fhl2b is upregulated in the EOM in response to desmin-related muscular dystrophy.a Expression of myofiber-related DEGs for the following comparisons: 5 months desma−/−:desmb−/−vs WT EOMs (I), 5 months desma−/−:desmb−/−vs WT trunk (II), 20 months desma−/−:desmb−/−vs WT EOMs (III), 20 months desma−/−:desmb−/−vs WT trunk (IV), 5 months WT EOMs vs WT trunk (V), 5 months desma−/−:desmb−/− EOMs vs desma−/−:desmb−/− trunk (VI), 20 months WT EOMs vs WT trunk (VII) and 20 months desma−/−:desmb−/− EOMs vs desma−/−:desmb−/− trunk (VIII). bfhl2b expression in the abovementioned comparisons I-VIII. FHL2 antibody labeling of WT and desma−/−;desmb−/− cross-sectioned EOMs at c 5 and d 20 months. e FHL2 positive myofibers quantification of desma−/−;desmb−/− versus WT control EOMs in 5 (p = 0.017) and 20-monthold zebrafish (p = 3.7e−7), respectively and 5-months versus 20-months-old WT (p = 0.014) and desma−/−:desmb−/− (p = 0.005), respectively. Data is presented as median (line) and quartiles (dashed lines). f WT, plecb−/− and obscnb−/− 12-month-old EOM cross sections immunolabeled with FHL2 antibodies. g Human EOM cross section immunolabeled with DAPI/FHL2/laminin antibodies, dashed square indicates area enlarged below. h Western blot on human EOMs showing FHL2. i Mouse EOM cross section immunolabeled using DAPI/FHL2/laminin antibodies, dashed boxes indicate area enlarged below. j Western blot of mouse EOMs showing FHL2. Statistical analysis in b: Two-sided Wald test with B/H-correction, e: Two-sided t-tests with Welch correction. Scale bars in c, d, f, g, i: 50 µm, g, i bottom panel: 25 µm. White dashed lines outline the entire cross-section of the EOMs. Schematic images were adapted from https://www.biorender.com.

nomo1 mutant induce brain inflammatory in the brain, MC treatment rescued the small circling behavior.(A) GO enrichment analysis of DEGs and (B) KEGG pathway analysis of DEGs. Advanced bubble chart showing the enrichment of DEGs in signaling pathways. The vertical axis indicates the pathway, and the horizontal axis indicates the gene ratio (gene ratio is the ratio of the number of DEGs to the total number of DEGs annotated to the KEGG pathway). The size and color of the bubble represent the number of DEGs enriched in a pathway and the significance of enrichment, respectively. (C–F) (C) Relative expression level of inflammatory cytokines in WT, mutant and MT treatment brains (technical replicates, N = 4). Locomotor activity (D) biological replicates, N = 24), shoaling behavior (E) (biological replicates, N = 24) and three kinds of repetitive behaviors (F) (biological replicates, N = 24)) of WT, mutant and MC treatment zebrafish. NT was short for no treatment. Data are analyzed using unpaired t test and presented as the mean ± SEM, *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001. Source data are available online for this figure.

Disruption of Zebrafish irf2bpl Leads to Spontaneous Seizures. A. Representative bright-field imaging of larval zebrafish at 5 days post fertilization (dpf). Top, cas9 injected control; bottom, irf2bpl F0 CRISPR (crispant). B-C. Measurements of eye distance and body length in cas9 injected control (n = 20 fish) vs. irf2bpl crispants (n = 26 fish; unpaired t test). Data were normalized to the average values of cas9 control group. D. Representative imaging of HuC: eGFP expressed larval zebrafish shows CNS fluorescence pattern at 5 dpf (dorsal view). Left, cas9 injected control; right, irf2bpl crispant. E. Normalized CNS area in cas9 injected controls vs. irf2bpl crispants. Data were normalized to the average CNS area of cas9 control group. F. CRISPR efficacy calculated via TIDE method of individual irf2bpl crispant used in phenotypic study (n = 26 fish). G. Representative local field potential (LFP) recording from cas9 injected control shows baseline activity. H. Representative LFP recording from irf2bpl crispants shows epileptiform events, and a magnified view of the orange box is shown. I. Occurrence of electrographic epileptiform events in cas9 injected control (n = 30 fish) and irf2bpl crispants (n = 24 fish; Chi-square test). Grey, number of fish showed baseline activity; Orange, number of fish showed epileptiform activity. Scale bars as indicated in the figure. Error bars indicate standard deviation (SD). Statistical significance is indicated as *p < 0.05, **p < 0.01, ***p < 0.001

foxj1 is expressed in ciliated OSNs of zebrafish and mice.(A) Schematic showing the larval zebrafish OE and OB; l: lateral, m: medial, d: dorsal, v: ventral. (B-J) Confocal images of the larval (3–5 dpf) zebrafish nose and OB. (B) foxj1b (Gt(foxj1b:GFP), green) is expressed in neurons labeled by HuC (magenta). Asterisks show foxj1-expressing neurons in 5 dpf larva. (C) Differential expression of foxj1a (Gt(foxj1a:2A-TagRFP), magenta) and foxj1b (Gt(foxj1b:GFP), green) paralogs in 5 dpf larvae. Note that foxj1a is expressed primarily at the outer rim of the OE. (D) foxj1b-positive cells (Gt(foxj1b:GFP), green) bear a brush of motile cilia marked by glutamylated-tubulin (magenta) in 5 dpf larvae. (E) foxj1b-positive OSNs (Gt(foxj1b:GFP), green) in the nasal pit bear cilia, indicated by the marker Gαolf (magenta) in 3 dpf larva. White arrowheads in insets show Gαolf marked cilia arising from foxj1b-positive OSNs. (F) Overlap of foxj1b-expressing cells (Gt(foxj1b:GFP), green) with the ciliated OSN marker OMP (Tg(OMP:gal4); Tg(UAS:NTR-mCherry), magenta) in 4 dpf larva. (G) Mutually exclusive localization of foxj1b-expressing cells (Gt(foxj1b:GFP), green) and microvilli OSNs in 4 dpf larvae, indicated by the marker trpc2 (Tg(trpc2:gal4); Tg(UAS:NTR-mCherry), magenta). (H) foxj1b-expressing OSNs (Gt(foxj1b:GFP), green) project mainly to Gαolf-labeled glomeruli (magenta) in 3 dpf larvae. Note that foxj1b:GFP and Gαolf do not overlap entirely (dotted region). (I, J) foxj1b-positive OSNs, in 4 dpf larvae, (I) project to more glomeruli than OMP-expressing OSNs (J) (Tg(OMP:ChR-YFP)). Glomeruli are shown by staining with the presynaptic vesicle marker SV2 (magenta). (K) Schematic showing mouse RE, OE, and OB; r: rostral, c: caudal, d: dorsal, v: ventral. (L) In the nasal epithelium of the adult mouse (P30), Foxj1 localizes to MCCs (arrowhead) within the RE and the layer of OSNs in the OE. Dashed line demarcates the lamina propria separating OE from the underlying tissue. (M) Foxj1 is absent from the nasal epithelium of the Foxj1 knockout mouse (P21). The residual puncta likely represent nonspecific staining of blood vessels or mesenchymal tissue located in the lamina propria under the basement membrane. (N) Foxj1 (magenta) localizes to the nuclei of mature OSNs immunostained for the OMP (P30). (O) Expression levels of Foxj1 in OSNs at different ages (newborn P0, day 5 P5, and adult P30) calculated by comparing immunofluorescence in OSNs relative to respiratory MCCs. No significant difference was found (MCC/OSN ratio of 10.87 ± 2.61, P0; 8.74 ± 0.53, P5; 6.83 ± 1.56, P30; fluorescence intensity was measured in 20–30 cells of each type per individual field of view and ratio was calculated per animal, 3 mice in each group) by one-way ANOVA with Kruskal–Wallis test. Scale bars = 10 μm (B-J), 50 μm (L, M), and 10 μm (N). Raw data files are available in Mendeley Data (https://data.mendeley.com/datasets/2pn963jn6y). dpf, days post fertilization; MCC, motile multiciliated cell; OB, olfactory bulb; OE, olfactory epithelium; OMP, olfactory marker protein; OSN, olfactory sensory neuron; RE, respiratory epithelium.