European Journal of Medical Genetics

Chemical screens in a zebrafish model of CHARGE syndrome identifies small molecules that ameliorate disease-like phenotypes in embryo
Zainab Asada,b,1, Chetana Sachidanandana,b,∗
a CSIR-Institute of Genomics and Integrative Biology (CSIR-IGIB), New Delhi, 110025, India
b Academy of Scientific and Innovative Research (AcSIR), New Delhi, India

A R T I C L E I N F O

Keywords:
CHARGE syndrome CHD7
Zebrafish
Drug discovery M344
CHIC-35 DAPT
Procainamide

A B S T R A C T

CHARGE syndrome is an autosomal dominant congenital disorder caused primarily by mutations in the CHD7 gene. Using a small molecule screen in a zebrafish model of CHARGE syndrome, we identified 4 compounds that rescue embryos from disease-like phenotypes. Our screen yielded DAPT, a Notch signaling inhibitor that could ameliorate the craniofacial, cranial neuronal and myelination defects in chd7 morphant zebrafish embryos. We discovered that Procainamide, an inhibitor of DNA methyltransferase 1, was able to recover the pattern of expression of isl2a, a cranial neuronal marker while also reducing the effect on craniofacial cartilage and myelination. M344, an inhibitor of Histone deacetylases had a strong recovery effect on craniofacial cartilage defects and could also modestly revert the myelination defects in zebrafish embryos. CHIC-35, a SIRT1 inhibitor partially restored the expression of isl2a in cranial neurons while causing a partial reversion of myelination and craniofacial cartilage defects. Our results suggest that a modular approach to phenotypic rescue in multi-organ syndromes might be a more successful approach to treat these disorders. Our findings also open up the possibility of using these compounds for other disorders with shared phenotypes.
1. Introduction
Mutations in CHD7 gene cause a rare genetic disorder known as CHARGE syndrome. CHARGE is an acronym for the defects present in the syndrome: Coloboma of eye, Heart defects, Atresia of choanae, Retardation of growth, Genital hypoplasia and Ear anomalies (Pagon et al., 1981; Zentner et al., 2010). It is the second most common cause for congenital deafness and visual impairment in U.S.A. after Usher syndrome (Gage et al., 2015).
CHD7 belongs to a family of chromatin remodelers called Chromodomain Helicase DNA binding proteins (Ho and Crabtree, 2010). The two distinctive features of this family are the two N-terminal chromodomains and an SNF-2 like ATPase domain. The chromodo- mains recognize methylated histone tails, putting CHD proteins in the class of epigenetic ‘readers’ (Flanagan et al., 2005). These proteins in- teract with methylated histones to cause unwinding of DNA (Flanagan et al., 2007). Schnetz and colleagues have shown that CHD7 pre- ferentially localizes to enhancer regions along chromatin marked by monomethylation of lysine 4 (H3K4me1) and acetylation of lysine 27 on histone 3 (H3K27Ac) (Feng et al., 2017; Schnetz et al., 2009). The SNF-2 like ATPase domain is an evolutionarily conserved, catalytic

domain present in all ATP-dependent chromatin-remodeling proteins and provides energy by ATP hydrolysis for nucleosome remodeling (Becker and Hörz, 2002; Dürr et al., 2006). CHD7 belongs to the tri- thorax group of chromatin factors. In general, Trithorax (TrXG) proteins maintain their targets in open state, by activating gene expression. CHD7 has been shown to activate expression of Sox 4 and Sox 11 by maintaining their promoters in open chromatin state to induce neuronal differentiation in mouse neural stem cells (Feng and Liu, 2013). In vitro studies using reconstituted chromatin have shown that CHD7 is able to slide nucleosomes on DNA in an ATP dependent manner (Bouazoune and Kingston, 2012). Additionally, subtle to complete loss of nucleo-
somal remodeling activity has been shown in CHD7 mutations present
in CHARGE syndrome patients (Bouazoune and Kingston, 2012), sug- gesting haploinsufficiency to be a cause for the disease.
Balow and colleagues have shown that in chd7 morphant zebrafish embryos, MO-mediated knockdown of histone demethylase fbxl10/ kdm2bb, a repressor of ribosomal RNA genes, rescues zebrafish embryos from patterning defects of craniofacial cartilage (Balow et al., 2013). In previous studies, we have shown that MO-mediated knockdown of sox10, a gene important for neural crest development, can rescue zeb- rafish embryos from defects in craniofacial cartilage and peripheral
∗ Corresponding author. CSIR-Institute of Genomics and Integrative Biology (CSIR-IGIB), New Delhi, 110025, India.
E-mail address: [email protected] (C. Sachidanandan).
1 Present address: Warwick Medical School, University of Warwick, Coventry CV4 7AL, United Kingdom.
Received 8 January 2019; Received in revised form 9 April 2019; Accepted 28 April 2019
1769-7212/©2019ElsevierMassonSAS.Allrightsreserved.
Z. Asad and C. Sachidanandan EuropeanJournalofMedicalGeneticsxxx(xxxx)xxx–xxx
myelination (Asad et al., 2016). In another study, the inhibition of to- poisomerase has been shown to rescue cerebellar defects in Chd7 mu- tant mice (Feng et al., 2017). In yet another approach for ther- apeutically targeting CHARGE syndrome, it was demonstrated that changes in RA signaling could partially rescue inner ear defects in a mouse model (Micucci et al., 2014). This indicates that modulation of CHD7 effectors might have beneficial effects in CHARGE syndrome patients. Thus, we postulated that chemically modulating epigenetic modifier enzymes or signaling pathway components might overcome the loss of CHD7 activity, as happens in CHARGE patients.
CHARGE syndrome models have been created in different organisms like Drosophila (Melicharek et al., 2010), Xenopus (Bajpai et al., 2010), mouse (Bosman et al., 2005; Hurd et al., 2007) and zebrafish (Asad et al., 2016; Patten et al., 2012; Jacobs-McDaniels and Albertson, 2011; Balasubramanian et al., 2014; Cloney et al., 2018; Liu et al., 2018). This demonstrates the functional conservation of CHD7 across species. Zebrafish (Danio rerio) has emerged as an excellent tool for high throughput genetic and chemical screens for human diseases (Asad et al., 2016; Babu et al., 2018; Basu et al., 2018 and reviewed in Lam and Peterson, 2019).
In this study, we performed a highly selective chemical screen with epigenetic and signaling modulators. Our chemical screen was focused on mapping the defects in neural crest-derived tissues such as cranio- facial cartilage, peripheral neurons in cranial and trunk region and myelinating Schwann cells that we have previously described in our zebrafish model for CHARGE Syndrome. Our study revealed that Notch signaling inhibitor, DAPT, ameliorates defects in myelination and cra- niofacial cartilage. Histone deacetylase inhibitor 3, M344, partially rescues embryos from myelination and craniofacial cartilage patterning defects but not neuronal defects. Procainamide, a DNMT1 inhibitor and CHIC-35, an inhibitor of the SIRT1 class of HDACs, were both able to partially revert the expression pattern of gene marking cranial neurons, craniofacial cartilage elements and myelinating Schwann cells. Thus, here, we demonstrate the amelioration of CHARGE syndrome-like phenotypes in an animal model using small molecules, for the first time.
2. Materials and methods
2.1. Zebrafish care and maintenance
Wild-type fish stocks of the TU strain were utilized in this study. Zebrafish were reared using standard procedures, at a constant tem- perature of 28 °C, fed three times daily with hatched shrimp, and maintained on a 14 h light and 10 h dark photoperiod. Fish embryos were kept in egg water (60μg/ml sea salt in distilled water).
Embryos were staged using both, time from fertilization (hours post fertilization, hpf) and morphological features like somites as previously described (Kimmel et al., 1995). Zebrafish experiments were carried out according to standard procedures approved by the Institutional Animal Ethics Committee (IAEC) of the CSIR-Institute of Genomics and In- tegrative Biology, India in accordance with the recommendations of the Committee for the Purpose of Control and Supervision of EXperiments on Animals (CPCSEA), Govt. of India. The zebrafish transgenic lines used in this study are Tg(NBT-dsRed) (Peri and Nüsslein-Volhard, 2008).
2.2. Morpholino knockdown
Antisense morpholino oligonucleotides were purchased from Genetools and microinjected in one-cell stage embryos to perturb en- dogenous gene expression as described in our previous study (Asad et al., 2016).
2.3. Chemical screen for modifiers of CHARGE syndrome phenotypes
Compounds for screening were purchased from Sigma, Calbiochem, Chembridge. Embryos were microinjected with morpholinos at one-cell

stage and incubated at 28.5 °C in embryo water. For screening, embryos were arrayed in 12 well plates with 25 embryos per well. DMSO was used as vehicle control since all small molecules reported in this study were dissolved in DMSO. The volume of DMSO administered to control was equal to the highest volume of drug administered for that particular experiment. All compounds were used at 5 μM concentration. Phenotypes were observed from 1 dpf to 4dpf. The details of con- centration, catalogue number and supplier of compounds is listed in Supplementary Table S1.
2.4. Wholemount RNA insitu hybridization
To visualize expression of genes, wholemount RNA in situ hy- bridization was performed as described (Asad et al., 2016). DigoX- igenin-labeled antisense RNA probes for the following genes foxd3, sox9a, sox9b, mbp, isl2a were prepared using RNA labeling reagents from Sigma as described. (Asad et al., 2016).
2.5. Imaging
Embryos between 1 and 4d pf were observed and imaged using Zeiss (Stemi, 2000®C) bright field microscope. Embryos older than 24hpf were anesthetized in Tricaine, dechorionated manually and treated with PTU to prevent pigmentation prior to imaging. Fluorescent images were captured using Zeiss AXioScope A1 microscope. Images were processed for alignment, balancing, and brightness/contrast using Adobe Photoshop CS6 and Adobe Illustrator.
2.6. Alcian blue staining
Alcian blue, which stains chondroitin sulphate in cartilage, was used to visualize the structure of zebrafish craniofacial skeleton using a previously described protocol (Dingerkus and Uhler, 1977).
2.7. Statistics
We have performed Fisher’s EXact test to compare the percentage of morphant embryos with defect in the compound treated set to the ve- hicle (DMSO) treated set in each case. p values > 0.05 were considered not significant. Each bar in the graph is marked with a single star (p ≤ 0.05), two stars (p ≤ 0.01), three stars (p ≤ 0.001) or four stars (p ≤ 0.0001).
3. Results
3.1. A small molecule screen to identify molecules that mitigate disease-like phenotypes in the zebrafish model
In our previous studies, we demonstrated that Chd7 has conserved roles in vertebrate development (Asad et al., 2016). Our study showed that perturbation of chd7 expression in zebrafish, using antisense morpholino oligonucleotides, elicits phenotypes reminiscent of the CHARGE syndrome in humans. Many aspects of CHARGE syndrome can be traced back to the multipotent and migratory cell population known as neural crest (Pauli et al., 2017). We observed defects in multiple neural crest derived cell types such as craniofacial cartilage, various cranial neurons (derived from both neural crest and ectodermal pla- codes) and peripheral neurons and glia or Schwann cells in our CHARGE syndrome model. We also discovered that modulation
of sox10 levels at early stages of development, when it is expressed in
the neural crest progenitors (Dutton et al., 2001), could ameliorate some phenotypes in the CHARGE syndrome model e.g. the craniofacial cartilage and myelination defects (Asad et al., 2016). In this study, we attempted to identify small molecules that could rescue chd7 morphant zebrafish embryos from these phenotypes.
Since Chd7 is a chromatin remodeler with known functions in 1. chd7 morphant zebrafish recapitulates features of CHARGE syndrome. (a–b) At 4dpf, control embryos show a well-formed jaw, a normal heart, and a normal eye, (black arrowheads, a), while chd7 morphants show reduced jaw, smaller eye and pericardial edema, (black arrowheads, b). RNA in situ hybridization in 24hpf embryos for
sox9a marks craniofacial cartilage precursors in pharyngeal arches of the control group (black dots, c) but does not detect signal in the chd7 morphants (d). (e–f) RNA in situ hybridization for sox9b marks the perichondrium bands in control embryos (black dots, e) while reduced number of bands in chd7 morphants (black dots, f). (g–h) At 3 dpf, RNA in situ hybridization for islet 2a marks the differentiated motor and sensory neurons in cranial region in
control embryos (black arrowheads, g) while the pattern of expression is disrupted in chd7 morphant (black arrowheads, h). (i–j) At 4dpf, Tg(NBT:dsRed) marks enteric neurons shown as red puncta on the gut tube in controls (white arrowheads, i). chd7 morphants have severe reduction in enteric neurons (white arrowheads, j). (k–l) At 4dpf, RNA in situ hybridization for myelin basic protein (mbp) marks myelinated glia in control embryos (black arrow- heads, k); this signal is reduced in chd7 morphants (l). The posterior-most extent of expression of mye- linating Schwann cells on the lateral line are shown
by the black arrowheads in both cases. All animals have anterior to the left. Lateral view (a-b,e-f, i-l), dorsal view (c-d, g-h). All scale bars are 100 μm.transcriptional gene regulation, we reasoned that small molecules, which modulate epigenetic players in the cell, could potentially modify the chd7 morphant phenotypes. In addition, we also selected mod- ulators of signaling pathways with known role in processes like mye- lination and neurogenesis. We assembled a library of 35 compounds and performed a chemical modifier screen for molecules that amelio- rate selected phenotypes in the zebrafish disease model. We treated embryos at early stages with 5 μM concentration of selected compounds and found that there was no significant difference in the survival of embryos treated with either DMSO or the drugs (DAPT, Procainamide, CHIC-35 or HDACi3) (Supplementary . S1). Thus, all further ex- periments were performed with 5 μM concentration of small molecules, except where mentioned otherwise. No gross phenotypic abnormality was observed in compound treated embryos at this concentration.
Zebrafish embryos were microinjected with 2.4 ng of chd7 MO and allowed to develop to different stages of development as will be de- scribed in the next sections. The embryos were then exposed to 5 μM of the compounds. Larvae were fiXed at 4dpf for visualization of cranio- facial cartilage by alcian blue staining, at 24hpf for cartilage markers, at 72hpf for neuronal marker and at 96hpf for Schwann cell marker staining respectively ( 2). Control embryos were microinjected with
2.4 ng scrambled MO and then exposed to DMSO.
3.2. Compounds that modify craniofacial cartilage defects in the zebrafish CHARGE model
To screen for compounds that rescue craniofacial cartilage defects, chd7 morphant embryos were treated with various compounds from 8hpf to 24hpf. The compounds were removed at 24hpf and embryos were incubated in fresh egg water until 4dpf. We performed alcian blue staining, which marks matriX and cartilage elements and found that the chd7 morphant larvae had a severely reduced and disrupted pattern of cartilage elements in comparison to the control, with 72% of the morphants having no Alcian blue staining ( 3a–b). However, larvae treated with 4 compounds, DAPT, Procainamide, CHIC-35 and M344

showed partial recovery in craniofacial cartilage elements (. 3c–f). We quantified the percentage of embryos with complete absence of Alcian blue staining in morphant embryos treated with DMSO and the test compounds. There was a significant reduction in embryos lacking cartilage staining when treated with each of the four compounds (Supplementary S2). The cartilage structures, however, did not translate to recovery of the gross jaw structure in most embryos
SoX9 proteins are transcription factors with established roles in craniofacial cartilage specification (Yan et al., 2005). The expression of sox9a, which marks pharyngeal arches 1 and 2 in 24hpf zebrafish embryos, showed clear recovery in morphant embryos treated with DAPT, Procainamide and M344; changes in CHIC-35 were not sig- nificant ( 3m–r and Supplementary S2). Interestingly, we did not see a rescue of sox9b expression, which marks the perichondrium, with any of the compounds ( 3s–X).
3.3. Compounds that rescue zebrafish embryos from cranial ganglia defects
Cranial neuronal defects are prevalent in 90% cases of CHARGE syndrome patients (Byerly and Pauli, 1993). Defects in several cranial nerves have been observed in CHARGE syndrome. We have shown previously (Asad et al., 2016) that knockdown of chd7 in zebrafish leads to severe effects in the expression pattern of isl2a, a transcription factor expressed mainly in the differentiated cells of the motor and sensory neurons of the trigeminal ganglia in the cranial region (Tokumoto et al., 1995). Therefore, we aimed to look for modifiers of isl2a expression. We treated chd7 morphant embryos with different compounds from 24hpf to 72hpf, and performed RNA in situ hybridization with the isl2a probe. Nearly 30% of chd7 morphant embryos showed a near complete loss of isl2a expression in the cranial region compared to 10% of the wildtype controls. We found that treatment with two compounds, procainamide and CHIC-35 could reduce this to 11.5% and 7.5% respectively . These changes were significant. However, none of the tested compounds show any discernible effect on

2. Schematic of the chemical screen. Embryos were collected im- mediately after fertilization, washed and incubated at 28 °C in egg water. For cartilage lineage, embryos were incubated with chemicals from 8hpf to 24hpf. The compounds were washed off and the embryos were incubated till 4dpf for alcian blue staining and imaging. For neuronal lineage, embryos were in-
cubated with chemicals from 24hpf to 72hpf, washed and fiXed for RNA in situ hybridization for isl2a or incubated in PTU for fluorescent imaging. For mye- lination, embryos were incubated with chemicals from 48 hpf to 96hpf washed and fiXed for RNA in situ hybridization for mbp and foxd3. (For interpretation of the references to colour in this legend, the reader is referred to the Web version of this article.)

the enteric neurons marked by Tg(NBT:dsred) ( 4e–h). These results highlight the differential requirement for Chd7 in development of dif- ferent neurons.

3.4. Compounds that ameliorate myelination defects in the CHARGE syndrome model
In our previous study we showed myelination defects in the CHARGE syndrome model for the first time (Asad et al., 2016). Schwann cells are the glial cells of peripheral nervous system that function to support and protect neurons (Bhatheja and Field, 2006; Topilko et al., 1994). Schwann cells are of two types, myelinating and non-myelinating. Myelinating Schwann cells wrap around neurons to form myelin sheath that helps in the faster conduction of nerve im- pulses. The glia that ensheath axons help in their long-term survival (Nave, 2010). Schwann cells have also been implicated in neuronal regeneration (Bhatheja and Field, 2006). Myelination occurs late, after the formation of large axons stimulated by interactions of glia with axons (Nave and Werner, 2014). Therefore, we started the chemical treatment of embryos at 2 dpf and observed the effect on myelination at 4dpf. At 4dpf, embryos were fiXed and the expression of myelin basic

protein mRNA (mbp) was detected by RNA in situ hybridization. 48% of chd7 morphants have a near complete loss of mbp expression along the lateral line and the optic cup ( 5a–b)). We discovered 3 compounds that significantly reduced this percentage of embryos. DAPT did not show a significant effect on the mbp expression ( 5a–f, and Supplementary S4).
Previously, we found that the peripheral glial population marked by foxd3 was increased in chd7 morphants compared to controls and that in chd7 and sox10 double morphants, there was a concomitant reduc- tion in the foxd3 levels with rescue in myelination (Asad et al., 2016). Therefore, we reasoned that the small molecule modifiers of the mye- lination phenotype might also lead to a reduction in expression of foxd3. 65% of chd7 morphant embryos had elevated levels of foxd3 expression. This percentage was completely reverted to wildtype levels in M344 treated embryos. DAPT and CHIC-35 showed significant but milder effects on foxd3 expression. Procainamide did not show reduc- tion in foxd3 expression in significant number of embryos (5g–l, and Supplementary S4).
4. Discussion
Embryonic development requires the expression of a number of gene products at the right time, in the right cells at the right levels. Birth defects are the result of breakdown of one or more of these ele- ments. Thus, discovering small molecules that rescue animals from developmental disorders are challenging because they have to recover the time, pattern and expression levels of target genes in multiple cells and cell types. Zebrafish is one of the best animal systems to assay the complex developmental patterning events in whole organism against small molecule diversity. Being an externally fertilized animal, zebra- fish allows easy access to all stages of embryonic development. With this in mind we developed a model for CHARGE syndrome in zebrafish. CHD7, the gene implicated in more than 60% cases of CHARGE
syndrome, is an ATPase dependent chromatin remodeler with essential role in gene regulation throughout life. Knockdown of chd7in zebrafish led to developmental defects in the embryo, affecting multiple tissues derived completely or partially from neural crest, such as craniofacial cartilage, cranial neurons, enteric and peripheral neurons and myeli- nating glia. We have previously demonstrated that downregulating the expression of sox10 in the background of Chd7 depletion can partially rescue zebrafish embryos from craniofacial defects as well as peripheral neuron myelination defects (Asad et al., 2016). This suggested that it might be possible to chemically compensate for the loss of Chd7 in the CHARGE syndrome model. Since Chd7 is an epigenetic modulator, known to be present at enhancer regions and is capable of opening the chromatin for transcriptional activation, we selected small molecules that modulate epigenetic enzymes for our chemical screen. We also
selected small molecule inhibitors of signaling pathways known to be important for neuronal, glial and cartilage development for screening. Our chemical screen led to the discovery of 4 compounds that could ameliorate multiple phenotypes in the zebrafish CHARGE model: (1) DAPT, a Notch signaling inhibitor, (2) M344, a histone deacetylase inhibitor, (3) CHIC-35, a SIRT1 inhibitor and (4) Procainamide, a DNA methyltransferase inhibitor.
Notch signaling is central to the development of many types of glial cells in the Central Nervous System (CNS) like astrocytes, Müller cells, oligodendrocytes, and its dysfunction has been implicated in a number
of CNS diseases (Woodhoo et al., 2009). DAPT is a small molecule that inhibits NOTCH by inhibiting ψ-secretase activity, thereby preventing the cleavage of intracellular domain and thus, disrupting activation of
downstream target genes (Yang et al., 2010). Our screen identified DAPT as a molecule that could rescue chd7 morphant embryos from defects in craniofacial cartilage and to a lesser extent in myelinating Schwann cells. Recent studies have shown that inhibition of Notch signaling in mouse condylar cartilage culture cells causes induction of Sox9 expression (Serrano et al., 2014). Our studies show a clear

3. chd7 morphant zebrafish embryos exhibit partial recovery of jaw elements on treatment with selected compounds. (a–f) Alcian blue staining at 4dpf marks well patterned cartilage ele- ments in control embryos (black arrowhead, a), while chd7 morphants show absence of cartilage elements (b). Treatment with DAPT (c), Procainamide (d), CHIC-35 (e) and M344 (f) show partial recovery of cartilage elements (black arrow- heads, c, d, f). (g–l) At 4dpf, zebrafish embryos show a well-formed lower jaw in controls (black dots, g), while chd7 morphants exhibit reduced lower jaw (h). Treatment with compounds did not show sig- nificant recovery of jaw in brightfield (black dots, i- l). (m–r) At 24hpf, sox9a marks craniofacial carti- lage precursors in control embryos (white dots, m)
and this expression was decreased in chd7 mor- phants (n). Treatment with DAPT Procainamide CHIC-35 and M344 rescued the expression of sox9a in chd7 morphants (shown by white dots o-r). (s–X) sox9b marks perichondrium in control embryos (black dots, s) at 24hpf, while chd7 morphants show reduced expression (t). Treatment with the com-
pounds did not show recovery of sox9b expression (black dots, u-X). (a–X) All embryos have anterior to the left. Ventral views (a–f) lateral view (g–l) (s–X), dorsal view (m–r). All scale bars are 100 μm. The numbers on bottom left corner indicate the ratio of embryos with the phenotype as represented in the
recovery of sox9a expression in chd7 morphants treated with DAPT suggesting the rescue of cartilage elements in the jaw may be a direct consequence of the effect of Notch signaling on chrondrocyte progeni- tors. DAPT treatment also caused reversal of the effects of chd7 mor- phants on the peripheral glial progenitors marked by foxd3. Notch signaling has complex regulatory roles in Schwann cell development and was shown to promote the formation of Schwann cells from pre- cursor cells (Woodhoo et al., 2009). However, it was also shown that Notch signaling inhibited myelination in perinatal nerves in mice (Woodhoo et al., 2009). This is in accordance with our observations that inhibition of Notch signaling induces increase in myelination in the chd7 morphant zebrafish embryos.
Histone deacetylases are enzymes that play an important role in epigenetic gene regulation by modulating the accessibility of DNA

(Choudhary et al., 2009). In our screen, we identified M344, a histone deacetylase inhibitor that could partially revert the defects in cranio- facial cartilage in chd7 morphant zebrafish, suggesting that inhibiting HDACs, and thus perhaps nonspecifically elevating the acetylation le- vels of chromatin, could aid the development of craniofacial cartilage. We have previously observed a similar effect in a zebrafish morphant for the histone acetyl transferase EP300 (Babu et al., 2018). Our present study also discovered a strong role for HDAC activity in myelination. We found a dramatic recovery of myelin basic protein expression in chd7 morphant embryos, which was accompanied by a near complete reversal of foxd3 upregulation in the glial precursors.
Histone deacetylases are classified into a number of different classes based on their enzymatic activities. Class 3 HDACs are different from other HDACs in that they require NAD + as a cofactor, while other

4. Treatment with procainamide and CHIC- 35 induces recovery of cranial neurons in chd7 morphants. (a–d) At 72hpf, RNA in situ hybridiza- tion of isl2a marks a well-patterned arrangement of sensory and motor neurons in cranial region in controls (marked by white dots, a). chd7 morphants had decreased and loss of patterning of isl2a ex- pression (white dots, b). (c–d) Treatment with pro- cainamide and CHIC-35 rescued isl2a expression pattern in chd7 morphants (white dots, c and d re- spectively). (e–h) At 4 dpf, Tg(NBT:dsred) marks enteric neurons shown as distinct dots (white ar-
rowheads, e) in control embryos while chd7 mor- phants had severely reduced enteric neurons (f). Treatment with Procainamide or CHIC-35 did not induce recovery of neurons (g,h) All embryos have anterior to the left. Dorsal view (a–d) lateral view (e–h). All scale bars are 100 μm. The numbers on bottom left corner indicate the ratio of embryos with the phenotype as represented
5. Rescue of myelination defects in chd7 morphant zebrafish embryos treated with com- pounds. (a–f) RNA in situ hybridization of mbp at 4dpf marks myelinated glia along the lateral line in controls (a), shown by black arrowheads up to the posterior extent of expression. The extent and levels of expression was reduced in chd7 morphants (b). Treatment with DAPT (c), Procainamide (d), CHIC- 35 (e) and M344 (f) rescued the extent and level of
expression of mbp in chd7 morphants to different degrees (black arrowheads, a–f). (g–l) Low levels of foxd3 expression marks glia in 4dpf control embryos (black arrowheads, g), while chd7 morphants show elevated expression of foxd3 in trunk region (black arrowheads, h). Treatment with DAPT (i), CHIC-35
(k) and M344 (l) rescued foxd3 overexpression compared to chd7 morphants treated with DMSO. Procainamide did not affect foxd3 expression. All embryos have anterior to the left, lateral views. All scale bars are 100 μm. The numbers on bottom left corner indicate the ratio of embryos with the phe- notype as represented in the .HDACs require zinc as a cofactor (Barneda-Zahonero and Parra, 2012). Class 3 HDACS are also known as Sirtuins. SIRT proteins have been implicated in a number of processes such as embryonic development, cell survival, longevity, apoptosis, senescence, proliferation, DNA re- pair and cell metabolism. There are 7 types of SIRTs and CHIC-35 is a small molecule inhibitor of SIRT1 (Lugrin et al., 2013). CHIC-35 oc- cupies the selectivity pocket to induce conformational changes at the hinge region of SIRT1 (Rumpf et al., 2015) and inhibits its activity. Our study shows that CHIC-35 could partially rescue defects in craniofacial cartilage, cranial neurons and Schwann cells. Mice with a mutation in SIRT1 have been shown to have defective cartilage (Gabay et al., 2012). A number of other studies have revealed a positive role for SIRT1 in
chondrogenesis and Sox9 activation (Bar Oz et al., 2016; Dvir-Ginzberg et al., 2008). Thus, the effect of CHIC-35 on sox9a expression was not significant in our studies, but paradoXically there was significant re- covery of jaw cartilage elements in CHIC-35 treated embryos. This suggests that CHIC-35 might influence the expression of other players involved in craniofacial cartilage development. Our study demonstrates that CHIC-35 induces the expression of mbp gene in chd7 morphants and significantly rescues embryos from myelination defects. Loss of Sirt1 has previously been shown to induce faster remyelination post injury in mouse sciatic nerves (Zuccaro and Arlotta, 2013), supporting our observations. CHIC-35 treatment was also able to partially ame- liorate the defects in cranial neurons in chd7 morphants. Sirt proteins have been studied extensively in neurodegenerative disorders. SIRT1 protein has been shown to regulate neurogenic potential of neural precursors in hippocampus and subventricular zone (Hisahara et al., 2008; Saharan et al., 2013), and suppress self-renewal of adult neural stem cells in hippocampus (Ma et al., 2014). Thus our observation, that CHIC-35 is able to partially rescue defects in craniofacial cartilage, myelinating glia and cranial neurons in CHARGE model, is in agree- ment with previous known roles of SIRT1, the most likely target of CHIC-35 in our system.
Our chemical screen also identified Procainamide, a specific in- hibitor of DNA methyltransferase 1, as a compound that could rescue the cranial neuron defects in chd7 morphant embryos. Procainamide, although more commonly known as a Na+ channel blocker and used in the treatment of arrhythmia (Shih et al., 2016), is known to induce the expression of neuronal markers and promote neurite development in human nasal olfactory stem and neural progenitor cells (NOS/PCs)

(Franco et al., 2017). Since our assay only involved the cranial neurons and enteric neurons, while CHARGE syndrome patients suffer from multiple neurological problems, it would be important to probe the effect of Procainamide overall on the nervous system in the CHARGE model.
5. Conclusion
CHARGE syndrome is primarily caused by dominant mutations in the chromatin remodeler, CHD7 that disrupt gene expression control and result in defective embryonic development. We used zebrafish embryos to screen for compounds that can ameliorate the defects in a zebrafish model for CHARGE syndrome that we had reported earlier (Asad et al., 2016). Our discovery of four compounds, one signaling inhibitor and three epigenetic modulators, suggests that the loss of Chd7 activity in development is at least partially compensated by these compounds, thus opening up the possibility of small molecule ther- apeutics for multi-organ syndromes such as CHARGE. However, our failure to discover a single compound that ameliorates or reverses all the phenotypes also suggests that the small molecules we have identi- fied are not chemically compensating for the Chd7 chromatin re- modeling activity, but rather affecting various individual processes of specification and differentiation in different tissue types. This also suggests that cocktails of small molecules, rather than single molecules, might be the way forward to such complex syndromes as CHARGE.
Conflicts of interest
The authors declare no conflict of interest.
Funding
This work was supported by Council of Scientific and Industrial Research (CSIR), New Delhi [BSC0118 and MLP1801 grants to C. S., Research Fellowship to Z. A].
Acknowledgements
We thank Shashi Ranjan for maintaining fish facility. We also thank Aditi Pandey for her inputs and Sarah Iqbal for her edits and comments.

Appendix A. Supplementary data
Supplementary data to this article can be found online
References

Asad, Z., Pandey, A., Babu, A., Sun, Y., Shevade, K., Kapoor, S., Ullah, I., Ranjan, S.,
Scaria, V., Bajpai, R., Sachidanandan, C., 2016. Rescue of neural crest-derived phe- notypes in a zebrafish CHARGE model by SoX10 downregulation. Hum. Mol. Genet.
25, 3539–3554.
Babu, A., Kamaraj, M., Basu, M., Mukherjee, D., Kapoor, S., Ranjan, S., Swamy, M.M.,
Kaypee, S., Scaria, V., Kundu, T.K., Sachidanandan, C., 2018. Chemical and genetic rescue of an ep300 knockdown model for Rubinstein Taybi Syndrome in zebrafish.
Biochim. Biophys. Acta (BBA) – Mol. Basis Dis. 1864, 1203–1215.
Bajpai, R., Chen, D.A., Rada-Iglesias, A., Zhang, J., Xiong, Y., Helms, J., Chang, C.-P., Zhao, Y., Swigut, T., Wysocka, J., 2010. CHD7 cooperates with PBAF to control
multipotent neural crest formation. Nature 463, 958–962.
Balasubramanian, R., Choi, J.H., Francescatto, L., Willer, J., Horton, E.R., Asimacopoulos,
E.P., Stankovic, K.M., Plummer, L., Buck, C.L., Quinton, R., Nebesio, T.D., Mericq, V., Merino, P.M., Meyer, B.F., Monies, D., Gusella, J.F., Al Tassan, N., Katsanis, N.,
Crowley Jr., W.F., 2014. Functionally compromised CHD7 alleles in patients with isolated GnRH deficiency. Proc. Natl. Acad. Sci. U. S. A. 16 (50), 17953–17958 111.
Balow, S.A., Pierce, L.X., Zentner, G.E., Conrad, P.A., Davis, S., Sabaawy, H.E.,
McDermott Jr., B.M., Scacheri, P.C., 2013. Knockdown of fbxl10/kdm2bb rescues chd7 morphant phenotype in a zebrafish model of CHARGE syndrome. Dev. Biol.
382, 57–69.
Barneda-Zahonero, B., Parra, M., 2012. Histone deacetylases and cancer. Mol. Oncol. 6, 579–589.
Bar Oz, M., Kumar, A., Elayyan, J., Reich, E., Binyamin, M., Kandel, L., Liebergall, M.,
Steinmeyer, J., Lefebvre, V., Dvir-Ginzberg, M., 2016. Acetylation reduces SOX9 nuclear entry and ACAN gene transactivation in human chondrocytes. Aging Cell 15,
499–508.
Basu, S., Jalodia, K., Ranjan, S., Yeh, J.J., Peterson, R.T., Sachidanandan, C., 2018. Small molecule inhibitors of NFkB reverse iron overload and hepcidin deregulation in a
zebrafish model for hereditary hemochromatosis type 3. ACS Chem. Biol. 13 (8), 2143–2152.
Becker, P.B., Hörz, W., 2002. ATP-dependent nucleosome remodeling. Annu. Rev.
Biochem. 71, 247–273.
Bhatheja, K., Field, J., 2006. Schwann cells: origins and role in axonal maintenance and regeneration. Int. J. Biochem. Cell Biol. 38, 1995–1999.
Bosman, E.A., Penn, A.C., Ambrose, J.C., Kettleborough, R., Stemple, D.L., Steel, K.P.,
2005. Multiple mutations in mouse Chd7 provide models for CHARGE syndrome. Hum. Mol. Genet. 14, 3463–3476.
Bouazoune, K., Kingston, R.E., 2012. Chromatin remodeling by the CHD7 protein is im- paired by mutations that cause human developmental disorders. Proc. Natl. Acad. Sci.
U.S.A. 109, 19238–19243.
Byerly, K.A., Pauli, R.M., 1993. Cranial nerve abnormalities in CHARGE association. Am.
J. Med. Genet. 45, 751–757.
Choudhary, C., Kumar, C., Gnad, F., Nielsen, M.L., Rehman, M., Walther, T.C., Olsen, J.V., Mann, M., 2009. Lysine acetylation targets protein complexes and Co-regulates major cellular functions. Science 325, 834–840.
Cloney, K., Steele, S.L., Stoyek, M.R., Croll, R.P., Smith, F.M., Prykhozhij, S.V., Brown,
M.M., Midgen, C., Blake, K., Berman, J.N., 2018. Etiology and functional validation of gastrointestinal motility dysfunction in a zebrafish model of CHARGE syndrome.
FEBS J. 285 (11), 2125–2140.
Dingerkus, G., Uhler, L.D., 1977. Enzyme clearing of alcian blue stained whole small vertebrates for demonstration of cartilage. Stain Technol. 52, 229–232.
Dürr, H., Flaus, A., Owen-Hughes, T., Hopfner, K.-P., 2006. Snf2 family ATPases and DEXX
boX helicases: differences and unifying concepts from high-resolution crystal struc- tures. Nucleic Acids Res. 34, 4160–4167.
Dutton, K.A., Pauliny, A., Lopes, S.S., Elworthy, S., Carney, T.J., Rauch, J., et al., 2001.
Zebrafish colourless encodes soX10 and specifies non-ectomesenchymal neural crest fates. Development 128 (21), 4113–4125.
Dvir-Ginzberg, M., Gagarina, V., Lee, E.-J., Hall, D.J., 2008. Regulation of cartilage- specific gene expression in human chondrocytes by SirT1 and nicotinamide phos-
phoribosyltransferase. J. Biol. Chem. 283, 36300–36310.
Feng, W., Kawauchi, D., Körkel-Qu, H., Deng, H., Serger, E., Sieber, L., Lieberman, J.A., Jimeno-González, S., Lambo, S., Hanna, B.S., Harim, Y., Jansen, M., Neuerburg, A.,
Friesen, O., Zuckermann, M., Rajendran, V., Gronych, J., Ayrault, O., Korshunov, A., Jones, D.T.W., Kool, M., Northcott, P.A., Lichter, P., Cortés-Ledesma, F., Pfister, S.M.,
Liu, H.-K., 2017. Chd7 is indispensable for mammalian brain development through activation of a neuronal differentiation programme. Nat. Commun. 8, 14758.
Feng, W., Liu, H.-K., 2013. Epigenetic regulation of neuronal fate determination: the role of CHD7. Cell Cycle 12, 3707–3708.
Flanagan, J.F., Blus, B.J., Kim, D., Clines, K.L., Rastinejad, F., Khorasanizadeh, S., 2007. Molecular implications of evolutionary differences in CHD double chromodomains. J.
Mol. Biol. 369, 334–342.
Flanagan, J.F., Mi, L.-Z., Chruszcz, M., Cymborowski, M., Clines, K.L., Kim, Y., Minor, W., Rastinejad, F., Khorasanizadeh, S., 2005. Double chromodomains cooperate to re-
cognize the methylated histone H3 tail. Nature 438, 1181–1185.
Franco, I., Ortiz-López, L., Roque-Ramírez, B., Ramírez-Rodríguez, G.B., Lamas, M., 2017.
Pharmacological inhibition of DNA methyltransferase 1 promotes neuronal differ- entiation from rodent and human nasal olfactory stem/progenitor cell cultures. Int. J.
Dev. Neurosci. 58, 65–73.
Gabay, O., Sanchez, C., Dvir-Ginzberg, M., Gagarina, V., Zaal, K.J., Song, Y., He, X.H., McBurney, M.W., 2012. Sirtuin 1 enzymatic activity is required for cartilage home- ostasis in vivo in a mouse model. Arthritis Rheum. 65, 159–166.

Gage, P.J., Hurd, E.A., Martin, D.M., 2015. Mouse models for the dissection of CHD7 functions in eye development and the molecular basis for ocular defects in CHARGE
syndrome. Investig. Ophthalmol. Vis. Sci. 56, 7923–7930.
Hisahara, S., Chiba, S., Matsumoto, H., Tanno, M., Yagi, H., Shimohama, S., Sato, M.,
Horio, Y., 2008. Histone deacetylase SIRT1 modulates neuronal differentiation by its nuclear translocation. Proc. Natl. Acad. Sci. U.S.A. 105, 15599–15604.
Ho, L., Crabtree, G.R., 2010. Chromatin remodelling during development. Nature 463, 474–484.
Hurd, E.A., Capers, P.L., Blauwkamp, M.N., Adams, M.E., Raphael, Y., Poucher, H.K.,
Martin, D.M., 2007. Loss of Chd7 function in gene-trapped reporter mice is em- bryonic lethal and associated with severe defects in multiple devel- oping tissues.
Mamm. Genome 18, 94–104.
Jacobs-McDaniels, N.L., Albertson, R.C., 2011. Chd7 plays a critical role in controlling
left-right symmetry during zebrafish somitogenesis. Dev. Dynam. 240 (10), 2272–2280.
Kimmel, C.B., Ballard, W.W., Kimmel, S.R., Ullmann, B., Schilling, T.F., 1995. Stages of embryonic development of the zebrafish. Dev. Dynam. 203, 253–310.
Lam, P.Y., Peterson, R.T., 2019. Developing zebrafish disease models for in vivo small molecule screens. Curr. Opin. Chem. Biol. 28 (50), 37–44.
Liu, Z.Z., Wang, Z.L., Choi, T.I., Huang, W.T., Wang, H.T., Han, Y.Y., Zhu, L.Y., Kim, H.T.,
Choi, J.H., Lee, J.S., Kim, H.G., Zhao, J., Chen, Y., Lu, Z., Tian, X.L., Pan, B.X., Li,
B.M., Kim, C.H., Xu, H.A., 2018. Chd7 is critical for early T-cell development and thymus organogenesis in zebrafish. Am. J. Pathol. 188 (4), 1043–1058.
Lugrin, J., Ciarlo, E., Santos, A., Grandmaison, G., dos Santos, I., Le Roy, D., Roger, T.,
2013. The sirtuin inhibitor cambinol impairs MAPK signaling, inhibits inflammatory and innate immune responses and protects from septic shock. Biochim. Biophys. Acta
1833, 1498–1510.
Ma, C.-Y., Yao, M.-J., Zhai, Q.-W., Jiao, J.-W., Yuan, X.-B., Poo, M.-M., 2014. SIRT1
suppresses self-renewal of adult hippocampal neural stem cells. Development 141, 4697–4709.
Melicharek, D.J., Ramirez, L.C., Singh, S., Thompson, R., Marenda, D.R., 2010. Kismet/
CHD7 regulates axon morphology, memory and locomotion in a Drosophila model of CHARGE syndrome. Hum. Mol. Genet. 19, 4253–4264.
Micucci, J.A., Layman, W.S., Hurd, E.A., Sperry, E.D., Frank, S.F., Durham, M.A., Swiderski, D.L., Skidmore, J.M., Scacheri, P.C., Raphael, Y., Martin, D.M., 2014.
CHD7 and retinoic acid signaling cooperate to regulate neural stem cell and inner ear development in mouse models of CHARGE syndrome. Hum. Mol. Genet. 23, 434–448.
Nave, K.-A., 2010. Myelination and the trophic support of long axons. Nat. Rev. Neurosci.
11, 275–283.
Nave, K.-A., Werner, H.B., 2014. Myelination of the nervous system: mechanisms and functions. Annu. Rev. Cell Dev. Biol. 30, 503–533.
Pagon, R.A., Graham, J.M., Zonana, J., Yong, S.-L., 1981. Coloboma, congenital heart
disease, and choanal atresia with multiple anomalies: CHARGE association. J. Pediatr. 99, 223–227.
Patten, S.A., Jacobs-McDaniels, N.L., Zaouter, C., Drapeau, P., Albertson, R.C., Moldovan, F., 2012. Role of Chd7 in zebrafish: a model for CHARGE syndrome. PLoS One 7 (2),
e31650.
Pauli, S., Bajpai, R., Borchers, A., 2017. CHARGEd with neural crest defects. Am. J. Med.
Genet. C Semin. Med. Genet. 175, 478–486.
Peri, F., Nüsslein-Volhard, C., 2008. Live imaging of neuronal degradation by microglia reveals a role for v0-ATPase a1 in phagosomal fusion in vivo. Cell 133, 916–927.
Rumpf, T., Gerhardt, S., Einsle, O., Jung, M., 2015. Seeding for sirtuins: microseed matriX seeding to obtain crystals of human Sirt3 and Sirt2 suitable for soaking. Acta
Crystallogr. Sect. F Struct. Biol. Cryst. Commun. 71, 1498–1510.
Saharan, S., Jhaveri, D.J., Bartlett, P.F., 2013. SIRT1 regulates the neurogenic potential of neural precursors in the adult subventricular zone and hippocampus. J. Neurosci. Res. 91, 642–659.
Schnetz, M.P., Bartels, C.F., Shastri, K., Balasubramanian, D., Zentner, G.E., Balaji, R.,
Zhang, X., Song, L., Wang, Z., Laframboise, T., Crawford, G.E., Scacheri, P.C., 2009. Genomic distribution of CHD7 on chromatin tracks H3K4 methylation patterns.
Genome Res. 19, 590–601.
Serrano, M.J., So, S., Hinton, R.J., 2014. Roles of notch signalling in mandibular condylar cartilage. Arch. Oral Biol. 59, 735–740.
Shih, C.-C., Hii, H.-P., Tsao, C.-M., Chen, S.-J., Ka, S.-M., Liao, M.-H., Wu, C.-C., 2016.
Therapeutic effects of procainamide on endotoXin-induced Rhabdomyolysis in Rats. PLoS One 11, e0150319.
Tokumoto, M., Gong, Z., Tsubokawa, T., Hew, C.L., Uyemura, K., Hotta, Y., Okamoto, H.,
1995. Molecular heterogeneity among primary motoneurons and within myotomes revealed by the differential mRNA expression of novel islet-1 homologs in embryonic
zebrafish. Dev. Biol. 171 (2), 578–589.
Topilko, P., Schneider-Maunoury, S., Levi, G., Baron-Van Evercooren, A., Chennoufi, A.B.,
Seitanidou, T., Babinet, C., Charnay, P., 1994. KroX-20 controls myelination in the peripheral nervous system. Nature 371, 796–799.
Woodhoo, A., Duran Alonso, M.B., Droggiti, A., Turmaine, M., D’Antonio, M., Parkinson,
D.B., Wilton, D.K., Al-Shawi, R., Simons, P., Shen, J., Guillemot, F., Radtke, F., Meijer, D., Laura Feltri, M., Wrabetz, L., Mirsky, R., Jessen, K.R., 2009. Notch con-
trols embryonic Schwann cell differentiation, postnatal myelination and adult plas- ticity. Nat. Neurosci. 12, 839–847.
Yang, T., Arslanova, D., Xu, X., Li, Y.-M., Xia, W., 2010. In vivo manifestation of Notch related phenotypes in zebrafish treated with Alzheimer’s amyloid reducing gamma-
secretase inhibitors. J. Neurochem. 113, 1200–1209.
Yan, Y.-L., Willoughby, J., Liu, D., Crump, J.G., Wilson, C., Miller, C.T., Singer, A., Kimmel, C., Westerfield, M., Postlethwait, J.H., 2005. A pair of SoX: distinct and
overlapping functions of zebrafish soX9 co-orthologs in M344 craniofacial and pectoral fin development. Development 132, 1069–1083.
Zentner, G.E., Layman, W.S., Martin, D.M., Scacheri, P.C., 2010. Molecular and pheno- typic aspects of CHD7 mutation in CHARGE syndrome. Am. J. Med. Genet. 152A,
674–686.
Zuccaro, E., Arlotta, P., 2013. The quest for myelin in the adult brain. Nat. Cell Biol. 15, 572–575.