Chromatin remodeller CHD7 is required for GABAergic neuron development by promoting PAQR3 expression
2021; Springer Nature; Volume: 22; Issue: 6 Linguagem: Inglês
10.15252/embr.202050958
ISSN1469-3178
AutoresPriyanka Jamadagni, Maximilian Breuer, Kathrin Schmeißer, Tatiana Cardinal, Betelhem Kassa, J. Alex Parker, Nicolas Pilon, Éric Samarut, Shunmoogum A. Patten,
Tópico(s)Neuroscience and Neuropharmacology Research
ResumoArticle26 April 2021Open Access Transparent process Chromatin remodeller CHD7 is required for GABAergic neuron development by promoting PAQR3 expression Priyanka Jamadagni Priyanka Jamadagni INRS- Centre Armand-Frappier Santé Biotechnologie, Laval, QC, Canada Search for more papers by this author Maximilian Breuer Maximilian Breuer INRS- Centre Armand-Frappier Santé Biotechnologie, Laval, QC, Canada Search for more papers by this author Kathrin Schmeisser Kathrin Schmeisser Centre de recherche du Centre Hospitalier de l'Université de Montréal (CRCHUM), Montréal, QC, Canada Search for more papers by this author Tatiana Cardinal Tatiana Cardinal Centre d'Excellence en Recherche sur les Maladies Orphelines - Fondation Courtois (CERMO-FC), Université du Québec à Montréal (UQAM), Montréal, QC, Canada Search for more papers by this author Betelhem Kassa Betelhem Kassa INRS- Centre Armand-Frappier Santé Biotechnologie, Laval, QC, Canada Search for more papers by this author J Alex Parker J Alex Parker orcid.org/0000-0002-3333-2445 Centre de recherche du Centre Hospitalier de l'Université de Montréal (CRCHUM), Montréal, QC, Canada Modelis inc., Montréal, QC, Canada Search for more papers by this author Nicolas Pilon Nicolas Pilon orcid.org/0000-0003-3641-0776 Centre d'Excellence en Recherche sur les Maladies Orphelines - Fondation Courtois (CERMO-FC), Université du Québec à Montréal (UQAM), Montréal, QC, Canada Département des sciences biologiques, Université du Québec à Montréal (UQAM), Montréal, QC, Canada Département de pédiatrie, Université de Montréal, Montréal, QC, Canada Search for more papers by this author Eric Samarut Eric Samarut Centre de recherche du Centre Hospitalier de l'Université de Montréal (CRCHUM), Montréal, QC, Canada Modelis inc., Montréal, QC, Canada Search for more papers by this author Shunmoogum A Patten Corresponding Author Shunmoogum A Patten [email protected] orcid.org/0000-0002-2782-3547 INRS- Centre Armand-Frappier Santé Biotechnologie, Laval, QC, Canada Centre d'Excellence en Recherche sur les Maladies Orphelines - Fondation Courtois (CERMO-FC), Université du Québec à Montréal (UQAM), Montréal, QC, Canada Search for more papers by this author Priyanka Jamadagni Priyanka Jamadagni INRS- Centre Armand-Frappier Santé Biotechnologie, Laval, QC, Canada Search for more papers by this author Maximilian Breuer Maximilian Breuer INRS- Centre Armand-Frappier Santé Biotechnologie, Laval, QC, Canada Search for more papers by this author Kathrin Schmeisser Kathrin Schmeisser Centre de recherche du Centre Hospitalier de l'Université de Montréal (CRCHUM), Montréal, QC, Canada Search for more papers by this author Tatiana Cardinal Tatiana Cardinal Centre d'Excellence en Recherche sur les Maladies Orphelines - Fondation Courtois (CERMO-FC), Université du Québec à Montréal (UQAM), Montréal, QC, Canada Search for more papers by this author Betelhem Kassa Betelhem Kassa INRS- Centre Armand-Frappier Santé Biotechnologie, Laval, QC, Canada Search for more papers by this author J Alex Parker J Alex Parker orcid.org/0000-0002-3333-2445 Centre de recherche du Centre Hospitalier de l'Université de Montréal (CRCHUM), Montréal, QC, Canada Modelis inc., Montréal, QC, Canada Search for more papers by this author Nicolas Pilon Nicolas Pilon orcid.org/0000-0003-3641-0776 Centre d'Excellence en Recherche sur les Maladies Orphelines - Fondation Courtois (CERMO-FC), Université du Québec à Montréal (UQAM), Montréal, QC, Canada Département des sciences biologiques, Université du Québec à Montréal (UQAM), Montréal, QC, Canada Département de pédiatrie, Université de Montréal, Montréal, QC, Canada Search for more papers by this author Eric Samarut Eric Samarut Centre de recherche du Centre Hospitalier de l'Université de Montréal (CRCHUM), Montréal, QC, Canada Modelis inc., Montréal, QC, Canada Search for more papers by this author Shunmoogum A Patten Corresponding Author Shunmoogum A Patten [email protected] orcid.org/0000-0002-2782-3547 INRS- Centre Armand-Frappier Santé Biotechnologie, Laval, QC, Canada Centre d'Excellence en Recherche sur les Maladies Orphelines - Fondation Courtois (CERMO-FC), Université du Québec à Montréal (UQAM), Montréal, QC, Canada Search for more papers by this author Author Information Priyanka Jamadagni1, Maximilian Breuer1, Kathrin Schmeisser2,†, Tatiana Cardinal3, Betelhem Kassa1, J Alex Parker2,4, Nicolas Pilon3,5,6, Eric Samarut2,4 and Shunmoogum A Patten *,1,3 1INRS- Centre Armand-Frappier Santé Biotechnologie, Laval, QC, Canada 2Centre de recherche du Centre Hospitalier de l'Université de Montréal (CRCHUM), Montréal, QC, Canada 3Centre d'Excellence en Recherche sur les Maladies Orphelines - Fondation Courtois (CERMO-FC), Université du Québec à Montréal (UQAM), Montréal, QC, Canada 4Modelis inc., Montréal, QC, Canada 5Département des sciences biologiques, Université du Québec à Montréal (UQAM), Montréal, QC, Canada 6Département de pédiatrie, Université de Montréal, Montréal, QC, Canada †Present address: Max-Planck Institute of Molecular Cell Biology and Genetics (MPI-CBG), Dresden, Germany *Corresponding author. Tel: +1 450 687 5010 ext 8864; E-mail: [email protected] EMBO Rep (2021)22:e50958https://doi.org/10.15252/embr.202050958 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Mutations in the chromatin remodeller-coding gene CHD7 cause CHARGE syndrome (CS). CS features include moderate to severe neurological and behavioural problems, clinically characterized by intellectual disability, attention-deficit/hyperactivity disorder and autism spectrum disorder. To investigate the poorly characterized neurobiological role of CHD7, we here generate a zebrafish chd7−/− model. chd7−/− mutants have less GABAergic neurons and exhibit a hyperactivity behavioural phenotype. The GABAergic neuron defect is at least in part due to downregulation of the CHD7 direct target gene paqr3b, and subsequent upregulation of MAPK/ERK signalling, which is also dysregulated in CHD7 mutant human cells. Through a phenotype-based screen in chd7−/− zebrafish and Caenorhabditis elegans, we show that the small molecule ephedrine restores normal levels of MAPK/ERK signalling and improves both GABAergic defects and behavioural anomalies. We conclude that chd7 promotes paqr3b expression and that this is required for normal GABAergic network development. This work provides insight into the neuropathogenesis associated with CHD7 deficiency and identifies a promising compound for further preclinical studies. SYNOPSIS Loss-of-function of chd7 causes defects in GABAergic neuron development and behavioural anomalies reminiscent of CHARGE syndrome, which are rescued by genetic and pharmacological interventions in zebrafish. Zebrafish chd7−/− larvae display aberrant GABAergic network development and hyperactivity behavioral phenotypes. Downregulation of paqr3b expression contributes to the GABAergic neuron defects in zebrafish chd7−/− larval brains. Ephedrine rescues the GABAergic neuron and behavioral defects. Introduction Heterozygous loss-of-function mutations in CHD7 are the major cause of a rare congenital disorder termed CHARGE syndrome (CS), which stands for coloboma of the eye, heart defects, atresia choanae, retardation of growth and/or development, genital abnormalities and ear abnormalities (Pagon et al, 1981; Zentner et al, 2010; Janssen et al, 2012). The mutations are equally distributed along the coding region of CHD7, and the most prevalent types are nonsense mutations (44%), followed by frameshift deletions or insertions (34%) (Janssen et al, 2012). Although neurological abnormalities are not considered for clinical diagnosis of CS, many individuals with CS display moderate to severe neurological deficits, which include autism-like behaviour, obsessive–compulsive disorder, attention-deficit/hyperactivity disorder, anxiety, aggressivity and seizures (Hartshorne et al, 2005; Souriau et al, 2005; Johansson et al, 2006; Bergman et al, 2011; Hartshorne et al, 2017). Along these lines, CHD7 mutations have been identified in patients with autism spectrum disorder (ASD) (O'Roak et al, 2012; Takata et al, 2018). These reports strongly suggest an important role for CHD7 in the development and functioning of the central nervous system. However, the precise mechanisms underlying the neurological deficits in CS remain poorly understood. A recent study reported anxious- and aggressive-like behaviours with increased expression of glycine transporters in adult chd7 heterozygous mutant zebrafish, leaving however unexplored the molecular and cellular mechanisms of brain circuitry (Liu & Liu, 2020). Also, noteworthy, there are no pharmacological and/or genetic treatments to ameliorate/rescue CS-related neurological features. Current treatment options primarily focus on behavioural management as well as educational and physical therapies. Development of successful therapeutic strategies would benefit from the identification and targeting of causative factors. Chd7−/− mice die in utero around embryonic day 10.5 (Van Nostrand et al, 2014), a stage incompatible for studying the role of CHD7 in the neuropathogenesis of CS. Additionally, Chd7+/− mice are viable and phenocopy a number of aspects of CS, but the full spectrum and severity of certain CS malformations are not seen (Payne et al, 2015). Yet, Chd7+/− mice, Chd7 conditional knockout mice and other cellular and animal (Drosophila, zebrafish and Xenopus) models have provided insights on the general function of CHD7 (Schnetz et al, 2010; Patten et al, 2012; Ohta et al, 2016; Feng et al, 2017; Whittaker et al, 2017; Belanger et al, 2018). For instance, it has been shown that CHD7 is capable of both enhancing and inhibiting expression of embryonic stem cell genes (Schnetz et al, 2010). In that respect, CHD7 facilitates neural stem cell maintenance and proliferation in the developing brain (Ohta et al, 2016) and quiescence in the adult (Jones et al, 2015). It is also required for the formation of migratory neural crest cells (Bajpai et al, 2010; Okuno et al, 2017). CHD7 coordinates with the transcription factor SOX10 to regulate the initiation of myelinogenesis (He et al, 2016) and is required for oligodendrocyte precursor cell survival (Marie et al, 2018). Genetic inactivation of Chd7 in cerebellar granule neuron (GN) progenitors leads to cerebellar hypoplasia in mice, due to impairment of GN differentiation (Feng et al, 2017; Whittaker et al, 2017) but these cerebellar defects did not alter the social behaviour in these mice (Whittaker et al, 2017). Although these recent findings point to an important role of CHD7 in brain development, the precise neural substrates that may contribute to CS-associated neurological deficits such as autistic traits and/or hyperactivity disorder remain poorly understood. Emerging evidence suggests that abnormalities in inhibitory GABAergic neurons development/function in the context of neurodevelopmental disorders are characterized by a shared symptomatology of ASD symptoms (Rubenstein & Merzenich, 2003; Coghlan et al, 2012). Whether such alterations in brain inhibitory neural networks underlie the neurological deficits in CHD7 mutation-positive cases of CS is currently unknown. The zebrafish is a powerful tool for studying neurological diseases including ASD (Stewart et al, 2014; Meshalkina et al, 2018). Here, we report the generation of a chd7−/− mutant zebrafish line and show that these animals exhibit altered number and positioning of GABAergic neurons in the brain and display a hyperactive behaviour phenotype. Using genetic, pharmacological and biochemical approaches, we unravel the molecular mechanisms by which chd7 regulates GABAergic network development and behaviour in zebrafish. Finally, through a chemical-genetic screen, we identified ephedrine that effectively ameliorates behavioural anomalies as well as the GABAergic defects in chd7 mutants. This study provides novel insights into the role of CHD7 in brain development and disease and has important translational implications. Results Zebrafish chd7 mutants display phenotypic characteristics of CHARGE syndrome To investigate the neurobiological function of CHD7, we generated a chd7 knockout zebrafish line using CRISPR/Cas9 to target the helicase domain of the chd7 gene (17th exon) for disruption. A positive founder transmitting a single nucleotide insertion causing a frame-shifting mutation was selected (Fig 1A). This mutation causes a premature stop codon 8 amino acids after the mutation site (Fig EV1A). To assess whether the mutant chd7 transcript underwent nonsense-mediated decay upon that mutation, we performed qPCR. The relative abundance of chd7 mRNA in mutant zebrafish was significantly decreased, suggesting a loss of mutant transcript via nonsense-mediated decay (Fig EV1B). While no major morphological differences were observed between wild-type (chd7+/+) and heterozygous (chd7+/−) fish (Fig 1B), the survival rate of homozygous (chd7−/−) larvae sharply declined after 10 days postfertilization (dpf) (Fig 1C). Remarkably, chd7−/− zebrafish larvae displayed a small head phenotype (Fig 1D) compared with controls but nevertheless all the brain regions were fairly well-preserved in mutant fish (Fig EV1C). Additionally, chd7−/− zebrafish larvae exhibited a low frequency of pericardial oedema (20 %) (Fig EV1D), cranial cartilage malformations (Fig EV1E) and cranial nerve defects (Fig 1E). Notably, there were less arborizations of peripheral projections from the Vth cranial nerve in chd7−/− fish. Precisely, chd7−/− fish had reduced growth and branching of the peripheral axons, resulting in a significant decrease in the mean total length of the axon projections as compared to controls (Fig 1E). Strikingly, these phenotypic characteristics are hallmarks of CS (Hsu et al, 2014). Additionally, this new chd7−/− mutant fish recapitulates other anomalies previously reported in chd7 morphants (Patten et al, 2012) and other chd7 mutant zebrafish lines (Cloney et al, 2018; Liu et al, 2018), but with less pronounced cardiac defects and no apparent blindness, thereby making it an ideal model to investigate the pathogenic mechanisms underlying CS-associated neurological deficits. Behaviourally, chd7−/− larvae were significantly hyperactive compared with wild-type and chd7+/− fish larvae at 5dpf (Fig 1F and G). This hyperactive phenotype was particularly prominent and persistent during the dark cycles (Fig 1G). Figure 1. Generation of a zebrafish chd7 mutant using CRISPR/Cas9 Chromatograms showing the confirmation a 1-nucleotide insertion mutation by Sanger sequencing. Gross morphological analyses of control (chd7+/+; top left image), heterozygous (chd7+/−; bottom left image) and knockout mutants (chd7−/−; images in right panel). Kaplan–Meier survival plot showing low survival of chd7 mutants after 12 dpf (N = 5). Measurement of head size of control (n = 12) and mutants (n = 14) showing significantly smaller head size in chd7−/− fish (****P < 0.0001, Student's t-test). Acetylated tubulin staining in 28 hpf controls (left) and mutants (right) showing severely affected outbranching of the trigeminal nerve (Vth cranial nerve). Notably, chd7−/− display reduced branching of the Vth cranial nerve (arrows) and axonal arborization in the tectal area. Graphs showing quantitative analyses of percentage (n = 5) and mean total length of peripheral projections (n = 6) per zebrafish in controls and mutants (***P < 0.001; **P < 0.005, Student's t-test). Locomotor activity of control (black), heterozygous (blue) and mutants (red) showing significant hyperactivity of mutants in dark and light cycles (N = 3, n = 48). Average activity per second during dark cycle (left) is significantly increased in chd7−/− mutants compared with chd7+/+ (n = 32; ****P < 0.0001, Student's t-test). Representative swimming tracks during dark cycle of control and mutant fish (right). Mutant chd7−/− larvae displayed hyperactive swimming. Data information: ****P < 0.0001; ***P < 0.001; **P < 0.005, Student's t-test. Data are presented as mean ± SEM. Scale bar = 50 μm. n represents number of fish. N represents number of experimental repeats. Download figure Download PowerPoint Click here to expand this figure. Figure EV1. Generation and characterization of chd7 mutants Translation following genome editing resulted in a premature stop codon (*) in chd7−/− fish. qPCR analysis of RNAs from 3dpf larvae shows a significant reduction of chd7 mRNA expression in both chd7+/− and chd7−/− compared with wild-type (N = 5). ****P < 0.0001, one-way ANOVA. Examination of brain tissues in chd7+/+ (left images) and chd7−/− (right images) by H&E staining at 5 dpf. Zebrafish brains were sectioned at telencephalic (1,1'), diencephalic (2, 2'), mesencephalic (3,3') and rhombencephalic levels (4,4'). Levels of sections are indicated in the sketch of a sagittal view of a 5 dpf zebrafish brain (top image). The scale bar is 0.12 mm. P: pallium, S: subpallium, Po:preoptic region, Tel: Telencephalon; TeO: tectum opticum (or OT: optic tectum), m: medial tectal proliferating zone, DT: dorsal thalamus, PTd: dorsal part of posterior tuberculum, PTv: ventral part of posterior tuberculum, MO: medulla oblongata, Hyp: hypothalamus, CeP: cerebellar plate. chd7−/− mutant fish displayed features of CS such as heart defects (red arrow) at a low penetrance (N = 3). ****P < 0.0001; Student's t-test. Alcian blue staining of 6dpf larvae showing craniofacial defects at Meckel's cartilage (red arrow). Data information: Data are presented as mean ± SEM. n is the number of fish used. N is the number of experimental repeats. Download figure Download PowerPoint GABAergic neuron differentiation is defective in chd7−/− mutants A hyperactivity behavioural phenotype in ASD mouse (Lee et al, 2018) and zebrafish (Hoffman et al, 2016) models has been reported to be due to alterations in GABAergic interneuron development. To test whether similar alterations in GABAergic neuron development occur in chd7−/− mutants, we analysed the inhibitory GABAergic neuronal populations in wild-type controls and chd7−/− mutants during early brain development (Fig 2), using a transgenic line that labels GABAergic interneurons (Tg(dlx5a/6a:GFP)). Compared with controls, chd7−/− larvae had a significant reduction in the density of GFP-positive GABAergic cells in the brain at 5 dpf (Fig 2A and B). Particularly, we observed a highly significant decrease in the number of GABAergic neurons in the optic tectum (OT) (Fig 2C and D) and a near-complete loss of GFP-positive cells in the cerebellum (CB) compared with the controls. Reduced number and malpositioning of GABAergic cells were also observed in the hypothalamus (HYP; Fig 2E and F) and telencephalon (TEL; Fig 2G and H). Figure 2. GABAergic neuron defects in zebrafish chd7 mutant brain A. Structural illustration of 5 dpf zebrafish brain from dorsal (top) and lateral (bottom) view (OB: Olfactory bulb, Tel: Telencephalon, OT: Optic tectum, CB: Cerebellum, HB: Hindbrain). B. 5 dpf dlx5a/6a transgenic line showing GFP+ GABAergic neurons are reduced in chd7−/− mutants (bottom) in comparison with controls (top) in both dorsal (left) and ventral (right) view. C–H. Total number of GABAergic neurons (GFP+ cells) in (C, D) the optic tectum (OT) and cerebellum (CB) regions of 5 dpf wild-type and chd7 mutant fish (n = 16; ****P < 0.0001; Student's unpaired t-test), (E, F) the hypothalamus (hyp) region (n = 10; *P = 0.0182; Student's t-test) and (G, H) the telencephalon (tel) (n = 7; *P = 0.0347; Student's t-test). I. Treatment of control (dark grey) and mutants (light grey) with GABA agonists Baclofen (N = 3, n = 24; ns, P = 0.1427; Student's t-test) and Muscimol (N = 3, n = 24; ns, P = 0.3987; Student's t-test) showing recovery of hyperactive locomotor activity in chd7−/− mutants (vehicle: N = 3, n = 24, ****P < 0.0001; Student's t-test). J. Functional analysis of GABAergic signalling shows increased responsiveness to GABA antagonist PTZ in both onset and overall locomotor activity (n = 24; ****P < 0.0001; one-way ANOVA). K. Average locomotor activity between 2 dpf controls (black) and chd7−/− mutants (red) shows increased activity after 3 mM PTZ exposure (n = 24; ***P < 0.001; two-way ANOVA). Data information: Data are presented as mean ± SEM. Scale bar = 50 μm. n represents number of fish used. N represents number of experimental repeats. Download figure Download PowerPoint We also examined the development of GABAergic neurons in chd7−/− brain throughout major developmental phases between 1 and 5 dpf (Fig EV2A). The reduced number of GFP-positive GABAergic neurons occurs very early in chd7−/− embryos, with a striking decrease of GABAergic neurons posteriorly between 1 and 2 dpf (Fig EV2A). We next tested whether the reduced number of GABAergic neurons in chd7−/− fish could be due to reduced proliferation, defects in neuronal differentiation and/or enhanced cell death. The zebrafish CNS proliferative profile is still very high at 2 dpf and is rapidly downregulated up to 5 dpf (Wullimann & Knipp, 2000). At 2 dpf, we did not observe a change in either the proliferation marker pH3 (Fig EV2B and C) or differences in the number of apoptotic cells (Fig EV2D). Additionally, we did not notice differences in the number of double-positive cells in pH3 and NeuroD1 (neuronal progenitor marker) co-staining (Fig EV2E). However, at 5 dpf, while no apoptosis was observed, a significant increase in pH3-positive cells was detected (Fig EV2-EV6), suggesting a failure in differentiation of progenitor cells into GABAergic neurons. Click here to expand this figure. Figure EV2. Proliferation and apoptosis analyses in wild-type and chd7 mutant zebrafish Analysis of GABAergic neurons network development between 1 dpf and 5 dpf between control (top row) and chd7 mutants (bottom row) (N = 3). Proliferation analysis by pH3 staining at 2 dpf in control and chd7 mutants. Bar graph showing no difference in pH3-positive cells in zebrafish brain at 2 dpf between control and mutants (n = 11; ns, P = 0.1816; Student's t-test). Cell death analysis by TUNEL assay in chd7−/− and chd7+/+ brains shows no change in apoptotic cells at 2 dpf (N = 3, n = 8; ns, P = 0.464; Student's t-test). Transverse sections of 2 dpf larvae after immunostaining with pH3 (red) and NeuroD1 (green). Bar graph showing no difference in pH3 and NeuroD1 double-positive cells (arrows) in zebrafish brain at 2 dpf between control and mutants (N = 3, n = 5; ns, P = 0.124; Student's t-test). P: pallium, S: subpallium, TeO: tectum opticum, m: medial tectal proliferating zone, DT: dorsal thalamus R: retina. Proliferation analysis by pH3 staining at 5 dpf in control and chd7 mutants. An increase in pH3-positive cells was noted in brains of mutant fish compared with controls at 5 dpf (N = 4, n = 10; ****P < 0.0001; Student's t-test). Transverse sections of 5 dpf larvae after immunostaining with pH3 (red) and HuC/D (green). pH3-positive cells (arrows) were observed at 5 dpf in the medial tectal proliferating zone of mutant fish brains but none in controls (N = 3). P: pallium, S: subpallium, TeO: tectum opticum, m: medial tectal proliferating zone, DT: dorsal thalamus. Data information: Data are presented as mean ± SEM. Scale bar = 50 μm and 10 μm for 2 dpf NeuroD1 and pH3 co-stain. Download figure Download PowerPoint Click here to expand this figure. Figure EV3. Aberrant GABAergic neuronal differentiation in chd7 mutant zebrafish A, B. Immunostaining with BrdU and HuC/D in brain sections of the zebrafish tectal region in chd7+/+ (A) and chd7−/− (B). Level of the sections is indicated in the sketch of a 5 dpf zebrafish brain (top right image in (A)). The scale bar is 10 μm. Tel: Telencephalon; TeO: tectum opticum, m: medial tectal proliferating zone, DT: dorsal thalamus, PTd: dorsal part of posterior tuberculum, PTv: ventral part of posterior tuberculum, l: lateral tectal proliferation zone. Asterisks (*) marks early migrated region of pretectum and proglomerular. C. The number of BrdU-positive cells in transverse sections of the zebrafish brain in chd7+/+ and chd7−/− (N = 3, chd7+/+: n = 8; chd7−/−: n = 4; **P < 0.05; Student's t-test). D. Immunostaining with BrdU and HuC/D in brain sections of the zebrafish medial tectal region. Scale bar = 10 μm. m: medial tectal. E. The percentage of BrdU and HuC/D-double positive cells among the BrdU-positive cells in the medial tectal zone (N = 3, n = 4; **P < 0.05; Student's t-test). F. Immunostaining with BrdU and GFP (to label dlx5a/6a-GFP + GABAergic neurons) in brain sections of the zebrafish medial tectal region. Scale bar = 10 μm. m: medial tectal. G. The percentage of BrdU and dlx5a/6a-GFP-double positive cells among the BrdU-positive cells in the medial tectal zone (N = 3, n = 3; **P < 0.05; Student's t-test). Data information: Data are presented as mean ± SEM. n is the number of fish used. N is the number of experimental repeats. Download figure Download PowerPoint Click here to expand this figure. Figure EV4. Impaired neurogenesis in chd7 mutant zebrafish A, B. Immunostaining with BrdU and NeuroD1 in brain sections of the zebrafish tectal region in chd7+/+ (A) and chd7−/− (B). The scale bar is 10 μm. Tel: Telencephalon; TeO: tectum opticum, m: medial tectal, DT: dorsal thalamus, PTd: dorsal part of posterior tuberculum, PTv: ventral part of posterior tuberculum, l: lateral tectal proliferation zone. C. Immunostaining with BrdU and NeuroD1 in brain sections of the zebrafish medial tectal region. Scale bar = 10 μm. m: medial tectal. D. The percentage of BrdU and NeuroD1-double positive cells among BrdU-positive cells in the medial tectal zone (N = 3, chd7+/+: n = 6; chd7−/−: n = 6; **P < 0.05; Student's t-test). E. Expression level of scl1a3 mRNA in chd7−/− relative to chd7+/+ (N = 4). ns, not significant; Student's t-test. Data information: Data are presented as mean ± SEM. n is the number of fish used. N is the number of experimental repeats. Download figure Download PowerPoint Click here to expand this figure. Figure EV5. Expression of paqr3b in wild-type and chd7 mutant zebrafish A, B. Expression profile of paqr3b in whole-mount zebrafish by in situ hybridization (A) and in tissues by qRT–PCR (B). N = 4. C. qRT–PCR validation of the downregulation of paqr3b (N = 4; ****P < 0.0001, Student's t-test). D. Images of gross morphology of 2 dpf zebrafish embryos with or without overexpression of paqr3b mRNA. Of note, neither abnormalities nor death were observed in zebrafish embryos upon overexpression of paqr3b mRNA. Data information: Data are presented as mean ± SEM. n is the number of fish used. N is the number of experimental repeats. Download figure Download PowerPoint Click here to expand this figure. Figure EV6. Pharmacological responses of chd7 mutants and amelioration of neuronal network development by ephedrine PCR proof of a 700 bp deletion in the chd-7 gene in chd-7(gk290) mutant worms. Lifespan analyses of chd-7(gk290) Caenorhabditis elegans mutants treated with ephedrine (green) compared with control DMSO (black). Log-rank test was performed for statistical analyses. (N = 3, n = 50; *P < 0.05). Survival rate of chd7−/− zebrafish mutants treated with ephedrine (blue) compared with untreated mutants (red). N = 3, n = 60. Acetylated tubulin staining in non-treated and ephedrine-treated chd7−/− zebrafish mutants showing rescue of the severely affected outbranching structure of Vth cranial nerves. Graphs showing quantitative analyses of percentage and mean total length of peripheral projections per zebrafish in controls and mutants without and with ephedrine treatment (n = 5; ***P < 0.001; **P < 0.005; one-way ANOVA). Data information: Data are presented as mean ± SEM. Scale bar = 50 μm. n is the number of fish or worms used. N is the number of experimental repeats. Download figure Download PowerPoint We, thus, next sought to evaluate further neurogenesis in chd7 mutants during brain development, with a focus on the midbrain region—the brain region where the reduced number of GABAergic neurons was more prominent in 5 dpf chd7−/− fish. Zebrafish larvae (4 dpf) were exposed to BrdU-containing media for 24 h and fixed. In both chd7+/+ and chd7−/− fish, BrdU-labelled cells were noted in the medial tectal proliferation zone (m), dorsal thalamus (DT), posterior tuberculum (PT) and the lateral tectal proliferation zone (l) of the midbrain (Fig EV3A and B). Interestingly, compared with chd7+/+, BrdU-labelled cells in chd7−/− did not migrate over long distances to reach the early migrated region of pretectum and proglomerular (Fig EV3A; asterisks), which are regions involved with visual and other sensorimotor circuits (ref). We also found an increased in the number of BrdU-labelled cells in chd7 mutant fish compared with wild-type controls (Fig EV3C). In order to determine the phenotype of BrdU-positive cells after the 24-h incubation period, brain sections were double-labelled to detect the colocalization of BrdU with HuC/D (a neuronal marker; Fig EV3A, B, D and E), dlx5a/6a-GFP (a GABAergic neuron marker; Fig EV3F and G) or NeuroD1 (a neuronal progenitor marker; Fig EV4-EV6) in the midbrain area of the 5 dpf zebrafish larvae. The number of cells positive for both BrdU and HuC/D (Fig EV3E), BrdU and dlx5a/6a-GFP (Fig EV3G) or BrdU and NeuroD1 (Fig EV4D) in the midbrain area, surrounding the proli
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