G2C::Proteomics

Figure 1. Transmission electron microscopy of zebrafish asymmetric synapses in four different brain areas.

a. Evolutionary tree of the vertebrate lineage with timescale in million years (my). The occurrence of the two whole genome duplication events common to all vertebrates and specific to teleost (TSGD) are indicated by blue lines.

b. Schematic representation of the zebrafish brain with the four regions studied (o: olfactory bulb, t: telencephalon, ot: optic tectum and cc: cerebellar corpus) and of an excitatory synapse. Synaptosomes are formed by the axon terminal and the dendritic spine, which are separated from their corresponding neurons during tissue processing. The location of the postsynaptic density (PSD) is also indicated.

c. Asymmetric synapse from the olfactory bulb. A red asterisk and a red arrow indicate the location of presynaptic vesicles and the PSD respectively. Scale bar 200 nm.

d. An asymmetric dendro-dendritic synapse of the olfactory bulb (framed by a red dotted square) is shown. Asterisks indicate pre- and post-synaptic vesicles. The PSD is indicated by a red arrow. Scale bar 500 nm.

e. Asymmetric synapses from the telencephalon. Red asterisks and arrows indicate the location of presynaptic vesicles and PSDs respectively. The area corresponding to postsynaptic spine-like structures is filled with pink. Scale bar 500 nm.

f. Asymmetric synapses from the optic tectum. Red asterisks and arrows indicate the location of presynaptic vesicles and the PSD respectively. Red arrowheads indicate microtubule location within thin dendritic-like projection. The area of a thin dendritic-like projection, where synapses are form, is filled with purple. Scale bar 500 nm.

g. Flat (standard) asymmetric synapse from the cerebellar corpus. A red asterisk and a red arrow indicate the location of presynaptic vesicles and the PSD respectively. Scale bar 500 nm.

h. Asymmetric synapse from the medial part of the cerebellar corpus showing the extent at which the presynaptic element (highlighted in purple) surrounds the dendritic spine. Scale bar 200 nm.

i. Micrograph displaying the morphology of most abundant asymmetric synapses from the cerebellar corpus. Red asterisks and arrows indicate the location of presynaptic vesicles and the PSD respectively. Scale bar 500 nm.

Figure 2. Mouse and zebrafish synaptic protoeme.

a. Venn diagrams of mouse (red) and zebrafish (yellow) proteins identified in synaptosomal and PSD preparations indicating the percentage of proteins found in both fractions. The total number of protein identified in each species is also indicated.

b/c. Volcano plots showing quantitative enrichment and depletion of proteins between synaptosomes and postsynaptic densities purified from mouse (b) and zebrafish brain (c). Enriched or depleted proteins were identified from statistical analysis of triplicate PSD and synaptosome datasets for each species using t-testing and a Permutation based False discovery rate of 0.05.

d. Venn diagrams of mouse (red) and zebrafish (yellow) proteins found depleted or enriched in the PSD when compared with the synaptosomal fraction. The percentage of proteins found with equal abundance at the synaptosomal and PSD fraction is also indicated.

e. Scheme indicating the number of mouse synaptosomal and PSD proteins found only in one of the two fractions, depleted or enriched at the PSD or found in equal abundance in both fractions.

f. Scheme indicating the number of zebrafish synaptosomal and PSD proteins found only in one of the two fractions, depleted or enriched at the PSD or found in equal abundance in both fractions.

Figure 3. Expansion and retention of synaptic proteins after teleost-specific whole genome duplication.

a. Cumulative frequency plot of proteins found per Ensemble Family amongst mouse, zebrafish and mouse orthologs of zebrafish SYN proteins. Kruskal-Wallis Test used to calculate significance between distributions (** p < 0.01 and *** p < 0.0001).

b. Cumulative frequency plot of proteins found per Ensemble Family amongst mouse, zebrafish and mouse orthologs of zebrafish PSD proteins. Kruskal-Wallis Test used to calculate significance between distributions (** p < 0.01 and *** p < 0.0001).

c. Distribution of orthology types between zebrafish and mouse. For each protein in the proteome, the corresponding gene was identified and orthologous genes between species were determined using the biomaRt bioconductor package1. For each gene the zebrafish:mouse ratio of orthologs was determined and characterised as 1:1, 1:Many, Many:1, Many:Many or unique to mouse or zebrafish.

d. Density of proteome by ortholog ratio (zebrafish:mouse). Statistically significant comparisons between pairs of distributions (two-tailed Kolmogorov–Smirnov test applied to distributions) are shown. *** p < 0.001 and * p < 0.05, non significant comparisons are not shown.

Figure 4. Functional similarity between mouse and zebrafish synaptic proteomes.

a. Fraction of mouse (red) and zebrafish (yellow) synaptic proteins annotated to IPA cell location categories.

b. Fraction of mouse (red) and zebrafish (yellow) synaptic proteins annotated to IPA molecular function categories.

c. Overlap between Biological Process and Molecular Function GO-slim terms and PANTHER Protein Classes enriched in mouse and zebrafish. Top row shows data for synaptosomes and bottom row for PSD proteomes. The exact percentage of overlap is indicated.

d. Cumulative frequency distribution plots of individual protein innovation index (number of unique domain types per protein) for each proteome in mouse. Statistically significant comparisons between pairs of distributions (two-tailed Kolmogorov–Smirnov test applied to distributions) are shown at the legend. *** p < 0.001, ** p < 0.01, * p < 0.05 non significant comparisons are not shown.

e. Cumulative frequency distribution plots of individual protein innovation index (number of unique domain types per protein) for each proteome in zebrafish. Statistically significant comparisons between pairs of distributions (two-tailed Kolmogorov–Smirnov test applied to distributions) are shown at the legend. *** p < 0.001, ** p < 0.01, * p < 0.05 non significant comparisons are not shown.

Figure 5. Differential expression at the mouse PSD of proteins involved in intracellular vesicle biogenesis.

For each protein the mouse ortholog gene name is given. A white squares denote that a protein has not been found in a given species, a blue square identifies proteins found in synaptosomes but not at the level of postsynaptic densities, and a red square identifies proteins found at the PSD.

a. Proteins involved in vesicle fusion with membranes, including SNARE complex proteins: Syntaxins, Vamps and SNAPs, syntaxin binding proteins (Sec1/Munc18) and synaptotagmins.

b. Proteins form the HOPS complex, involved in membrane vesicle tethering to membranes.

c. Proteins involved in membrane bending/budding and scission to form vesicles, including proteins from ESCRT complexes (ESCRT I, II, III and Vps4-Vta1/ALIX), dynamins and small GTPases from the Rab family.

Figure 6. Conservation of the core vertebrate PSD machinery.

a. Pie chart of main GO-Slim Molecular Function categories enriched amongst vPSD proteins.

b. Box plots for the percentage of protein identity since the last common ancestor between zebrafish and mouse of zebrafish PSD proteins, PSD components found in both species (vPSD), zebrafish-specific PSD proteins (Zf-sPSD) and a zebrafish brain proteom. Distributions compared using the Mann-Whitney U test (*** p < 0.001).

c. Box plots for the percentage of protein identity since the last common ancestor between zebrafish and mouse of mouse PSD proteins, PSD components found in both species (vPSD), mouse-specific PSD proteins (Mm-sPSD) and a mouse brain proteome. Distributions compared using the Mann-Whitney U test (*** p < 0.001).

d. Percentage of protein identity for individual proteins found only in zebrafish PSD (yellow), in both species PSD (white) and only in mouse PSD (red). Red lines represent the median percentage of protein identity for two zebrafish (a and b) and a mouse (c) whole brain proteomes.

Table 1. Protein Families among mouse and zebrafish SYN and PSD proteins.

Table 2. Number of proteins in families characteristic of the synapse.

Supplementary Figure 1. Ultrastructure of Zebrafish PSD morphologies.

Transmission electron microscopy images taken from sections of four different zebrafish brain regions.

a. Olfactory bulb: (a') Schematic representation of the zebrafish brain. The black line indicates the position of the olfactory bulb. (a1-a8) PSDs found in different areas of the olfactory bulb. Arrowheads indicate PSDs from dendro-dendritic synapses.

b. Telencephalon: (b') Schematic representation of the zebrafish brain. The black line indicates the position of the telencephalon. (b'') Coronal semi-thin section through telencephalon stained with toluidine blue. (b1-b8) PSDs found in different areas of the telencephalon.

c. Optic tectum: (c') Schematic representation of the zebrafish brain. The black line indicates the position of the midbrain. (c'') Coronal semi-thin section through midbrain stained with toluidine blue. The red encircled area corresponds to the optic tectum. The orange line indicates the area where asymmetric synapses were identified. (c1-c8) PSDs found in different sections of the optic tectum. Arrowheads denote PSDs.

d. Cerebellar corpus: (d') Schematic representation of the zebrafish brain. The black line indicates the position of the hindbrain. (d'') Coronal semi-thin section through hindbrain stained with toluidine blue. The red encircled area corresponds to the cerebellar corpus. The orange line indicates the area where asymmetric synapses were identified. (d1-d8) PSDs found in different sections of the cerebellum. Arrows indicate PSDs showing the curved morphology with the presynaptic element surrounding the postsynaptic spine.

Supplementary Figure 2. Morphological characteristics of zebrafish PSDs.

a. Synapses from the cerebellar corpus with morphological characteristics similar to those observed in mammals and other zebrafish brain regions, including flat PSDs.

b. Synapses from the cerebellar corpus with presynaptic elements engulfing the postsynaptic terminal and highly curved PSDs.

c. Proportion of flat (light grey) and curved PSDs found at the cerebellar corpus of zebrafish brain.

d. Representation of the three measures taken on cerebellar synapses as in (b): PSD arch length, PSD transversal length and spine longitudinal length.

e. Representative images of the two types of PSD found amongst curved ones: type 1 (left image) and Type 2 (right image).

f. PSD arch length differences between the three types of PSDs found at the cerebellar corpus (p < 0.05). Type 1 n = 24; type 2 n = 40; standard n = 10.

g. PSD transversal length differences between the three types of PSDs found at the cerebellar corpus. Type 1 n = 24; type 2 n = 40; standard n = 10.

h. Spine longitudinal length differences between the three types of PSDs found at the cerebellar corpus. Type 1 n = 24; type 2 n = 40; standard n = 10.

i. Representation of the two measures taken on PSD from all brain areas: length and area.

j. Differences in PSD length between brain regions (p < 0.05). Optic tectum n = 55; Cerebellum type 1 n = 25; Olfactory bulb n = 52; Cerebellum standard n = 10; Telencephalon n = 86; Cerebellum type 2 n = 40.

k. Differences in PSD area between brain regions (p < 0.05). Olfactory bulb = 53; Optic tectum = 53; Cerebellum type 1 = 26; Cerebellum standard = 10; Telencephalon = 87; Cerebellum type 2 = 37.

Supplementary Figure 3. PSD morphological metrics.

Supplementary Figure 4. Isolation of postsynaptic densities from mouse and zebrafish brains.

a. SDS-PAGE electrophoresis of the three PSD fractions isolated from mouse brain (lanes 1-3) and zebrafish brain (lanes 4-6).

b. Protein yield for synaptosomal (SYN) preparations from mouse and zebrafish brains.

c. Protein yield for postsynaptic density (PSD) preparations from mouse and zebrafish brains.

d. Immunoblot of synaptosomal (S) and PSD (P) fractions for PSD95 protein in triplicate samples from mouse and zebrafish brains. Representative image of 3 western blots.

Supplementary Figure 5. Label Free Quantification (LFQ) intensities of zebrafish and mouse synaptic proteins.

a. Multiscatter plot of protein LFQ intensities calculated from data obtained from proteomic analysis of Zebrafish synaptosome and PSD samples. Pearson correlation coefficients are shown for each comparison and were >0.90 for biological replicates in each group.

b. Multiscatter plot of protein LFQ intensities calculated from data obtained from proteomic analysis of mouse synaptosome and PSD samples. Pearson correlation coefficients are shown for each comparison and were >0.90 for biological replicates in each group.

Supplementary Figure 6. Conservation of SYN and PSD proteins along vertebrate evolution.

a. Box plots for the percentage of protein identity since the last common ancestor between zebrafish and mouse in the following zebrafish protein groups: all proteins encoded in the genome, brain synaptosomes (Zf-SYN) and brain postsynaptic densities (Zf-PSD).

b. Box plots for the percentage of protein identity since the last common ancestor between zebrafish and mouse in the following mouse protein groups: all proteins encoded in the genome, brain synaptosomes (Mm-SYN) and brain postsynaptic densities (Mm-PSD).

c. Box plots for the percentage of protein identity since the last common ancestor between mouse and human in the following protein groups: all mouse proteins encoded in the genome, mouse synaptosomal (Mm-SYN) and postsynaptic density (Mm-PSD) proteins.

d. Box plots for the percentage of protein identity since the last common ancestor between mouse and human of of proteins found only in the mouse synaptosomal fraction, with equal abundance in both fractions or depleted, enriched or only found at the PSD.

e. Box plots for the percentage of protein identity since the last common ancestor between zebrafish and mouse of proteins identified in the zebrafish brain divided into those found (SYN) or absent (non-SYN) in our synaptosomal preparation. Data from two independent zebrafish brain proteomes are used2,5.

f. Box plots for the percentage of protein identity since the last common ancestor between zebrafish and mouse of proteins identified in the zebrafish brain divided into those found (PSD) or absent (non-PSD) in our postsynaptic density preparation. Data from two independent zebrafish brain proteomes are used2,5.

g. Box plots for the percentage of protein identity since the last common ancestor between zebrafish and mouse of proteins identified in a mouse brain proteome3 and a human frontal cortex proteome6 divided into those found (SYN) or absent (non-SYN) in our synaptosomal preparation.

h. Box plots for the percentage of protein identity since the last common ancestor between zebrafish and mouse of proteins identified in a mouse brain proteome3 and a human frontal cortex proteome6 divided into those found (PSD) or absent (non-PSD) in our postsynaptic density preparation.

All distribution compared using the Mann-Whitney U test (*** p < 0.001; ** p < 0.01 and * p< 0.05).

Supplementary Table 1a. Synaptosomal proteins identified in mouse.

Proteins identified in mouse whole brain synaptosomal preparations. For each protein the following database identifiers are given: Ensembl Gene, Associated Gene Name, Description and MGI ID.