Fig. 3: The microglial metalloproteinase Mmp14b restricted ECM accumulation and destabilized newborn synapses.
From: Extracellular matrix proteolysis maintains synapse plasticity during brain development
a, Schematic of microglial ablation by adding 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 5 mM Mtz in fish water for 24 h. Experiment at 14 dpf. c, Quantification of brevican intensity in synaptic region normalized to mean of vehicle control (vehicle, n = 15 fish; Mtz, n = 12 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 RNA sequencing at 28 dpf in optic tectum (OT), midbrain (MB) and hindbrain (HB). Marker genes hexb and p2ry12 are shown for comparison. Reanalyzed from ref. 35. 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 an mpeg-GFP microglia expressing mpeg:mmp14b-HA. Inset shows HA signals colocalized with microglial processes (arrowheads). Similar results were observed from n = 2 individual experiments. g, Quantification of Mmp14b-HA intensity in microglial processes versus 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. 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-PSD95FingR;mmp14b+/+ and ;mmp14−/− fish were injected with mpeg:mmp14b-HA construct at one-cell-stage embryos and analyzed at 14 dpf. k, Quantification of synapse density (Chat-PSD95FingR 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 n = ~1–4 dendritic segments analyzed per fish (mmp14+/+ no rescue, n = 33 dendrites from n = 16 fish; mmp14b+/+ rescue, n = 31 dendrites from n = 14 fish; mmp14b−/− no rescue, n = 27 dendrites from n = 16 fish; mmp14b−/− rescue, n = 40 dendrites from n = 18 fish; two-way ANOVA for fish, **F(1,60) interaction effect P = 0.0022, asterisks showing Tukey’s multiple comparisons). MG, microglia. l, Schematic of time-lapse imaging to measure synapse turnover in Chat-PSD95FingR;mmp14b+/+ and ;mmp14b−/− fish. m, Representative merged images of synapses in Chat-PSD95FingR;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. Nonmerged images are shown in Extended Data Fig. 8d. n, Quantification of newly observed, lost and stable synapses between t = 0 and t = 24 in mmp14b+/+ versus mmp14b−/− fish (mmp14b+/+, n = 14 fish; mmp14b−/−, n = 16 fish; ****P < 0.0001 for newly observed, P = 0.058 for lost, P = 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-h time point set as t = 0 (lower time course). 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. Dashed circle, site of newborn synapse. q, Kaplan–Meier plot of survival of individual synapses over time in mmp14b+/+ control (from Fig. 1m) and mmp14b−/− fish (for mmp14b−/−, data from n = 15 fish, n = 331 stable synapses and n = 26 new synapses). Values were plotted as mean ± s.e.m. ****P < 0.0001; **P < 0.01; *P < 0.05. Scale bars, 20 µm (b,h), 5 µm (f), 5 µm (j,m), 2 µm (p). Illustrations in a and e created using BioRender.com.
