 6 hours after hyaluronidase injection at 14 dpf (vehicle, n=7 fish; Hyal, n=8 fish; p=0.003**, Welch’s t-test). SV2 intensity normalized to mean of vehicle control. Scale: 100 µm. e) Non-merged images of time lapse imaging from . Circles indicate stable synapses. Boxes indicate the region shown in . Scale: 5 µm. f) Expression of bcan gene in bcan -/- fish assessed by RT-qPCR from whole larvae. Expression normalized to ef1a housekeeper gene (n=3/group; p=0.0064**, Welch’s t-test). g) Representative images and quantification of brevican protein in bcan -/- fish ( bcan +/+ , n=10 fish; bcan -/- , n=12 fish; p<0.0001****, Welch’s t-test). Brevican intensity normalized to mean of bcan +/+ control. Scale: 100 µm. h) Representative images and quantification of SV2 presynapse marker in bcan -/- fish at 14 dpf ( bcan +/+ , n=15 fish; bcan -/- , n=15 fish, p=0.049*, Welch’s t-test). SV2 intensity normalized to mean of bcan +/+ control. Scale: 100 µm. i) Non-merged images of time lapse imaging experiment from . Circles indicate stable synapses. Boxes indicate the region shown in . Scale: 5 µm. Values were plotted as mean ±SEM. ****: p<0.0001; **: p<0.01; *: p<0.05; ns: not significant.

Article Snippet: The following primary antibodies were used: anti-GFP chicken polyclonal antibody (Aves Labs GFP-1020, 1:1000), anti-brevican mouse monoclonal antibody (1:100) , Living Colors DsRed polyclonal antibody (Clontech 632496, 1:1000), anti-SV2 mouse monoclonal antibody (DSHB, 1:500), anti-4C4 mouse monoclonal antibody (Gift from Hitchcock lab, 1:200), anti-HA rabbit monoclonal antibody (Cell Signaling Technology 3724T, 1:500).

Techniques: Injection, Fluorescence, Control, Marker, Imaging, Expressing, Quantitative RT-PCR

Journal: bioRxiv

Article Title: Extracellular matrix proteolysis maintains synapse plasticity during brain development

doi: 10.1101/2025.02.27.640672

Figure Lengend Snippet: a) Schematic of microglial ablation by adding metronidazole (Mtz) to fish water in fish that express nitroreductase ( ntr ) in microglia and macrophages ( Tg(mpeg:gal4);Tg(UAS:NTR-mCherry) ). b) Representative images of brevican in synaptic region after vehicle (DMSO) or microglial ablation with 5mM metronidazole (Mtz) in fish water for 24 hours. Experiment at 14 dpf. Scale: 20 µm. c) Quantification of brevican intensity in synaptic region normalized to mean of vehicle control (vehicle, n=15 fish; Mtz, n=11 fish; p=0.043*, Welch’s t-test). d) Heatmap of absolute expression (counts) of mmp , adam , and adamts metalloproteinase genes in zebrafish microglia from bulk RNAsequencing at 28 dpf in optic tectum (OT), midbrain (MB) and hindbrain (HB). Marker genes hexb and p2ry12 shown for comparison. Reanalyzed from . e) Schematic of a dimer of the membrane-associated metalloproteinase MMP14 and design of the mmp14b-HA expression construct for epitope tagging of the C-terminal end with HA. Catalytic domain, transmembrane domain and cytoplasmic domain are indicated. f) Mmp14b-HA transgene expression in a mpeg-GFP microglia expressing mpeg:mmp14b-HA. Inset shows HA signals colocalized with microglial processes (arrow heads). Scale: 5 µm. g) Quantification of Mmp14b-HA intensity in microglial processes vs soma. Lines connect data from the same microglia (n= 6 microglia from 3 fish; p=0.015*, Paired t-test per cell). h) Representative images of brevican staining in synaptic regions of mmp14b -/- and mmp14b +/+ control at 14 dpf. Scale: 20 µm. i) Quantification of brevican intensity in synaptic region normalized to mean of mmp14b +/+ control ( mmp14b +/+ , n=10 fish; mmp14b -/- , n=11 fish; p=0.0014**, Welch’s t-test). j) Representative images of microglia-specific Mmp14b rescue experiment. Chat-PSD95 FingR ;mmp14b +/+ and ;mmp14 -/- fish were injected with mpeg:mmp14b-HA construct at one-cell stage embryos and analyzed at 14 dpf. Scale: 5 µm. k) Quantification of synapse density (Chat-PSD95 FingR puncta per µm of dendrite length) in mmp14b +/+ and mmp14 -/- with or without microglial-specific rescue with mpeg:mmp14b-HA. Dots show means per fish from at least 1 dendritic segment analyzed per fish ( mmp14 +/+ no rescue, n=16 fish; mmp14b +/+ rescue, n=14 fish; mmp14b -/- no rescue, n=16 fish; mmp14b -/- rescue, n=18 fish; Two-way ANOVA, F (1,60) interaction effect p=0.0022**, asterisks show Tukey’s multiple comparisons). l) Schematic of time lapse imaging to measure synapse turnover in Chat-PSD95 FingR ;mmp14b +/+ and ;mmp14b -/- fish. m) Representative merged images of synapses in Chat-PSD95 FingR ;mmp14b -/- and ; mmp14b +/+ fish at t=0 (pink) and t=24 (green). Experiments at 10-12 dpf. Pink arrowheads: lost synapses, green arrowheads: newly observed synapses. Non-merged images in . Scale: 5 µm. n) Quantification of newly observed, lost, and stable synapses between t=0 and t=24 in mmp14b +/+ vs mmp14b -/- fish ( mmp14b +/+ , n=14 fish; mmp14b -/- , n=16 fish; p<0.0001**** for newly observed, p=0.058 for lost, 0.61 for stable, Welch’s t-test). o) Schematic of time lapse imaging to determine the fate of individual synapses in mmp14b -/- fish. Synapses at t=0 were defined as “stable” synapses. Synapses born between t=0 and t=6 were defined as “new” synapses and subsequently followed with the 6 hour timepoint set as t=0 (lower timecourse). Experiments performed at 10-12 dpf. p) Representative image of a single excitatory synapse imaged at t=0, 6, 12, and 24 shows a “new synapse” born between t=0 and t=6 in mmp14b -/- fish. Raw fluorescence images on top and fluorescence overlaid with 3D reconstruction of synapse on bottom. Arrowheads: newborn synapse. Circle: site of newborn synapse. Scale: 2 µm. q) Kaplan-Meier plot of survival of individual synapses over time in mmp14b +/+ control (from ) and mmp14b -/- fish (For mmp14b -/- , Data from n=15 fish, n=331 stable synapses and n=26 new synapses). Values were plotted as mean ±SEM. ****: p<0.0001; **: <0.01; *: p<0.05; ns: not significant.

Article Snippet: The following primary antibodies were used: anti-GFP chicken polyclonal antibody (Aves Labs GFP-1020, 1:1000), anti-brevican mouse monoclonal antibody (1:100) , Living Colors DsRed polyclonal antibody (Clontech 632496, 1:1000), anti-SV2 mouse monoclonal antibody (DSHB, 1:500), anti-4C4 mouse monoclonal antibody (Gift from Hitchcock lab, 1:200), anti-HA rabbit monoclonal antibody (Cell Signaling Technology 3724T, 1:500).

Techniques: Control, Expressing, Marker, Comparison, Membrane, Construct, Staining, Injection, Imaging, Fluorescence

Journal: bioRxiv

Article Title: Extracellular matrix proteolysis maintains synapse plasticity during brain development

doi: 10.1101/2025.02.27.640672

Figure Lengend Snippet: a) Representative images of mCherry and the microglial marker 4C4 and quantification of microglia number in the Tg(mpeg:gal4);Tg(UAS:NTR-mCherry) with Mtz or vehicle (DMSO) treatment at 14 dpf (vehicle, n=3 fish; Mtz, n=3 fish; p=0.015*, Welch’s t-test). non-Tg= non-transgenic fish. Scale: 100 µm. b) Representative images and quantification of brevican staining in Mtz-treated non-transgenic siblings at 14 dpf (vehicle, n=6 fish; Mtz, n=6 fish; p=0.412, Welch’s t-test). Brevican intensity normalized to the mean of vehicle control. Scale: 20 µm. Values were plotted as mean ±SEM.*: p<0.05; ns: not significant.

Article Snippet: The following primary antibodies were used: anti-GFP chicken polyclonal antibody (Aves Labs GFP-1020, 1:1000), anti-brevican mouse monoclonal antibody (1:100) , Living Colors DsRed polyclonal antibody (Clontech 632496, 1:1000), anti-SV2 mouse monoclonal antibody (DSHB, 1:500), anti-4C4 mouse monoclonal antibody (Gift from Hitchcock lab, 1:200), anti-HA rabbit monoclonal antibody (Cell Signaling Technology 3724T, 1:500).

Techniques: Marker, Transgenic Assay, Staining, Control

Journal: bioRxiv

Article Title: Extracellular matrix proteolysis maintains synapse plasticity during brain development

doi: 10.1101/2025.02.27.640672

Figure Lengend Snippet: a) Scatter plot of gene expression (sorted by rank) of iPSC derived microglia and human fetal microglia. MMPs, ADAMs, ADAMTSs families that have transmembrane domain in dark green; MMPs, ADAMs, ADAMTSs without transmembrane domains in light green; microglial marker genes in violet. Data reanalyzed from the previous study . b) Schematic of the iPSC derived astrocyte-neuron-microglia tri-culture system. c) Schematic of the proteomics analysis with iPSC derived cells. Cell supernatants were collected from co-culture (neurons and astrocytes; no microglia) and tri-culture (neurons, astrocytes and microglia; with microglia) and analyzed. d) Venn diagram of proteins detected in cell culture supernatants with or without microglia. Proteins only detected in the absence of microglia are listed on the left and proteins only detected in the presence of microglia are listed on the right. ECM related proteins as defined by the matrisome database MatrisomeDB2.0 are labeled in red. e) Volcano plot of proteins in the intersection of venn diagram in d. The fold change is calculated by comparing the “with microglia” condition to the “no microglia” condition. Thresholds: p-value<0.05 (horizontal dashed line), and the vertical grey dash lines show where the average log 2 fold change >1 (vertical dashed lines). ECM related proteins as defined by the matrisome database Matrisome DB2.0 in red. Significantly matrisome proteins are outlined in blue. f) Strategy for MMP14 knockdown (KD) by shRNA (short hairpin RNA interference) with a scrambled shRNA control, and MMP14 rescue by expression of MMP14 fused to GFP, vs a GFP only control in microglia (MG). The rescue construct has silent mutations in the MMP-GFP to prevent knockdown by shMMP14. All constructs were delivered by lentivirus at the iPSC stage and used the ubiquitous promoter EF1a. g) Representative images of the tri-culture system with MMP14 KD and rescue in microglia. Iba1 (microglia), S100β (astrocyte), and neurons (MAP2) stainings are shown. Scale: 100 µm. h) Quantification of the process length of microglia in the tri-culture system with MMP14 KD and rescue. Dots show mean microglial process length per field of view from 18 fields of view over 3 independent experiments. Box-and-whiskers plot: box shows 25-75th percentile, whiskers show min to max. Statistics performed on means per field of view. (Kruskal-Wallis test, asterisks in the figure show results of Dunn’s multiple comparisons). i) Representative image of western blotting for brevican in cell supernatant and MAP2 loading control in cell lysate without microglia or with microglia after MMP14 KD and rescue. j) Quantification of brevican in the cell supernatant without microglia or with microglia after MMP14 KD and rescue. Each dot represents mean brevican intensity normalized to no microglia control from n=4 independent experiments (One-way ANOVA, asterisks in the figure show results of Tukey’s multiple comparisons). Values were plotted as mean ±SEM. ****: p<0.0001; *: p<0.05; ns: not significant.

Article Snippet: The following primary antibodies were used: anti-GFP chicken polyclonal antibody (Aves Labs GFP-1020, 1:1000), anti-brevican mouse monoclonal antibody (1:100) , Living Colors DsRed polyclonal antibody (Clontech 632496, 1:1000), anti-SV2 mouse monoclonal antibody (DSHB, 1:500), anti-4C4 mouse monoclonal antibody (Gift from Hitchcock lab, 1:200), anti-HA rabbit monoclonal antibody (Cell Signaling Technology 3724T, 1:500).

Techniques: Gene Expression, Derivative Assay, Marker, Co-Culture Assay, Cell Culture, Labeling, Knockdown, shRNA, Control, Expressing, Construct, Western Blot

Journal: bioRxiv

Article Title: Extracellular matrix proteolysis maintains synapse plasticity during brain development

doi: 10.1101/2025.02.27.640672

Figure Lengend Snippet: a) MMP14 mRNA levels from MMP14 KD microglia analyzed by RT-qPCR showing two independent shRNAs targeting MMP14, with scrambled shRNA as control. Expression normalized to GAPDH housekeeping gene (n=3 independent experiments; One-way ANOVA with Tukey’s multiple comparison). b) MMP14 mRNA levels from MMP14 KD and MMP14-GFP rescue microglia analyzed by RT-qPCR. Expression normalized to GAPDH (n=3 independent experiments; one-way ANOVA, asterisks in the figure show results of Tukey’s multiple comparison). c) Western blot of MMP14 showing MMP14 KD and rescue with MMP14-GFP in microglia. Note higher molecular weight of MMP14-GFP vs. endogenous MMP14. d) Quantification of the microglia cell number in the triculture system with MMP14 KD and MMP14-GFP rescue (n=3 independent experiments, p=0.209, one-way ANOVA). e) Representative western blot and quantification of Brevican in cell supernatant vs. MAP2 loading control in cell lysate from the triculture system with MMP14 KD (n=4 independent experiments, One-way ANOVA, asterisks show results of Tukey’s multiple comparison). Values were plotted as mean ±SEM.****: p<0.0001; *: p<0.05; ns: not significant.

Article Snippet: The following primary antibodies were used: anti-GFP chicken polyclonal antibody (Aves Labs GFP-1020, 1:1000), anti-brevican mouse monoclonal antibody (1:100) , Living Colors DsRed polyclonal antibody (Clontech 632496, 1:1000), anti-SV2 mouse monoclonal antibody (DSHB, 1:500), anti-4C4 mouse monoclonal antibody (Gift from Hitchcock lab, 1:200), anti-HA rabbit monoclonal antibody (Cell Signaling Technology 3724T, 1:500).

Techniques: Quantitative RT-PCR, shRNA, Control, Expressing, Comparison, Western Blot, Molecular Weight

Journal: bioRxiv

Article Title: Extracellular matrix proteolysis maintains synapse plasticity during brain development

doi: 10.1101/2025.02.27.640672

Figure Lengend Snippet: a) Schematic of the forced swim paradigm performed in this study. Forced swim (FS) groups were put on a petri dish with a stirrer bar and exposed to water current for 8 hours per day at 650 rpm, for two consecutive days. Control (CT) groups were put in a petri dish without stirrer bars. Experiments were performed at 13-14 dpf. b) Representative images of brevican staining in control and forced swim groups. Scale: 20 µm. c) Quantification of brevican intensity in synaptic region normalized to mean of control group (CT, n=16 fish; FS, n=17 fish; p=0.0021**, Welch’s t-test). d) Representative images of Chat-PSD95 FingR dendrites from fixed sections stained for TdT in forced swim or control group. Scale: 5 µm. e) Quantification of excitatory synapse density (Chat-PSD95 FingR puncta) per µm of dendrite length in forced swim or control group. Dots represent means per fish from at least one dendritic segment analyzed per fish (CT, n=21 fish; FS, n=21 fish; p=0.026*, Welch’s t-test). f) Schematic of time lapse synapse imaging performed before (t=0) and after (t=30) forced swim paradigm. Experiments were performed at 10-12 dpf. g) Representative merged images of time lapse assay in the Chat-PSD95 FingR before (t=0, pink) and after (t=30, green) forced swim paradigm or control. Pink arrowheads: lost synapses, green arrowheads: new synapses. Non-merge images in Extended Data Fig.10a. Scale: 5 µm. h) Quantification of newly observed, lost, and stable synapses after forced swim paradigm (CT, n=8 fish; FS, n=9 fish; p=0.0060** for newly observed, p=0.140 for lost, p=0.507 for stable, Welch’s t-test). i) Representative images of brevican staining in mmp14b -/- fish from forced swim or control group. Scale: 20 µm. j) Quantification of brevican intensity in synaptic region in mmp14b -/- fish normalized to control group (CT, n=13 fish; FS, n=11 fish; p=0.397, Welch’s t-test). k) Representative images of Chat-PSD95 FingR dendrite in mmp14b -/- fish from forced swim or control group. Scale: 5 µm. l) Quantification of synapse density (Chat-PSD95 FingR puncta) per µm of dendrite length from forced swim or control group in mmp14b -/- . Dots represent means per fish from at least 1 dendritic segment analyzed per fish (CT, n=13 fish; FS, n=16 fish; p=0.48, Welch’s t-test). m) Representative merged images of time lapse assay in the Chat-PSD95 FingR ;mmp14b -/- fish before (t=0, pink) and after (t=30, green) forced swim paradigm or control. Pink arrowheads: lost synapses, green arrowheads: new synapses. Non-merged images in . Scale: 5 µm. n) Quantification of newly observed, lost, stable synapses after forced swim in the mmp14b -/- fish (CT, n=14 fish; FS, n=13 fish; p=0.542 for newly observed, p=0.621 for lost, p=0.690 for stable, Welch’s t-test). Values were plotted as mean ±SEM. **: p<0.01; *: p<0.05; ns: not significant.

Article Snippet: The following primary antibodies were used: anti-GFP chicken polyclonal antibody (Aves Labs GFP-1020, 1:1000), anti-brevican mouse monoclonal antibody (1:100) , Living Colors DsRed polyclonal antibody (Clontech 632496, 1:1000), anti-SV2 mouse monoclonal antibody (DSHB, 1:500), anti-4C4 mouse monoclonal antibody (Gift from Hitchcock lab, 1:200), anti-HA rabbit monoclonal antibody (Cell Signaling Technology 3724T, 1:500).

Techniques: Control, Staining, Imaging

Journal: bioRxiv

Article Title: Piccolo Regulates Secretion of the Extracellular Matrix Components Brevican and Tenascin R from Astrocytes to Drive Synapse Formation: Implications for Pontocerebellar Hypoplasia Type 3 (PCH3)

doi: 10.1101/2025.07.03.662734

Figure Lengend Snippet: A, Immunohistological staining for BCAN was performed on cerebellar brain sections from Pclo wt/wt and Pclo gt/gt rats. In both genotypes, BCAN is localized in GFAP-positive astrocytes, from which it is secreted. In Pclo wt/wt slices, BCAN is also found in the surrounding tissue, indicating effective secretion. In contrast, in Pclo gt/gt sections, significantly less BCAN was detected outside astrocytes, suggesting impaired secretion of BCAN from astrocytes. B , Quantification of a. BCAN intensity is slightly greater inside astrocytes in Pclo gt/gt cerebellar sections than in Pclo wt/wt cerebellar slices; however, this difference is not significant ( Pclo wt/wt : mean± SEM= 1± 0.05, n=40 astrocytes, 3 independent animals; Pclo gt/gt : mean± SEM= 1.151± 0.06, 2 independent animals; p=0.082, t test). C, Quantification of a. BCAN intensity is significantly lower outside astrocytes in Pclo gt/gt slices than in Pclo wt/wt slices ( Pclo wt/wt : mean± SEM= 1± 0.04, 3 independent animals; Pclo gt/gt : mean± SEM= 0,831± 0.06, 2 independent animals; p=0.018, t test). Scale bar, 10 μm.

Article Snippet: The following antibodies were used: Synaptophysin (1:1000; guinea pig; synaptic systems, Göttingen, Germany; Cat# 101 004, RRID:AB_1210382), PSD95 (1:500; mouse; Abcam, Cambridge, UK; Cat# ab2723, RRID:AB_303248), MAP2 (1:1000; chicken; Millipore, Darmstadt, Germany; Cat# AB5543, RRID:AB_571049), Piccolo (1:1000; rabbit; Synaptic Systems, Göttingen, Germany; Cat# 142002, RRID:AB_887759), GFAP (1:1000; chicken; Millipore, Burlington, US, Cat# AB5541, RRID:AB_177521), BCAN (1:200; rabbit; Proteintech, Illinois, US, Cat# 19017-1-AP, RRID:AB_10643526), TNR (1:200; rabbit; Synaptic Systems, Göttingen, germany; Cat# 217 008, RRID:AB_3083013), GM130 (1:300; mouse; BD Biosciences, Franklin Lakes, New Jersey, US, Cat# 610822, RRID:AB_398141), PRA1 (1:200; rabbit; Abcam, Cambridge, UK; Cat# ab213569), VGlut1 (1:1000; guinea pig; Synaptic Systems, Göttingen, germany; Cat# 135 304, RRID:AB_887878).

Techniques: Staining

Journal: bioRxiv

Article Title: Piccolo Regulates Secretion of the Extracellular Matrix Components Brevican and Tenascin R from Astrocytes to Drive Synapse Formation: Implications for Pontocerebellar Hypoplasia Type 3 (PCH3)

doi: 10.1101/2025.07.03.662734

Figure Lengend Snippet: A, Immunocytochemical staining for BCAN on primary Pclo wt/wt and Pclo gt/gt cortical astrocytes. In Pclo gt/gt astrocytes, BCAN intensity accumulates around the nucleus and extends along a line from the center toward the periphery. In Pclo wt/wt astrocytes, BCAN intensity was distributed more evenly. B , Immunocytochemical staining for TNR on Pclo wt/wt and Pclo gt/gt cortical astrocytes. In Pclo gt/gt astrocytes, TNR intensity accumulates around the nucleus and extends along a line from the cell center toward the cell periphery. In contrast, in Pclo wt/wt astrocytes, the TNR intensity was distributed more evenly. C , Quantification of A. A line was drawn from the nucleus to the periphery and divided into 10 bins. BCAN intensity was subsequently measured within each bin and graphically represented. BCAN intensity is greater close to the nucleus in Pclo gt/gt primary cortical astrocytes than in Pclo wt/wt primary cortical astrocytes. D , Quantification of B. A line was drawn from the nucleus to the periphery and divided into 10 bins, subsequently TNR intensity was measured within each bin. TNR intensity is greater close to the nucleus in Pclo gt/gt cortical astrocytes than in Pclo wt/wt . E , Western blot (WB) analysis of the supernatants of Pclo wt/wt and Pclo gt/gt cortical astrocyte cultures. Significantly less BCAN was detected in Pclo gt/gt cortical supernatants ( Pclo wt/wt : mean= 1; Pclo gt/gt : mean± SEM= 0.81± 0.045, n=3 independent experiments; p=0.0024, t test). F , Western blot (WB) analysis of the supernatants of Pclo wt/wt and Pclo gt/gt cortical astrocyte cultures. Significantly more TNR was detected in supernatant from Pclo gt/gt cortical astrocytes ( Pclo wt/wt : mean=1, n=3 independent experiments; Pclo gt/gt : mean± SEM= 1.653± 0.283, n=3 independent experiments; p=0.082, t test). Scale bar, 10 μm.

Article Snippet: The following antibodies were used: Synaptophysin (1:1000; guinea pig; synaptic systems, Göttingen, Germany; Cat# 101 004, RRID:AB_1210382), PSD95 (1:500; mouse; Abcam, Cambridge, UK; Cat# ab2723, RRID:AB_303248), MAP2 (1:1000; chicken; Millipore, Darmstadt, Germany; Cat# AB5543, RRID:AB_571049), Piccolo (1:1000; rabbit; Synaptic Systems, Göttingen, Germany; Cat# 142002, RRID:AB_887759), GFAP (1:1000; chicken; Millipore, Burlington, US, Cat# AB5541, RRID:AB_177521), BCAN (1:200; rabbit; Proteintech, Illinois, US, Cat# 19017-1-AP, RRID:AB_10643526), TNR (1:200; rabbit; Synaptic Systems, Göttingen, germany; Cat# 217 008, RRID:AB_3083013), GM130 (1:300; mouse; BD Biosciences, Franklin Lakes, New Jersey, US, Cat# 610822, RRID:AB_398141), PRA1 (1:200; rabbit; Abcam, Cambridge, UK; Cat# ab213569), VGlut1 (1:1000; guinea pig; Synaptic Systems, Göttingen, germany; Cat# 135 304, RRID:AB_887878).

Techniques: Staining, Western Blot

Journal: bioRxiv

Article Title: Piccolo Regulates Secretion of the Extracellular Matrix Components Brevican and Tenascin R from Astrocytes to Drive Synapse Formation: Implications for Pontocerebellar Hypoplasia Type 3 (PCH3)

doi: 10.1101/2025.07.03.662734

Figure Lengend Snippet: A, Immunocytochemical staining BCAN, GM130, GFAP, and DAPI in Pclo wt/wt and Pclo gt/gt cerebellar astrocytes. BCAN fluorescence intensity is diffusely distributed in Pclo wt/wt astrocytes, whereas it accumulates along a defined path in Pclo gt/gt astrocytes . B, Immunocytochemical staining of tenascin-R (TNR), GM130, GFAP, and DAPI in Pclo wt/wt and Pclo gt/gt cerebellar astrocytes. TNR fluorescence intensity is concentrated near the nucleus and extends toward the cell periphery in Pclo gt/gt astrocytes, in contrast to a more uniform distribution in Pclo wt/wt astrocytes. C, Schematic illustration of the cargo distribution analysis method. A line from the nucleus to the cell periphery was segmented into 10 equal bins (0–9), and the fluorescence intensity of the cargo was measured in each bin and normalized to the mean intensity per bin in Pclo wt/wt astrocytes. D, Quantification of a. Normalized BCAN intensity decreases progressively from the nucleus to the periphery in Pclo wt/wt astrocytes but slightly increases in Pclo gt/gt astrocytes. E, Quantification of B. Normalized TNR intensity is greater near the nucleus in Pclo gt/gt astrocytes than in Pclo wt/wt cells. F, Western blot (WB) analysis of BCAN levels in the supernatants of Pclo wt/wt and Pclo gt/gt cerebellar astrocytes. BCAN levels are reduced in Pclo gt/gt astrocytes ( Pclo wt/wt : mean= 1; Pclo gt/gt : mean± SEM= 0.641± 0.141, n=3 independent experiments; p=0.064, t test). G, Western blot (WB) analysis of TNR levels in the supernatants of Pclo wt/wt and Pclo gt/gt cerebellar astrocytes. TNR levels are increased in Pclo gt/gt astrocytes ( Pclo wt/wt : mean= 1; Pclo gt/gt : mean± SEM= 1.246± 0.179, n=3 independent experiments; p=0.242, t test). Scale bar, 10 μm.

Article Snippet: The following antibodies were used: Synaptophysin (1:1000; guinea pig; synaptic systems, Göttingen, Germany; Cat# 101 004, RRID:AB_1210382), PSD95 (1:500; mouse; Abcam, Cambridge, UK; Cat# ab2723, RRID:AB_303248), MAP2 (1:1000; chicken; Millipore, Darmstadt, Germany; Cat# AB5543, RRID:AB_571049), Piccolo (1:1000; rabbit; Synaptic Systems, Göttingen, Germany; Cat# 142002, RRID:AB_887759), GFAP (1:1000; chicken; Millipore, Burlington, US, Cat# AB5541, RRID:AB_177521), BCAN (1:200; rabbit; Proteintech, Illinois, US, Cat# 19017-1-AP, RRID:AB_10643526), TNR (1:200; rabbit; Synaptic Systems, Göttingen, germany; Cat# 217 008, RRID:AB_3083013), GM130 (1:300; mouse; BD Biosciences, Franklin Lakes, New Jersey, US, Cat# 610822, RRID:AB_398141), PRA1 (1:200; rabbit; Abcam, Cambridge, UK; Cat# ab213569), VGlut1 (1:1000; guinea pig; Synaptic Systems, Göttingen, germany; Cat# 135 304, RRID:AB_887878).

Techniques: Staining, Fluorescence, Western Blot

Journal: bioRxiv

Article Title: Extracellular matrix proteolysis maintains synapse plasticity during brain development

doi: 10.1101/2025.02.27.640672

Figure Lengend Snippet: a) Schematic of the perisynaptic ECM which consists of a hyaluronan sugar matrix connected by proteoglycans including brevican, linking proteins like tenascins, and others. b) Representative images of zebrafish hindbrain at 14 and 60 days post fertilization (dpf) showing brevican (antibody staining) and hyaluronan labeled with a genetically encoded sensor ubi:ssncan-GFP . Scales: 100 µm. c) Quantification of fluorescence intensity of brevican and hyaluronan ( ubi:ssncan-GFP ) over development from 7-90 dpf. Mean fluorescence intensity for brevican and hyaluronan were normalized to the maximum intensity measured for each (90 dpf and 28 dpf, respectively). Brevican quantification, number of fish: 7 dpf, n=9; 14 dpf, n=8; 21 dpf, n=8; 28 dpf, n=7; 60 dpf, n=7; 90 dpf, n=6. For hyaluronan, number of fish: 7 dpf, n=11; 14 dpf, n=10; 21 dpf, n=6; 28 dpf, n=10; 60 dpf, n=7; 90 dpf, n=3. d) Schematic of zebrafish larval hindbrain. Gray dots indicate cell bodies and the synaptic region is shown by pink. A cholinergic neuron with a cell body and dendrites is shown by black. e) Dorsal view of zebrafish hindbrain at 10 dpf shows sparsely labeled cholinergic neurons expressing a TdTomato (TdT) tagged FingR construct that binds to the excitatory postsynaptic marker PSD-95. Tg(chata:gal4);Tg(zcUAS:PSD95.FingR-TdT-CCR5TC-KRAB(A)), hereafter abbreviated Chat-PSD95 FingR . Scale: 20 µm. f) Strategy for quantification of synapses using Chat-PSD95 FingR . Insets of region in e shows (i) several sparsely labeled neurons and (ii) a dendritic segment from one cholinergic neuron with synapses indicated by asterisks. Scales: 20 µm. g) Immunostaining for brevican protein and Chat-PSD95 FingR at 14 dpf. Synaptic region is indicated in the image. Scale: 5 µm. h) Schematic of 24 hours time lapse imaging assay to quantify changes of synapse density. i) Representative images show a single Chat-PSD95 FingR dendrite imaged at 7 dpf (t=0) and 8 dpf (t=24). Pink arrowheads: synapses present at t=0 and absent at t=24 “lost synapses”. Green arrowheads: synapses that appear at t=24 “new synapses”. White circles: present at both time points. Scale: 5 µm. j) Quantification of total excitatory synapse density and dynamics over the live imaging window of hindbrain development (5-14 dpf), based on Chat-PSD95 FingR-TdT puncta normalized to µm of dendrite length. Black line indicates static synapse density per day (p=0.3437, One-way ANOVA). Pink bars indicate lost synapses (F (4,78) p=0.0040**, One-way ANOVA). Green bars indicate new synapses (F (4,78) p=0.018*, One-way ANOVA). Asterisks in the figure represent results of Tukey’s multiple comparisons with respect to the 5-6 dpf. Number of fish: 5-6 dpf, n=15; 7-8 dpf, n=18; 9-10 dpf, n=18; 11-12 dpf, n=15; 13-14 dpf, n=17. Results from individual fish in . k) Schematic of time lapse imaging to determine the fate of individual synapses at the indicated timepoints. Synapses at t=0 were defined as “stable” synapses. Synapses born between t=0 and t=6 were defined as “new” synapses and subsequently followed with the 6 hour timepoint set as t=0 (lower timecourse). Experiments were performed at 10-12 dpf. l) Representative image of a single excitatory synapse imaged at t=0, 6, 12, and 24 shows a “new synapse” born between t=0 and t=6, which disappeared between t=12 and t=24. Left shows a low power image of a Chat-PSD95 FingR cholinergic neuron. Dashed square indicates inset. Inset shows raw fluorescence (top) and fluorescence overlaid with 3D reconstruction of synapses (bottom). Arrowheads: newborn synapse. Circle: site of newborn synapse. Scale: 5 µm in low power image and 2 µm in inset. m) Kaplan-Meier plot of survival of individual synapses over time (Data from n=19 fish, n=427 stable synapses and n=25 new synapses). n) Quantification of the distance from the nearest synapse for stable and new synapses at t=6 (Stable, n=116 inter-synapse intervals from n=14 fish; New, n=25 inter-synapse intervals from n=14 fish; p<0.0001****, Welch’s t-test, performed for synapses). The inter-synapse intervals were normalized by the mean of stable synapses for each cell. Values were plotted as mean ±SEM. ****: p<0.0001; **: p<0.01; *: p<0.05; ns: not significant.

Article Snippet: To obtain anti-brevican antibody, SI10-brevican (Addgene, #46300) was transfected into HEK293 cells by Lipofectamine 3000 Transfection reagent (Thermo Scientific) and collected culture media at 48 or 72 hours after transfection.

Techniques: Staining, Labeling, Fluorescence, Expressing, Construct, Marker, Immunostaining, Imaging

Journal: bioRxiv

Article Title: Extracellular matrix proteolysis maintains synapse plasticity during brain development

doi: 10.1101/2025.02.27.640672

Figure Lengend Snippet: a) Representative images of brevican and hyaluronan ( ubi:ssncan-GFP ) in the hindbrain at 7, 14, 28, and 60 dpf. Scales: 100 µm. b) Representative image of PNN (perineuronal net)-like brevican staining at 60 dpf. Square indicates the region of inset on the right. Arrowheads indicate PNN-like brevican signals. Scale: 100 µm.

Article Snippet: To obtain anti-brevican antibody, SI10-brevican (Addgene, #46300) was transfected into HEK293 cells by Lipofectamine 3000 Transfection reagent (Thermo Scientific) and collected culture media at 48 or 72 hours after transfection.

Techniques: Staining

Journal: bioRxiv

Article Title: Extracellular matrix proteolysis maintains synapse plasticity during brain development

doi: 10.1101/2025.02.27.640672

Figure Lengend Snippet: a) Schematic of strategy for ECM digestion by hyaluronidase injection into the hindbrain ventricle. Tissues fixed or imaged 6 hours after injection with hyaluronidase or vehicle (PBS). b) Representative images of Chat-PSD95 FingR dendrites at 14 dpf from fixed section stained for ΤdT after vehicle or hyaluronidase injection. Scale: 5 µm. c) Quantification of synapse density at 14 dpf (Chat-PSD95 FingR puncta) per µm of dendrite length. Dots represent means per fish, with at least 1 dendrite quantified per fish (vehicle, n=14 fish; hyaluronidase, n=17 fish; p=0.020*, Welch’s t-test). d) Representative merged images of Chat-PSD95 FingR collected before injection (t=0, pink) and 6 hours after (t=6, green) hyaluronidase or vehicle injection. Overlap of pink and green appears white. Pink arrowheads: lost synapses, green arrowheads: newly observed synapses. Experiment performed at 10-12 dpf. Non-merged images in . Scale: 5 µm. e) Quantification of newly observed, lost, or stable synapses between t=0 and t=6 after hyaluronidase vs. vehicle injection (vehicle, n=17 fish; Hyal, n=16 fish; p=0.017* for newly observed, p=0.117 for lost, p=0.722 for stable, Welch’s t-test). f) Schematic of generation of brevican knock-out ( bcan -/- ) fish by CRISPR genome editing. Guide RNAs targeting exon 3 and exon 14 were injected to delete 19 kbp of the bcan gene. The truncation resulted in loss of bcan mRNA and brevican protein (see Extended Data Fig 3e,f). g) Representative images of Chat-PSD95 FingR dendrites at 14 dpf from fixed section stained for ΤdT from bcan +/+ vs. bcan -/- fish. Scale: 5 µm. h) Quantification of synapse density at 14 dpf (Chat-PSD95 FingR puncta) per µm of dendrite length. Dots represent means per fish, with at least 1 dendrite quantified per fish ( bcan +/+ , n=16 fish; bcan -/- , n=14 fish; p=0.031*, Welch’s t-test). i) Representative merged images of Chat-PSD95 FingR collected from bcan +/+ and bcan -/- at t=0 (pink) and t=24 (green). Pink arrowheads: lost synapses, green arrowheads: new synapses. Experiment performed at 10-12 dpf. Non-merged images in . Scale: 5 µm. j) Quantification of newly observed, lost and stable synapses between t=0 and t=24 bcan +/+ vs. bcan -/- fish ( bcan +/+ , n=13 fish; bcan -/- , n=15 fish; p=0.010* for newly observed, p=0.288 for lost, p=0.085 for stable, Welch’s t-test). Values were plotted as mean ±SEM. *: p<0.05; ns: not significant.

Article Snippet: To obtain anti-brevican antibody, SI10-brevican (Addgene, #46300) was transfected into HEK293 cells by Lipofectamine 3000 Transfection reagent (Thermo Scientific) and collected culture media at 48 or 72 hours after transfection.

Techniques: Injection, Staining, Knock-Out, CRISPR

Journal: bioRxiv

Article Title: Extracellular matrix proteolysis maintains synapse plasticity during brain development

doi: 10.1101/2025.02.27.640672

Figure Lengend Snippet: a) Representative images and quantification of hyaluronan depletion after hyaluronidase injection at 14 dpf, measured as mean fluorescence intensity of GFP ( ubi:ssncan-GFP ) at 6 hours post injection (hpi) (vehicle, n=7 fish; Hyal, n=8 fish; p=0.0023**, Welch’s t-test) and 24 hpi (vehicle, n=5 fish; Hyal, n=4 fish; p=0.392, Welch’s t-test). GFP intensity normalized to mean of vehicle control. Dashed lines indicate hindbrain regions. Scale: 100 µm. b) Representative images and quantification of hyaluronan depletion at 6 hpi at 60 dpf, measured as mean fluorescence intensity of GFP ( ubi:ssncan-GFP ) nomalized to vehicle control (vehicle, n=3 fish; Hyal, n=3 fish; p=0.049*, Welch’s t-test). Scale: 100 µm. c) Representative images and quantification of brevican depletion at 6 hours after hyaluronidase injection at 14 dpf (vehicle, n=19 fish; Hyal, n=18 fish; p=0.0025**, Welch’s t-test). Brevican intensity normalized to mean of vehicle control. Dashed lines indicate hindbrain regions. Scale: 20 µm. d) Representative images and quantification of SV2 presynapse marker 6 hours after hyaluronidase injection at 14 dpf (vehicle, n=7 fish; Hyal, n=8 fish; p=0.003**, Welch’s t-test). SV2 intensity normalized to mean of vehicle control. Scale: 100 µm. e) Non-merged images of time lapse imaging from . Circles indicate stable synapses. Boxes indicate the region shown in . Scale: 5 µm. f) Expression of bcan gene in bcan -/- fish assessed by RT-qPCR from whole larvae. Expression normalized to ef1a housekeeper gene (n=3/group; p=0.0064**, Welch’s t-test). g) Representative images and quantification of brevican protein in bcan -/- fish ( bcan +/+ , n=10 fish; bcan -/- , n=12 fish; p<0.0001****, Welch’s t-test). Brevican intensity normalized to mean of bcan +/+ control. Scale: 100 µm. h) Representative images and quantification of SV2 presynapse marker in bcan -/- fish at 14 dpf ( bcan +/+ , n=15 fish; bcan -/- , n=15 fish, p=0.049*, Welch’s t-test). SV2 intensity normalized to mean of bcan +/+ control. Scale: 100 µm. i) Non-merged images of time lapse imaging experiment from . Circles indicate stable synapses. Boxes indicate the region shown in . Scale: 5 µm. Values were plotted as mean ±SEM. ****: p<0.0001; **: p<0.01; *: p<0.05; ns: not significant.

Article Snippet: To obtain anti-brevican antibody, SI10-brevican (Addgene, #46300) was transfected into HEK293 cells by Lipofectamine 3000 Transfection reagent (Thermo Scientific) and collected culture media at 48 or 72 hours after transfection.

Techniques: Injection, Fluorescence, Control, Marker, Imaging, Expressing, Quantitative RT-PCR

Journal: bioRxiv

Article Title: Extracellular matrix proteolysis maintains synapse plasticity during brain development

doi: 10.1101/2025.02.27.640672

Figure Lengend Snippet: a) Schematic of microglial ablation by adding metronidazole (Mtz) to fish water in fish that express nitroreductase ( ntr ) in microglia and macrophages ( Tg(mpeg:gal4);Tg(UAS:NTR-mCherry) ). b) Representative images of brevican in synaptic region after vehicle (DMSO) or microglial ablation with 5mM metronidazole (Mtz) in fish water for 24 hours. Experiment at 14 dpf. Scale: 20 µm. c) Quantification of brevican intensity in synaptic region normalized to mean of vehicle control (vehicle, n=15 fish; Mtz, n=11 fish; p=0.043*, Welch’s t-test). d) Heatmap of absolute expression (counts) of mmp , adam , and adamts metalloproteinase genes in zebrafish microglia from bulk RNAsequencing at 28 dpf in optic tectum (OT), midbrain (MB) and hindbrain (HB). Marker genes hexb and p2ry12 shown for comparison. Reanalyzed from . e) Schematic of a dimer of the membrane-associated metalloproteinase MMP14 and design of the mmp14b-HA expression construct for epitope tagging of the C-terminal end with HA. Catalytic domain, transmembrane domain and cytoplasmic domain are indicated. f) Mmp14b-HA transgene expression in a mpeg-GFP microglia expressing mpeg:mmp14b-HA. Inset shows HA signals colocalized with microglial processes (arrow heads). Scale: 5 µm. g) Quantification of Mmp14b-HA intensity in microglial processes vs soma. Lines connect data from the same microglia (n= 6 microglia from 3 fish; p=0.015*, Paired t-test per cell). h) Representative images of brevican staining in synaptic regions of mmp14b -/- and mmp14b +/+ control at 14 dpf. Scale: 20 µm. i) Quantification of brevican intensity in synaptic region normalized to mean of mmp14b +/+ control ( mmp14b +/+ , n=10 fish; mmp14b -/- , n=11 fish; p=0.0014**, Welch’s t-test). j) Representative images of microglia-specific Mmp14b rescue experiment. Chat-PSD95 FingR ;mmp14b +/+ and ;mmp14 -/- fish were injected with mpeg:mmp14b-HA construct at one-cell stage embryos and analyzed at 14 dpf. Scale: 5 µm. k) Quantification of synapse density (Chat-PSD95 FingR puncta per µm of dendrite length) in mmp14b +/+ and mmp14 -/- with or without microglial-specific rescue with mpeg:mmp14b-HA. Dots show means per fish from at least 1 dendritic segment analyzed per fish ( mmp14 +/+ no rescue, n=16 fish; mmp14b +/+ rescue, n=14 fish; mmp14b -/- no rescue, n=16 fish; mmp14b -/- rescue, n=18 fish; Two-way ANOVA, F (1,60) interaction effect p=0.0022**, asterisks show Tukey’s multiple comparisons). l) Schematic of time lapse imaging to measure synapse turnover in Chat-PSD95 FingR ;mmp14b +/+ and ;mmp14b -/- fish. m) Representative merged images of synapses in Chat-PSD95 FingR ;mmp14b -/- and ; mmp14b +/+ fish at t=0 (pink) and t=24 (green). Experiments at 10-12 dpf. Pink arrowheads: lost synapses, green arrowheads: newly observed synapses. Non-merged images in . Scale: 5 µm. n) Quantification of newly observed, lost, and stable synapses between t=0 and t=24 in mmp14b +/+ vs mmp14b -/- fish ( mmp14b +/+ , n=14 fish; mmp14b -/- , n=16 fish; p<0.0001**** for newly observed, p=0.058 for lost, 0.61 for stable, Welch’s t-test). o) Schematic of time lapse imaging to determine the fate of individual synapses in mmp14b -/- fish. Synapses at t=0 were defined as “stable” synapses. Synapses born between t=0 and t=6 were defined as “new” synapses and subsequently followed with the 6 hour timepoint set as t=0 (lower timecourse). Experiments performed at 10-12 dpf. p) Representative image of a single excitatory synapse imaged at t=0, 6, 12, and 24 shows a “new synapse” born between t=0 and t=6 in mmp14b -/- fish. Raw fluorescence images on top and fluorescence overlaid with 3D reconstruction of synapse on bottom. Arrowheads: newborn synapse. Circle: site of newborn synapse. Scale: 2 µm. q) Kaplan-Meier plot of survival of individual synapses over time in mmp14b +/+ control (from ) and mmp14b -/- fish (For mmp14b -/- , Data from n=15 fish, n=331 stable synapses and n=26 new synapses). Values were plotted as mean ±SEM. ****: p<0.0001; **: <0.01; *: p<0.05; ns: not significant.

Article Snippet: To obtain anti-brevican antibody, SI10-brevican (Addgene, #46300) was transfected into HEK293 cells by Lipofectamine 3000 Transfection reagent (Thermo Scientific) and collected culture media at 48 or 72 hours after transfection.

Techniques: Control, Expressing, Marker, Comparison, Membrane, Construct, Staining, Injection, Imaging, Fluorescence

Journal: bioRxiv

Article Title: Extracellular matrix proteolysis maintains synapse plasticity during brain development

doi: 10.1101/2025.02.27.640672

Figure Lengend Snippet: a) Representative images of mCherry and the microglial marker 4C4 and quantification of microglia number in the Tg(mpeg:gal4);Tg(UAS:NTR-mCherry) with Mtz or vehicle (DMSO) treatment at 14 dpf (vehicle, n=3 fish; Mtz, n=3 fish; p=0.015*, Welch’s t-test). non-Tg= non-transgenic fish. Scale: 100 µm. b) Representative images and quantification of brevican staining in Mtz-treated non-transgenic siblings at 14 dpf (vehicle, n=6 fish; Mtz, n=6 fish; p=0.412, Welch’s t-test). Brevican intensity normalized to the mean of vehicle control. Scale: 20 µm. Values were plotted as mean ±SEM.*: p<0.05; ns: not significant.

Article Snippet: To obtain anti-brevican antibody, SI10-brevican (Addgene, #46300) was transfected into HEK293 cells by Lipofectamine 3000 Transfection reagent (Thermo Scientific) and collected culture media at 48 or 72 hours after transfection.

Techniques: Marker, Transgenic Assay, Staining, Control

Journal: bioRxiv

Article Title: Extracellular matrix proteolysis maintains synapse plasticity during brain development

doi: 10.1101/2025.02.27.640672

Figure Lengend Snippet: a) Scatter plot of gene expression (sorted by rank) of iPSC derived microglia and human fetal microglia. MMPs, ADAMs, ADAMTSs families that have transmembrane domain in dark green; MMPs, ADAMs, ADAMTSs without transmembrane domains in light green; microglial marker genes in violet. Data reanalyzed from the previous study . b) Schematic of the iPSC derived astrocyte-neuron-microglia tri-culture system. c) Schematic of the proteomics analysis with iPSC derived cells. Cell supernatants were collected from co-culture (neurons and astrocytes; no microglia) and tri-culture (neurons, astrocytes and microglia; with microglia) and analyzed. d) Venn diagram of proteins detected in cell culture supernatants with or without microglia. Proteins only detected in the absence of microglia are listed on the left and proteins only detected in the presence of microglia are listed on the right. ECM related proteins as defined by the matrisome database MatrisomeDB2.0 are labeled in red. e) Volcano plot of proteins in the intersection of venn diagram in d. The fold change is calculated by comparing the “with microglia” condition to the “no microglia” condition. Thresholds: p-value<0.05 (horizontal dashed line), and the vertical grey dash lines show where the average log 2 fold change >1 (vertical dashed lines). ECM related proteins as defined by the matrisome database Matrisome DB2.0 in red. Significantly matrisome proteins are outlined in blue. f) Strategy for MMP14 knockdown (KD) by shRNA (short hairpin RNA interference) with a scrambled shRNA control, and MMP14 rescue by expression of MMP14 fused to GFP, vs a GFP only control in microglia (MG). The rescue construct has silent mutations in the MMP-GFP to prevent knockdown by shMMP14. All constructs were delivered by lentivirus at the iPSC stage and used the ubiquitous promoter EF1a. g) Representative images of the tri-culture system with MMP14 KD and rescue in microglia. Iba1 (microglia), S100β (astrocyte), and neurons (MAP2) stainings are shown. Scale: 100 µm. h) Quantification of the process length of microglia in the tri-culture system with MMP14 KD and rescue. Dots show mean microglial process length per field of view from 18 fields of view over 3 independent experiments. Box-and-whiskers plot: box shows 25-75th percentile, whiskers show min to max. Statistics performed on means per field of view. (Kruskal-Wallis test, asterisks in the figure show results of Dunn’s multiple comparisons). i) Representative image of western blotting for brevican in cell supernatant and MAP2 loading control in cell lysate without microglia or with microglia after MMP14 KD and rescue. j) Quantification of brevican in the cell supernatant without microglia or with microglia after MMP14 KD and rescue. Each dot represents mean brevican intensity normalized to no microglia control from n=4 independent experiments (One-way ANOVA, asterisks in the figure show results of Tukey’s multiple comparisons). Values were plotted as mean ±SEM. ****: p<0.0001; *: p<0.05; ns: not significant.

Article Snippet: To obtain anti-brevican antibody, SI10-brevican (Addgene, #46300) was transfected into HEK293 cells by Lipofectamine 3000 Transfection reagent (Thermo Scientific) and collected culture media at 48 or 72 hours after transfection.

Techniques: Gene Expression, Derivative Assay, Marker, Co-Culture Assay, Cell Culture, Labeling, Knockdown, shRNA, Control, Expressing, Construct, Western Blot

Journal: bioRxiv

Article Title: Extracellular matrix proteolysis maintains synapse plasticity during brain development

doi: 10.1101/2025.02.27.640672

Figure Lengend Snippet: a) MMP14 mRNA levels from MMP14 KD microglia analyzed by RT-qPCR showing two independent shRNAs targeting MMP14, with scrambled shRNA as control. Expression normalized to GAPDH housekeeping gene (n=3 independent experiments; One-way ANOVA with Tukey’s multiple comparison). b) MMP14 mRNA levels from MMP14 KD and MMP14-GFP rescue microglia analyzed by RT-qPCR. Expression normalized to GAPDH (n=3 independent experiments; one-way ANOVA, asterisks in the figure show results of Tukey’s multiple comparison). c) Western blot of MMP14 showing MMP14 KD and rescue with MMP14-GFP in microglia. Note higher molecular weight of MMP14-GFP vs. endogenous MMP14. d) Quantification of the microglia cell number in the triculture system with MMP14 KD and MMP14-GFP rescue (n=3 independent experiments, p=0.209, one-way ANOVA). e) Representative western blot and quantification of Brevican in cell supernatant vs. MAP2 loading control in cell lysate from the triculture system with MMP14 KD (n=4 independent experiments, One-way ANOVA, asterisks show results of Tukey’s multiple comparison). Values were plotted as mean ±SEM.****: p<0.0001; *: p<0.05; ns: not significant.

Article Snippet: To obtain anti-brevican antibody, SI10-brevican (Addgene, #46300) was transfected into HEK293 cells by Lipofectamine 3000 Transfection reagent (Thermo Scientific) and collected culture media at 48 or 72 hours after transfection.

Techniques: Quantitative RT-PCR, shRNA, Control, Expressing, Comparison, Western Blot, Molecular Weight

Journal: bioRxiv

Article Title: Extracellular matrix proteolysis maintains synapse plasticity during brain development

doi: 10.1101/2025.02.27.640672

Figure Lengend Snippet: a) Schematic of the forced swim paradigm performed in this study. Forced swim (FS) groups were put on a petri dish with a stirrer bar and exposed to water current for 8 hours per day at 650 rpm, for two consecutive days. Control (CT) groups were put in a petri dish without stirrer bars. Experiments were performed at 13-14 dpf. b) Representative images of brevican staining in control and forced swim groups. Scale: 20 µm. c) Quantification of brevican intensity in synaptic region normalized to mean of control group (CT, n=16 fish; FS, n=17 fish; p=0.0021**, Welch’s t-test). d) Representative images of Chat-PSD95 FingR dendrites from fixed sections stained for TdT in forced swim or control group. Scale: 5 µm. e) Quantification of excitatory synapse density (Chat-PSD95 FingR puncta) per µm of dendrite length in forced swim or control group. Dots represent means per fish from at least one dendritic segment analyzed per fish (CT, n=21 fish; FS, n=21 fish; p=0.026*, Welch’s t-test). f) Schematic of time lapse synapse imaging performed before (t=0) and after (t=30) forced swim paradigm. Experiments were performed at 10-12 dpf. g) Representative merged images of time lapse assay in the Chat-PSD95 FingR before (t=0, pink) and after (t=30, green) forced swim paradigm or control. Pink arrowheads: lost synapses, green arrowheads: new synapses. Non-merge images in Extended Data Fig.10a. Scale: 5 µm. h) Quantification of newly observed, lost, and stable synapses after forced swim paradigm (CT, n=8 fish; FS, n=9 fish; p=0.0060** for newly observed, p=0.140 for lost, p=0.507 for stable, Welch’s t-test). i) Representative images of brevican staining in mmp14b -/- fish from forced swim or control group. Scale: 20 µm. j) Quantification of brevican intensity in synaptic region in mmp14b -/- fish normalized to control group (CT, n=13 fish; FS, n=11 fish; p=0.397, Welch’s t-test). k) Representative images of Chat-PSD95 FingR dendrite in mmp14b -/- fish from forced swim or control group. Scale: 5 µm. l) Quantification of synapse density (Chat-PSD95 FingR puncta) per µm of dendrite length from forced swim or control group in mmp14b -/- . Dots represent means per fish from at least 1 dendritic segment analyzed per fish (CT, n=13 fish; FS, n=16 fish; p=0.48, Welch’s t-test). m) Representative merged images of time lapse assay in the Chat-PSD95 FingR ;mmp14b -/- fish before (t=0, pink) and after (t=30, green) forced swim paradigm or control. Pink arrowheads: lost synapses, green arrowheads: new synapses. Non-merged images in . Scale: 5 µm. n) Quantification of newly observed, lost, stable synapses after forced swim in the mmp14b -/- fish (CT, n=14 fish; FS, n=13 fish; p=0.542 for newly observed, p=0.621 for lost, p=0.690 for stable, Welch’s t-test). Values were plotted as mean ±SEM. **: p<0.01; *: p<0.05; ns: not significant.

Article Snippet: To obtain anti-brevican antibody, SI10-brevican (Addgene, #46300) was transfected into HEK293 cells by Lipofectamine 3000 Transfection reagent (Thermo Scientific) and collected culture media at 48 or 72 hours after transfection.

Techniques: Control, Staining, Imaging