Phenotypic impact of individual conserved neuronal microexons and their master regulators in zebrafish
- Centre for Genomic Regulation (CRG), The Barcelona Institute of Science and Technology, Spain
- Department of Medicine and Life Sciences (MELIS), Universitat Pompeu Fabra, Spain
- Champalimaud Research, Champalimaud Foundation, Portugal
- Instituto de Biología Molecular y Celular del Cáncer, CSIC and Universidad de Salamanca, Spain
- Centro de Investigación Biomédica en Red de Cáncer (CIBERONC), Instituto de Salud Carlos III, Spain
- ICREA, Spain
Figures
Figure 1 with 1 supplement
Neural microexon program in zebrafish.
(A) Heatmap showing relative inclusion levels of neural microexons identified in zebrafish (Supplementary file 1). The value for each tissue type corresponds to the average inclusion in the samples for that tissue in VastDB (Supplementary file 1). Only events with sufficient read coverage in >10 tissue groups are plotted (N=157) and missing values were imputed. (B) Inclusion of neural microexons along embryo development (egg to 7 days post-fertilization [dpf]). Thick line corresponds to the median PSI and the shades to the first and third quartile of the PSI distribution. (C) Enriched Gene Ontology (GO) categories among genes harboring neural microexons in zebrafish. GO terms were grouped into networks by ClueGO. (D) Evolutionary conservation of zebrafish neural exons of different length group at the genomic and tissue-regulatory level compared with human. Exons conserved at the regulatory level (blue) are those with enriched inclusion in neural samples (ΔPSI ≥15) also in human. Those with no neural regulation (black) are conserved at the genomic level (Irimia et al., 2009), but are not neurally enriched. Those conserved but with insufficient coverage to assess regulation are indicated as "No coverage" (dark grey). P-value corresponds to a two-sided Fisher’s Exact test for neural conservation vs. others between exons >51 nts and 3–27 nts. (E) Distribution of exons of different length by the level of srrm3/4 misregulation in larva or retina (see Methods). P-value corresponds to a two-sided Fisher’s Exact test for non-regulated exons (ΔPSI ≥ –15) vs. others between exons >51 nts and 3–27 nts. (F) Change in inclusion levels [ΔPSI (enhancer of microexons domain (eMIC) mutant-WT)] for all exons shorter than 300 bp. Dots with different blue colors correspond to neural exons of different length.
Figure 1—figure supplement 1
Identification and validation of neural microexons in zebrafish.
(A) Distribution of neural exons in zebrafish and human according to their length group. (B) RT-PCR assays assessing the inclusion of 21 selected microexons across zebrafish tissues. Primer sequences are provided in Supplementary file 4. (C) Evolutionary conservation of human neural exons of different length group at the genomic and tissue-regulatory level compared with zebrafish. Exons conserved at the regulatory level (blue) are those with enriched inclusion in neural samples (ΔPSI ≥15) also in zebrafish. Those with no neural regulation (black) are conserved at the genomic level (Irimia et al., 2009), but are not neurally enriched. Those conserved but with insufficient coverage to assess regulation are indicated as ‘No coverage’ (dark grey). p-Value corresponds to a two-sided Fisher’s Exact test for neural conservation vs. others between exons >51 nts and 3–27 nts. (E) Distribution of exons of different length by the level of upregulation upon overexpression of the regulator in HEK cells (see Materials and methods). p-Value corresponds to a two-sided Fisher’s Exact test for non-regulated exons (ΔPSI ≤15) vs. others between exons >51 nts and 3–27 nts.
Figure 2 with 1 supplement
Selected neural microexons and experimental design.
(A) Inclusion levels of the 21 selected conserved microexons across zebrafish tissues as well as differential inclusion in neural vs other tissues (ΔPSI neural) in zebrafish and human, change in inclusion in response to eMIC depletion in zebrafish (ΔPSI eMIC) or eMIC overexpression in human (ΔPSI eMIC OE), and change in inclusion between ASD patients and control individuals (ΔPSI ASD). The three microexons with asterisks were excluded from phenotypic analyses. (B) Schematic representation of the CRISPR-Cas9 based deletions of individual microexons. A pair of guide RNAs flanking each microexon were designed, which is expected to lead to normal gene expression without the microexon in all cells. (C) Examples of two RT-PCRs testing the inclusion of the targeted microexon upon CRISPR-Cas9 removal. itsn1_1 shows the expected clean deletion in the homozygous, while pus7_1 exhibits inclusion of a cryptic sequence of higher length (red block) (see Figure 2—figure supplement 1). (D) Schematic representation of srrm3 and srrm4 protein domains and the impact of the CRISPR-Cas9 derived mutations (from Ciampi et al., 2022). (E) Distribution of body lengths at 30 dpf for heterozygous (green) and homozygous (yellow) fish for each microexon deletion or the srrm4 mutation. For srrm3, the values are shown for 90 dpf. P-value corresponds to a two-sided t-test. Error bars correspond to standard errors. (F) Top: two representative images from WT and srrm3 homozygous mutant larvae showing staining for 3A10 in Mauthner cells (schematized on the right side). Bottom: quantification of normal and altered number of larvae with respect to Mauthner cell morphology in homozygous mutants for each microexon or regulator line.
Figure 2—figure supplement 1
Validation of microexon deletion lines.
RT-PCR validations of embryo siblings for the main founder of each zebrafish microexon deletion line and genotype are shown. Wildtype (+/+), heterozygous (+/-) and homozygous (-/-). Red font indicates the three lines where the deletion of the microexon leads to the inclusion of cryptic sequences, potentially producing a loss of function of the gene. These microexons were excluded from the subsequent analyses.
Figure 3 with 2 supplements
Impact of microexon deletion on neurite outgrowth.
(A) Schematic representation of the experimental design used to assess neurite outgrowth in zebrafish neuronal primary cultures (see Methods). (B) Confocal images of example microexon deletions at 24 hours post-plating (hap). ×20 magnification images, white squares indicate the zoom region amplified in the right panel (digital zoom). Scale bar 50 µm. (C) Heatmap showing the median percent of change in neurite length at 10 and 24 hap of the homozygous mutant with respect to the matched control neurons (HuC:GFP line) for each main microexon deletion line (data for all tested lines in Figure 3—figure supplement 2). Significance is based on the median of p-value distribution of 10,000 bootstrap resampling Wilcoxon tests for each main founder. * 0.01<p ≤ 0.001, ** 0.001<p ≤ 0.0001, *** p<0.0001. (D) Boxplots of the distribution of the length of the longest neurite (one neuron per data point) for both founder lines generated for either evi5b or vav2 (_1 and _2), as well as the length distributions for neurons of siblings that are either WT and homozygous deletion (Del) for the main founder lines (evi5b_1 and vav2_1). For the regulators, neurite length distributions for neurons of WT and homozygous mutants (Del) siblings are shown for srrm3, srrm4 and the double mutant srrm3/4, for which the control sibling corresponds to the WT of srrm3 but homozygous mutant for srrm4 (i.e., srrm4 Del). p-Values correspond to ANOVA tests for evi5b and vav2 founders and for siblings the median of 10,000 bootstrap Wilcoxon tests. * 0.01<p ≤ 0.001, ** 0.001<p ≤ 0.0001, *** p<0.0001. (E) Schematic representation of the domain architecture of EVI5B in zebrafish and region encoded by the microexon and upstream and downstream exons (upper blocks). Microexon inclusion/exclusion leads to different C-termini. (F) NanoBRET quantification of protein-protein interaction between EVI5B with and without the microexon and RAB11 proteins from zebrafish. Error bars correspond to standard errors. (G) Schematic representation of the domain architecture of VAV2 in zebrafish and region encoded by the microexon and upstream and downstream exons (upper blocks). (H) Luciferase readout of the activation of SRF upon ectopic expression of zebrafish VAV2 proteins with and without the microexon, a negative control and an oncogenic VAV2 mutant protein in COS7 cells. P-values correspond to Student’s t-test after Holm-Sidak’s multiple test correction. Error bars correspond to standard errors. (I) Western blot for each overexpressed protein tagged to GFP, detected by anti-GFP antibody. Bottom blot: β-actin is shown as loading control.
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Figure 3—source data 1
Unedited uncropped western blots with bands highlighted.
- https://cdn.elifesciences.org/articles/104275/elife-104275-fig3-data1-v1.pdf
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Figure 3—source data 2
Raw, uncropped western blot gel images displayed in Figure 3I using an anti-GFP or an anti-β-actin antibody.
- https://cdn.elifesciences.org/articles/104275/elife-104275-fig3-data2-v1.zip
Figure 3—figure supplement 1
Zebrafish neuronal primary culture.
(A) Schematic summary of the experimental steps in the generation of zebrafish neuronal primary cultures from FACS-sorted HuC:GFP cells. (B) Confocal images of immunofluorescence anti GFP and acetylated tubulin to study zebrafish neuronal outgrowth over time (4, 8, 10, 24, 48, 72 hr after plating [hap]). Top panel: ×20 x confocal images. Bottom panel: digital zoom to show details of neural development progression. Red asterisk shows the two timepoints selected for this study. Scale bars 50 µm. (C) Neurite length manual quantifications of the corresponding confocal images.
Figure 3—figure supplement 2
Quantification of neurite outgrowth across zebrafish mutant lines.
(A) Boxplots showing neurite length distributions at 10 and 24 hap for each founder of the microexon deletion lines. The main founder (shown in Figure 3C) is highlighted in bold. Red boxplots correspond to neurons from a Del x Del cross for each microexon line. Dark boxplots correspond to matched control neurons from WT HuC:GFP fish processed in parallel in each experiment (see Materials and methods). (B) Boxplots showing neurite length distributions at 10 and 24 hap for the main founder of the microexon deletion lines. The second founder was not tested since no significant differences were found in any of the initial replicates with the exception of reln, for which no second founder could be obtained. (C) Boxplots showing neurite length distributions at 10 and 24 hap for siblings of WT (blue), heterozygous (yellow), or homozygous (red) genotype. These samples were generated through Het x Het crosses, fin clip genotyping of each larva and processing of pools of larvae of the same genotype. p-Value corresponds to ANOVA tests for each of the groups. * 0.01<p ≤ 0.001, ** 0.001<p ≤ 0.0001, *** p<0.0001.
Figure 4 with 13 supplements
Impact of microexon misregulation in larval activity and response to stimuli.
(A) Experimental design to assess larval activity patterns and response to stimuli using the DanioVision system and the EthoVision XT - Video tracking software. The protocol we implemented consists of 5' of habituation, 25' of baseline recording, five alternating 10' intervals of dark-light, 10' of re-habituation and the tapping experiment (30 taps at 1 Hz). (B) Heatmap showing the percent of change with respect to the WT value for the homozygous (Del) of each main microexon deletion line as well as the single and double regulator mutants for features related to activity and response to stimuli (full plots in Figure 4—figure supplements 1–13). For visualization purposes, the percent change for median WT values equal to zero was computed using the minimum median WT value of the relative category across founders. p-Values correspond to the median p-value from 100 permutation tests selecting 10 observations per genotype across replicates of the main founder. (C) Left: Activity (percentage of Δpixels/min) plots for a representative clutch of srrm3 and evi5b main lines. Traces represent mean ± SEM across larvae from the same clutch and genotype. Dark and light periods are shown with gray or white background, respectively. Right: boxplots showing the distribution of the mean baseline activity for each larva of each genotype. p-Values correspond to two-sided Wilcoxon Rank-Sum tests of each genotype against the WT. (D) Left: Activity (percentage of Δpixels/min) plots for a representative clutch of srrm3/4 double mutant line. Traces represent mean ± SEM across larvae from the same clutch and genotype. Dark and light periods are shown with gray or white background, respectively. Right: boxplots showing the mean difference in activity during the dark-to-light transition for each larva of each genotype. p-Values correspond to two-sided Wilcoxon Rank-Sum tests. (E) Left: Activity (percentage of Δpixels/s) after each of the 30 consecutive taps representative clutch of srrm3/4 double mutant line. Traces represent mean ± SEM across larvae from the same clutch and genotype. (F) Left: schematic representation of a well, divided between center and periphery regions. Right: two representative tracks of WT and Del srrm3/4 larvae for 60 s at baseline. (G) Boxplots showing the median percentage total distance moved (TDM) (mm) in the periphery for larvae of different genotypes of the srrm3/4 double mutant line, under baseline, dark and light conditions. p-Values correspond to two-sided Wilcoxon Rank-Sum tests of each genotype against the WT. In D-G, W/D denotes srrm3+/+,srrm4-/- fish, H/D srrm3+/-,srrm4-/-, and D/D srrm3-/-,srrm4-/-. For all panels: * 0.05<p ≤ 0.001, ** 0.001<p ≤ 0.0001, *** p<0.0001.
Figure 4—figure supplement 1
Larval activity over the time course of the experiment.
Activity (percentage of Δpixels/min) for each microexon and regulator deletion line and biological replicate (clutch). Traces represent mean ± SEM across larvae from the same clutch and genotype. Dark and light periods are shown with gray or white background, respectively. For simplicity, for the srrm3/4 double mutant, WT denotes srrm3+/+,srrm4-/-, Het srrm3+/-,srrm4-/-, and DEL srrm3-/-,srrm4-/-.
Figure 4—figure supplement 2
Activity response and habituation to tapping stimuli.
Log-scaled activity (percentage of Δpixels/s) at each of the 30 consecutive taps for each microexon and regulator deletion line and biological replicate (clutch). A+1 pseudocount was added to the activity data for plotting purposes only. Traces represent mean ± SEM across larvae from the same clutch and genotype. For simplicity, for the srrm3/4 double mutant, WT denotes srrm3+/+,srrm4-/-, Het srrm3+/-,srrm4-/-, and DEL srrm3-/-,srrm4-/-.
Figure 4—figure supplement 3
Baseline activity.
Boxplots showing baseline (B) activity (percentage of Δpixels/min) for each microexon and regulator deletion line and biological replicate (clutch). p-Values correspond to Wilcoxon Rank-Sum tests between WT and each of the other genotypes. * 0.05<p ≤ 0.001, ** 0.001<p ≤ 0.0001, *** p<0.0001. For simplicity, for the srrm3/4 double mutant, WT denotes srrm3+/+,srrm4-/-, Het srrm3+/-,srrm4-/-, and DEL srrm3-/-,srrm4-/-.
Figure 4—figure supplement 4
Activity during the light intervals.
Boxplots showing activity (percentage of Δpixels/min) during the light intervals for each microexon and regulator deletion line and biological replicate (clutch). p-Values correspond to Wilcoxon Rank-Sum tests between WT and each of the other genotypes. * 0.05<p ≤ 0.001, *** p<0.0001. For simplicity, for the srrm3/4 double mutant, WT denotes srrm3+/+,srrm4-/-, Het srrm3+/-,srrm4-/-, and DEL srrm3-/-,srrm4-/-.
Figure 4—figure supplement 5
Activity during the dark intervals.
Boxplots showing activity (percentage of Δpixels/min) during the dark intervals for each microexon and regulator deletion line and biological replicate (clutch). p-Values correspond to Wilcoxon Rank-Sum tests between WT and each of the other genotypes. * 0.05<p ≤ 0.001, ** 0.001<p ≤ 0.0001. For simplicity, for the srrm3/4 double mutant, WT denotes srrm3+/+,srrm4-/-, Het srrm3+/-,srrm4-/-, and DEL srrm3-/-,srrm4-/-.
Figure 4—figure supplement 6
Activity during the dark-to-light transitions.
Boxplots showing activity (percentage of Δpixels/min) during the dark-to-light (DL) transitions for each microexon and regulator deletion line and biological replicate (clutch). p-Values correspond to Wilcoxon Rank-Sum tests between WT and each of the other genotypes. * 0.05<p ≤ 0.001, ** 0.001<p ≤ 0.0001. For simplicity, for the srrm3/4 double mutant, WT denotes srrm3+/+,srrm4-/-, Het srrm3+/-,srrm4-/-, and DEL srrm3-/-,srrm4-/-.
Figure 4—figure supplement 7
Activity during the light-to-dark transitions.
Boxplots showing activity (percentage of Δpixels/min) during the light-to-dark (LD) transitions for each microexon and regulator deletion line and biological replicate (clutch). p-Values correspond to Wilcoxon Rank-Sum tests between WT and each of the other genotypes. * 0.05<p ≤ 0.001, *** p<0.0001.
Figure 4—figure supplement 8
Median activity after the first tap.
Boxplots showing activity (percentage of Δpixels/min) after the first tap for each microexon and regulator deletion line and biological replicate (clutch). p-Values correspond to Wilcoxon Rank-Sum tests between WT and each of the other genotypes. * 0.05<p ≤ 0.001, ** 0.001<p ≤ 0.0001. For simplicity, for the srrm3/4 double mutant, WT denotes srrm3+/+,srrm4-/-, Het srrm3+/-,srrm4-/-, and DEL srrm3-/-,srrm4-/-.
Figure 4—figure supplement 9
Difference in activity between the first tap and taps 3–5.
Boxplots showing the change in activity in the first tap vs. taps 3–5 for each microexon and regulator deletion line and biological replicate (clutch). p-Values correspond to Wilcoxon Rank-Sum tests between WT and each of the other genotypes. * 0.05<p ≤ 0.001. For simplicity, for the srrm3/4 double mutant, WT denotes srrm3+/+,srrm4-/-, Het srrm3+/-,srrm4-/-, and DEL srrm3-/-,srrm4-/-.
Figure 4—figure supplement 10
Difference in activity between the first tap and taps 21–30.
Boxplots showing the change in activity in the first tap vs. taps 21–30 for each microexon and regulator deletion line and biological replicate (clutch). p-Values correspond to Wilcoxon Rank-Sum tests between WT and each of the other genotypes. * 0.05<p ≤ 0.001, ** 0.001<p ≤ 0.0001. For simplicity, for the srrm3/4 double mutant, WT denotes srrm3+/+,srrm4-/-, Het srrm3+/-,srrm4-/-, and DEL srrm3-/-,srrm4-/-.
Figure 4—figure supplement 11
Thigmotaxis during the baseline condition.
Boxplots showing activity (percentage of Δpixels/min) during the baseline condition for each microexon and regulator deletion line and biological replicate (clutch). p-Values correspond to Wilcoxon Rank-Sum tests between WT and each of the other genotypes. * 0.05<p ≤ 0.001, *** p<0.0001. For simplicity, for the srrm3/4 double mutant, WT denotes srrm3+/+,srrm4-/-, Het srrm3+/-,srrm4-/-, and DEL srrm3-/-,srrm4-/-.
Figure 4—figure supplement 12
Thigmotaxis during the light intervals.
Boxplots showing activity (percentage of Δpixels/min) during the light intervals for each microexon and regulator deletion line and biological replicate (clutch). p-values correspond to Wilcoxon Rank-Sum tests between WT and each of the other genotypes. * 0.05<p ≤ 0.001. For simplicity, for the srrm3/4 double mutant, WT denotes srrm3+/+,srrm4-/-, Het srrm3+/-,srrm4-/-, and DEL srrm3-/-,srrm4-/-.
Figure 4—figure supplement 13
Thigmotaxis during the dark intervals.
Boxplots showing activity (percentage of Δpixels/min) during the dark intervals for each microexon and regulator deletion line and biological replicate (clutch). p-Values correspond to Wilcoxon Rank-Sum tests between WT and each of the other genotypes. * 0.05<p ≤ 0.001.
Figure 5 with 10 supplements
Impact of microexon misregulation on social behavior.
(A) Schematic representation of the behavioral station (details in Figure 5—figure supplement 1) and experimental design for each tested pair of 30 dpf juveniles. (B) Heatmap showing the percent of change with respect to the control value for each main microexon and regulator deletion line for nine different parameters related to locomotion, anxiety or social behavior (full plots in Figure 5—figure supplements 2–10). For parameters referring to individual (locomotion, anxiety, and leadership), two comparisons are shown: the average of the fish from Del-Del pairs vs the control Het-Het pairs (homotypic pairs, same ‘S’), or the value of the Del fish with respect to the Het one within each Het-Del pair (heterotypic pairs, different ‘D’). For group parameters (polarization order and distance to neighbors), the following two comparisons are shown: Het-Het vs Het-Del (HM) and Het-Het vs Del-Del (HD). While ‘ratio neighbor in front’ (D) does not reach statistical significance under our cut-offs for srrm3 mutants due to the small sample size (N=5; p=0.016), their strongly increased leadership can be observed in panels C and D. (C) Relative position map showing the position of the other fish of the pair (right genotype) with respect to the focal fish located at the center of the map (left genotype). For instance, (+/-) vs (-/-) shows the position of the Del fish respect to the focal Het one in a Het-Del pair. The merge plot of all fish pairs for vti1a, kif1b, and srrm3 are shown. (D) Boxplots for the fish pairs shown in (C). Left: ratio in front of the non-focal fish. Lower values indicate higher leadership (i.e. more time in front). ‘Same’ corresponds to either Het-Het or Del-Del pairs and ‘Different to Het-Del pairs, with values of individual fish plotted by genotype. Right: median of the distances between the two fish throughout the time course for each genotype pair combination. p-Values correspond to Wilcoxon Rank-Sum tests for the indicated comparisons. * 0.01<p ≤ 0.001, ** 0.001<p ≤ 0.0001, *** p<0.0001. Note that for all experiment, srrm3 KO mutants (Del) were sized-matched (not aged-matched) with Het controls (see Materials and methods).
Figure 5—figure supplement 1
Custom behavioral station.
(A) Overview of the behavioral system, based on Hinz and de Polavieja, 2017. (B–D) Details of the arena used to assess social interactions between pairs of juvenile fish.
Figure 5—figure supplement 2
Median speed by genotype for each main microexon and regulator line.
(A) Median speed (cm/s) for each fish over the 10' time course by pair type. ‘Same’ corresponds to either Het-Het or Del-Del pairs, and each data point is the median of the pair, and ‘Different’ to Het-Del pairs and each data point corresponds to an individual fish. None of the comparisons between Het and Del individuals were significant at a threshold of p<0.01 using Wilcoxon Rank-Sum tests.
Figure 5—figure supplement 3
Median absolute normal acceleration by genotype for each main microexon and regulator line.
(A) Median absolute normal acceleration (cm/s2) for each fish over the 10' time course by pair type. ‘Same’ corresponds to either Het-Het or Del-Del pairs, and each data point is the median of the pair, and ‘Different’ to Het-Del pairs and each data point corresponds to an individual fish. None of the comparisons between Het and Del individuals were significant at a threshold of p<0.01 using Wilcoxon Rank-Sum tests.
Figure 5—figure supplement 4
Median absolute tangential acceleration by genotype for each main microexon and regulator line.
(A) Median absolute tangential acceleration (cm/s2) for each fish over the 10' time course by pair type. ‘Same’ corresponds to either Het-Het or Del-Del pairs and ‘Different’ to Het-Del pairs. None of the comparisons between Het and Del individuals were significant at a threshold of p<0.01 using Wilcoxon Rank-Sum tests.
Figure 5—figure supplement 5
Median distance traveled by genotype for each main microexon and regulator line.
(A) Median distance traveled (cm) for each fish over the 10' time course by pair type. ‘Same’ corresponds to either Het-Het or Del-Del pairs and ‘Different’ to Het-Del pairs. None of the comparisons between Het and Del individuals were significant at a threshold of p<0.01 using Wilcoxon Rank-Sum tests.
Figure 5—figure supplement 6
Time in the periphery by genotype for each main microexon and regulator line.
(A) Median normalized frames in periphery (from 0 to 1) for each fish over the 10' time course by pair type. ‘Same’ corresponds to either Het-Het or Del-Del pairs, and each data point is the median of the pair, and ‘Different’ to Het-Del pairs and each data point corresponds to an individual fish. None of the comparisons between Het and Del individuals were significant at a threshold of p<0.01 using Wilcoxon Rank-Sum tests.
Figure 5—figure supplement 7
Median normalized distance to origin by genotype for each main microexon and regulator line.
(A) Median normalized distance to origin/center (from 0 to 1) for each fish over the 10' time course by pair type. ‘Same’ corresponds to either Het-Het or Del-Del pairs, and each data point is the median of the pair, and ‘Different’ to Het-Del pairs and each data point corresponds to an individual fish. None of the comparisons between Het and Del individuals were significant at a threshold of p<0.01 using Wilcoxon Rank-Sum tests.
Figure 5—figure supplement 8
Ratio neighbor in front by genotype for each main microexon and regulator line.
(A) Median ratio neighbor (nb) in front (from 0 to 1) for each fish over the 10' time course by pair type. ‘Same’ corresponds to either Het-Het or Del-Del pairs and ‘Different’ to Het-Del pairs. In both cases, each data point corresponds to an individual fish. None of the comparisons between Het and Del individuals were significant at a threshold of p<0.01 using Wilcoxon Rank-Sum tests.
Figure 5—figure supplement 9
Polarization by genotype pair combination for each main microexon and regulator line.
(A) Median value of the polarization order parameter (1=fully concordant vectors) for each fish pair over the 10' time course by pair type (Het-Het, Het-Del and Del-Del). p-Values correspond to Wilcoxon Rank-Sum tests. *** p<0.0001.
Figure 5—figure supplement 10
Interindividual distance by genotype pair combination for each main microexon and regulator line.
(A) Median distance to neighbor (cm) for each fish pair over the 10' time course by pair type (Het-Het, Het-Del and Del-Del). p-Values correspond to Wilcoxon Rank-Sum tests. *** p<0.0001.
Figure 6 with 2 supplements
Transcriptomic analyses of 5 dpf larvae suggest potential compensatory changes.
(A) Schematic representation of the experimental design. (B) Distribution of changes in gene expression (Δ corrected variance stabilizing transformation [VST] expression) between the Del and WT larvae for each main microexon deletion line, as well as change of the host gene (barplots) and of closely related paralogs (chordate or younger origin according to Biomart) (dots; Mic-hosting genes' paralogs). The color scale indicates the percentile of the expression change within the overall distribution. Asterisk on bars indicates change in the bottom or top decile. (C) Left: Gene Ontology (GO) categories (dots) clustered in related groups (rows) that are enriched among genes globally changing upon individual microexon deletions. Middle: Normalized Enrichment Scores (NES; X-axis) and adjusted p-values (color code) for the same GO categories in the comparison of srrm3 mutant larvae and WT siblings. Right: heatmap showing the NES for the gene sets comprising the union of all GO categories within each group for each specific microexon comparison. Stars indicate adjusted p-value <0.01. (D) NES for specific gene sets (Supplementary file 7) with relevance in neurobiology and/or development for each microexon deletion comparison.
Figure 6—figure supplement 1
Batch correction of RNA-seq data.
Principal component analysis (PCA) on raw log10 transformed gene counts (left) and after (right) degradation correction, normalization, and batch correction using DegNorm, DESeq2 (VST), and WCGNA (Empirical Bayes-moderate adjustment), Bioconductor-packages, respectively.
Figure 6—figure supplement 2
Enriched GO categories.
Enrichment of individual GO categories from category groups highlighted in Figure 6C for each microexon deletion. Each block is a group of related GO categories, and the block is named after the underlined category. Values obtained from Gene Set Enrichment Analyses (GSEA).
Videos
Video 1
Behavior of adult srrm3 mutants.
Also accessible at https://data.mendeley.com/datasets/3b3zx4cfg9/1.
Video 2
Group of five srrm3 mutant juvenile fish.
Also accessible at https://data.mendeley.com/datasets/3b3zx4cfg9/1.
Video 3
Group of five WT juvenile fish.
Also accessible at https://data.mendeley.com/datasets/3b3zx4cfg9/1.
Tables
Appendix 1—key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background (Danio rerio) | Tg(HuC:GFP) | Park et al., 2000 | Tg(HuC:GFP) | |
| Strain, strain background (Danio rerio) | Tg(HuC:GFP; Gli3_EX0035009_9) | This paper | gli3_9 | |
| Strain, strain background (Danio rerio) | Tg(HuC:GFP; Gli3_EX0035009_7) | This paper | gli3_7 | |
| Strain, strain background (Danio rerio) | Tg(HuC:GFP; Apbb1_EX0013846_1) | This paper | apbb1_1 | |
| Strain, strain background (Danio rerio) | Tg(HuC:GFP; Mef2ca_EX0045884_2) | This paper | mef2ca_2 | |
| Strain, strain background (Danio rerio) | Tg(HuC:GFP; Evi5b_EX0030978_3) | This paper | evi5b_3 | |
| Strain, strain background (Danio rerio) | Tg(HuC:GFP; Evi5b_EX0030978_6) | This paper | evi5b_6 | |
| Strain, strain background (Danio rerio) | Tg(HuC:GFP; Ap1g1_EX0013683_1) | This paper | ap1g1_1 | |
| Strain, strain background (Danio rerio) | Tg(HuC:GFP; Vdac3_EX0084235_5) | This paper | vdac3_5 | |
| Strain, strain background (Danio rerio) | Tg(HuC:GFP; kif1b_EX0041536_1) | This paper | kif1b_1 | |
| Strain, strain background (Danio rerio) | Tg(HuC:GFP; kif1b_EX0041536_2) | This paper | kif1b_2 | |
| Strain, strain background (Danio rerio) | Tg(HuC:GFP; Itsn1_EX0040386_1) | This paper | itsn1_1 | |
| Strain, strain background (Danio rerio) | Tg(HuC:GFP; Itsn1_EX0040386_3) | This paper | itsn1_3 | |
| Strain, strain background (Danio rerio) | Tg(HuC:GFP; Cdon_EX0020538_1) | This paper | cdon_1 | |
| Strain, strain background (Danio rerio) | Tg(HuC:GFP; Asap1b_EX0015073_3) | This paper | asap1b_3 | |
| Strain, strain background (Danio rerio) | Tg(HuC:GFP; Asap1b_EX0015073_7) | This paper | asap1b_7 | |
| Strain, strain background (Danio rerio) | Tg(HuC:GFP; Vav2_EX008416_1) | This paper | vav2_1 | |
| Strain, strain background (Danio rerio) | Tg(HuC:GFP; Vav2_EX008416_2) | This paper | vav2_2 | |
| Strain, strain background (Danio rerio) | Tg(HuC:GFP; Shank3b_EX0066093_1) | This paper | shank3b_1 | |
| Strain, strain background (Danio rerio) | Tg(HuC:GFP; Shank3b_EX0066093_2) | This paper | shank3b_2 | |
| Strain, strain background (Danio rerio) | Tg(HuC:GFP; Src_EX0075047_1) | This paper | src_1 | |
| Strain, strain background (Danio rerio) | Tg(HuC:GFP; Src_EX0075047_2) | This paper | src_2 | |
| Strain, strain background (Danio rerio) | Tg(HuC:GFP; Gli2b_EX0035002_1) | This paper | gli2b_1 | |
| Strain, strain background (Danio rerio) | Tg(HuC:GFP; Gli2b_EX0035002_2) | This paper | gli2b_2 | |
| Strain, strain background (Danio rerio) | Tg(HuC:GFP; Madd_EX0044304_1) | This paper | madd_1 | |
| Strain, strain background (Danio rerio) | Tg(HuC:GFP; Madd_EX0044304_2) | This paper | madd_2 | |
| Strain, strain background (Danio rerio) | Tg(HuC:GFP; Vti1a_EX0084665_1) | This paper | vti1a_1 | |
| Strain, strain background (Danio rerio) | Tg(HuC:GFP; Vti1a_EX0084665_2) | This paper | vti1a_2 | |
| Strain, strain background (Danio rerio) | Tg(HuC:GFP; Shank3a_EX0066086_1) | This paper | shank3a_1 | |
| Strain, strain background (Danio rerio) | Tg(HuC:GFP; Shank3a_EX0066086_2) | This paper | shank3a_2 | |
| Strain, strain background (Danio rerio) | Tg(HuC:GFP; Reln_EX0007444_1) | This paper | reln_1 | |
| Strain, strain background (Danio rerio) | Tg(HuC:GFP; Reln_EX0007444_2) | This paper | reln_2 | |
| Antibody | mouse anti-acetylated tubulin | BioLegend | 802001 | 1:400 |
| Antibody | rabbit anti- GFP | Millipore | AB3080P | 1:500 |
| Antibody | Alexa Fluor 594 goat anti-mouse | Thermo Fisher | A-11005 | 1:500 |
| Antibody | Alexa Fluor 488 goat anti-rabbit | Thermo Fisher | A-11034 | 1:500 |
| Antibody | Hoechst | Sigma | B2261 | 1:1000 |
| Antibody | anti-3A10 | Developmental Studies Hybridoma Bank | AB_531874 | |
| Antibody | anti-GFP | Covance, Princeton, NJ, USA | MMS-118P | 1:2000 |
| Antibody | anti-β-actin | Santa Cruz | sc-47778 | 1:1000 |
| Strain, strain background (Danio rerio) | srrm3 | Ciampi et al., 2022 | srrm3_17 | |
| Strain, strain background (Danio rerio) | srrm4 | Ciampi et al., 2022 | srrm4_12D | |
| Gene (Danio rerio) | gli3_mic | VastDB | EX0035009 | |
| Gene (Danio rerio) | mef2ca_mic | VastDB | EX0045884 | |
| Gene (Danio rerio) | evi5b_mic | VastDB | EX0030978 | |
| Gene (Danio rerio) | ap1g1_mic | VastDB | EX0030978 | |
| Gene (Danio rerio) | vdac3_mic | VastDB | EX0084235 | |
| Gene (Danio rerio) | kif1b_mic | VastDB | EX0041536 | |
| Gene (Danio rerio) | itsn1_mic | VastDB | EX0040386 | |
| Gene (Danio rerio) | apbb1_mic | VastDB | EX0013846 | |
| Gene (Danio rerio) | cdon_mic | VastDB | EX0020538 | |
| Gene (Danio rerio) | asap1b_mic | VastDB | EX0015073 | |
| Gene (Danio rerio) | vav2_mic | VastDB | EX008416 | |
| Gene (Danio rerio) | shank3b_mic | VastDB | EX0066093 | |
| Gene (Danio rerio) | src_mic | VastDB | EX0075047 | |
| Gene (Danio rerio) | gli2b_mic | VastDB | EX0035002 | |
| Gene (Danio rerio) | madd_mic | VastDB | EX0044304 | |
| Gene (Danio rerio) | vti1a_mic | VastDB | EX0084665 | |
| Gene (Danio rerio) | shank3a_mic | VastDB | EX0066086 | |
| Gene (Danio rerio) | reln_mic | VastDB | EX0007444 | |
| Gene (Danio rerio) | srrm3 | Ensembl | ENSDARG00000096920 | |
| Gene (Danio rerio) | srrn4 | Ensembl | ENSDARG00000086327 | |
| Cell line (Chlorocebus aethiops) | COS-7 | Created by Yakov Gluzman in the early 1980s. | NA | Cell line maintained in X. Bustelo lab; male. |
| Transfected construct (Homo sapiens) | PathDetect SRF cis-Reporting System | Agilent | 219081 | |
| Transfected construct (NA) | pRL-SV40 | Promega | E2231 | Renilla luciferase |
| Transfected construct (Homo sapiens) | VAV2_onco | Lorenzo-Martín et al., 2020 | Positive control VAV2 activity | pNM115 |
| Transfected construct (Danio rerio) | VAV2_MIC+ | This paper | Zebrafish vav2 cDNA with microexon | |
| Transfected construct (Danio rerio) | VAV2_MIC- | This paper | Zebrafish vav2 cDNA without microexon |
Additional files
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Supplementary file 1
Neural exons and microexons in zebrafish.
- https://cdn.elifesciences.org/articles/104275/elife-104275-supp1-v1.xlsx
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Supplementary file 2
Selected microexons for phenotypic characterization and associated regulatory data.
- https://cdn.elifesciences.org/articles/104275/elife-104275-supp2-v1.xlsx
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Supplementary file 3
Sequence of guide RNAs used to generate microexon deletion lines.
- https://cdn.elifesciences.org/articles/104275/elife-104275-supp3-v1.xlsx
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Supplementary file 4
Primers for genotyping and for mRNA validation of microexon inclusion.
- https://cdn.elifesciences.org/articles/104275/elife-104275-supp4-v1.xlsx
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Supplementary file 5
Config file used for the Get_Tissue_Specific_AS.pl script.
- https://cdn.elifesciences.org/articles/104275/elife-104275-supp5-v1.xlsx
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Supplementary file 6
RNA-seq samples generated in this study.
- https://cdn.elifesciences.org/articles/104275/elife-104275-supp6-v1.xlsx
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Supplementary file 7
Gene sets of special interest used in this study.
- https://cdn.elifesciences.org/articles/104275/elife-104275-supp7-v1.xlsx
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MDAR checklist
- https://cdn.elifesciences.org/articles/104275/elife-104275-mdarchecklist1-v1.pdf
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Source data 1
Spreadsheet containing all the numerical values used to generate the plots in each figure panel not covered in Source code 1 and Source data 2.
- https://cdn.elifesciences.org/articles/104275/elife-104275-data1-v1.xlsx
-
Source data 2
Raw data for the figures.
- https://cdn.elifesciences.org/articles/104275/elife-104275-data2-v1.zip
-
Source code 1
Code used to generate the plots from the raw data of Source data 22.
- https://cdn.elifesciences.org/articles/104275/elife-104275-code1-v1.zip
Download links
A two-part list of links to download the article, or parts of the article, in various formats.
Downloads (link to download the article as PDF)
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Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
Phenotypic impact of individual conserved neuronal microexons and their master regulators in zebrafish
eLife 13:RP104275.
https://doi.org/10.7554/eLife.104275.3
