Mutations in dock1 disrupt early Schwann cell development
In the peripheral nervous system (PNS), specialized glial cells called Schwann cells produce myelin, a lipid-rich insulating sheath that surrounds axons and promotes rapid action potential propagation. During development, Schwann cells must undergo extensive cytoskeletal rearrangements in order to become mature, myelinating Schwann cells. The intracellular mechanisms that drive Schwann cell development, myelination, and accompanying cell shape changes are poorly understood.
Through a forward genetic screen in zebrafish, we identified a mutation in the atypical guanine nucleotide exchange factor, dock1, that results in decreased myelination of peripheral axons. Rescue experiments and complementation tests with newly engineered alleles confirmed that mutations in dock1 cause defects in myelination of the PNS. Whole mount in situ hybridization, transmission electron microscopy, and live imaging were used to fully define mutant phenotypes.
We show that Schwann cells in dock1 mutants can appropriately migrate and are not decreased in number, but exhibit delayed radial sorting and decreased myelination during early stages of development.
Together, our results demonstrate that mutations in dock1 result in defects in Schwann cell development and myelination. Specifically, loss of dock1 delays radial sorting and myelination of peripheral axons in zebrafish.
Keywordsdock1 Schwann cell development Myelination Zebrafish
Central nervous system
Days post fertilization
Guanine nucleotide exchange factor
Hours post fertilization
Myelin basic protein
- MF 20
Myosin heavy chain antibody
Phosphate buffered saline
Polymerase chain reaction
Posterior lateral line nerve
Peripheral nervous system
Schwann cell precursor
Transmission electron microscopy
Whole mount in situ hybridization
Myelin, a lipid-rich multi-membrane structure, is an innovation of jawed vertebrates that enables the efficient conduction of action potentials. Schwann cells are the myelinating glia of the peripheral nervous system (PNS), and one Schwann cell myelinates one axonal segment. Schwann cells are derived from the neural crest and undergo a distinct series of developmental stages [1, 2]. These developmental stages of Schwann cells require migration as well as unique and substantial changes in cell shape. Schwann cell precursors (SCPs) migrate great distances longitudinally down peripheral nerves. SCPs develop into immature Schwann cells, which undergo a unique process called radial sorting in which Schwann cells extend processes into axon bundles and select an axon to myelinate . Prior to myelination, Schwann cells wrap themselves 1–1.5 times around a selected axon segment in what is termed the pro-myelinating state. A mature Schwann cell extends and wraps its membrane to form a myelin sheath around an axonal segment. Cytoskeletal dynamics are needed to facilitate these different stages of Schwann cell development and extensive changes in cell shape, but the intracellular intermediates between extracellular signals and the remodeling of the Schwann cell cytoskeleton are not well defined.
The Rho-GTPase Rac1 is well known for its role in facilitating cell shape changes through regulating polymerization of the actin cytoskeleton and mediates Schwann cell development . In Schwann cells, differential levels of Rac1 direct when a Schwann cell stops migrating and begins radial sorting and myelination . Schwann cell-specific ablation of Rac1 in a mouse model causes delays in radial sorting and myelination, as well as aberrant Schwann cell process extension [5, 6, 7]. Furthermore, Rac1 can function downstream of β1-integrin in Schwann cells performing radial sorting ; however, the intracellular mechanisms that influence the temporal and spatial activation of Rac1 following extracellular signaling during Schwann cell development are not well understood.
Guanine nucleotide exchange factors (GEFs) have the ability to temporally and spatially regulate the activation of RhoGTPases, such as Rac1, because many GEFs can regulate the same RhoGTPase . Roles of specific GEFs during distinct stages of Schwann cell development are beginning to be understood and help to broaden our knowledge of how extracellular signals are translated to intracellular signals in order to facilitate alterations in Schwann cell shape and movement [9, 10, 11, 12]. In addition to canonical GEFs, atypical GEFs also have the ability to activate RhoGTPases. One such family of atypical GEFs, the Dock1-related GEFs, is composed of 11 family members, including Dock1 (also known as Dock180). Dock1 is highly evolutionarily conserved across species and can specifically bind and activate Rac1 [13, 14, 15]. In vitro and in vivo studies in various model organisms have shown that Dock1 influences a variety of cytoskeletal-related cell processes such as phagocytosis and cell migration [16, 17, 18, 19]. Thus, Dock1 represents an ideal intracellular candidate to study for a role in cell shape regulation.
Although Dock1 has been studied in several biological contexts and is expressed in Schwann cells , a role for Dock1 has not yet been described in Schwann cell myelination. The ability of Dock1 to initiate changes in cell shape to facilitate phagocytosis and cell migration makes Dock1 an attractive candidate to investigate for a role in regulated cell shape changes throughout the development of Schwann cells, particularly during stages of radial sorting and myelination, when Rac1 levels most influence Schwann cell biology . Two other members of the Dock1 family, Dock7, which activates the RhoGTPase Cdc42 , and Dock8 , which can activate Cdc42 and Rac1, have been shown to influence SCP migration through in vitro and in vivo knockdown experiments. Therefore, other members of the Dock1 family may also be key intracellular signals regulating the timing of Schwann cell development.
In this study, we utilized zebrafish to study Schwann cell myelination , and we identify and characterize Dock1 as a regulator of early Schwann cell myelination. Although previous morpholino experiments in zebrafish have implicated dock1 in myoblast development and vasculature morphogenesis [23, 24, 25], a role for Dock1 in Schwann cell development has not been examined. In a screen for genetic regulators of myelination, we identified an early stop codon in dock1 that causes decreased expression of a mature myelin marker, myelin basic protein (mbp), in the PNS. Transmission electron microscopy (TEM) revealed that fewer axons are myelinated in mutants during early stages of myelin development, while axon number is not affected. We determined that SCP cell number and migration is not affected in dock1 mutants. Instead, radial sorting is delayed and early markers of myelination are reduced. These data suggest that Dock1 may contribute to the timely process extension of Schwann cells required for radial sorting and myelination.
Methods and materials
Zebrafish lines and rearing conditions
Zebrafish were reared in accordance with the Washington University IRB and animal protocols and were raised in the Washington University Zebrafish Consortium (http://zebrafish.wustl.edu/husbandry.htm). Zebrafish were crossed as either pairs or harems, and embryos were subsequently raised at 28.5 °C in egg water (5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl2, 0.33 mM MgSO4). Larvae were staged at hours post fertilization (hpf) and days post fertilization (dpf). The following mutant and transgenic strains were utilized in this study: dock1stl145, dock1stl365, dock1stl366, Tg(sox10(4.9):nls-eos) , Tg(foxd3:gfp) , and Tg(kdlr:mcherry) . Homozygous dockstl145 fish are viable as adults, therefore maternal zygotic (MZ) dock1stl145 animals were generated by crossing a dockstl145/stl145 female with a dock1stl145/+ male.
To identify adult and larval zebrafish for either rearing or phenotypic analyses, the following primers were used to amplify a region of interest by PCR: stl145 F: 5’-CATAGGCGTTCTTCACTGAG-3′ and R: 5’-CGTATTTCCCACTAAACAGC-3′, stl365 F: 5’-GCAGCCACTTTAAAGCTTCCCG-3′ and R: 5’-GCTGCTTACCTTGCCCTTGTC-3′, and stl366 F: 5’-CCAGTGCCTCACTTCATATCTCC-3′ and R: 5’ CTCTTAGTCTCACGCAACACTCATG-3′. After PCR, a restriction enzyme digest assay was performed and the resulting fragments were analyzed on a 3% agarose gel. The stl145 C-to-T mutation disrupts a BstNI site so that the wild-type PCR product is cleaved into 48 and 527 base pair (bp) products, and the mutant PCR product is 575 bp. The stl365 allele contains a one bp insertion that disrupts an EcoRV binding site so that the wild-type PCR product is cleaved into 86 and 159 bp products, and the mutant PCR product is 245 bp. The stl366 allele contains a 13-bp deletion that disrupts a HpyCH4III site so that the wild-type PCR product is cleaved into 323 bp and 165 bp products, and the mutant PCR product is 488 bp.
Zebrafish mutant strain generation
dock1stl145 was identified in a forward genetic screen described previously in [29, 30]. Phenotypically wild-type and mutant 5 dpf larvae were pooled and extracted DNA was sent for whole genome sequencing at the Genome Technology Access Center (GTAC) at Washington University. The wild-type to mutant allele ratio was determined using a bioinformatics pipeline generated in-house, and a SNP subtraction analysis suggested that dock1 was most likely the gene of interest . dock1 was confirmed as the gene responsible for the stl145 mutant phenotype through rescue experiments and complementation tests using two other dock1 mutant alleles, dock1stl365 and dock1stl366, which were generated by TALENs. The TALEN targeter tool (https://tale-nt.cac.cornell.edu/) and GoldyTALEN kit  were utilized to build each TALEN in a pCS2+ backbone. The repeat variable domains chosen for each stl365 TALEN arm and stl366 TALEN arm were: stl365 left arm: NN HD NG HD NI HD HD NG NN NI HD NN HD NI NN NI NN NI NN NI; stl365 right arm: HD NG NG NG NN NI NN NG NG NN NI HD HD HD NG NN NI NN NG; stl366 left arm: NN NG NG NI NG NI NG NG HD NI NG HD NG NN NI NI NN NN NI NN; stl366 right arm: NN HD NG NG NI NI NI HD NI NG NI HD NG NN NI HD HD HD NN HD. The TALEN constructs were transcribed with the mMESSAGE mMACHINE SP6 ULTRA Kit (Ambion) and equal concentrations (~ 50 pg) of left and right arm mRNA were injected into 1-cell stage wild-type embryos. Lesions that were successfully transmitted to the F0 germline were identified by restriction enzyme digest analysis as described above. Mutant bands were gel extracted using a QIAquick Gel Extraction kit (Qiagen) and then Sanger sequenced to identify the lesion.
Posterior lateral line nerve (PLLn) dissection and RNA isolation
Posterior lateral line nerves (PLLn) were dissected from 6-month-old adult zebrafish. Animals were euthanized in ice water until gill motion ceased for 5 min, followed by transection of the hindbrain. Using angled forceps, the skin was pulled back from behind the operculum on both sides of the animal to expose the PLLn. Small spring-loaded dissection scissors were used to cut the PLLn near the operculum and then forceps were used to gently remove the nerve by slowly pulling the nerve toward the anterior of the fish. Both nerves were transferred to microcentrifuge tubes sitting on dry ice and then flash frozen in liquid nitrogen and stored at − 80 °C. To isolate RNA, 40 nerves were pooled from 20 different 6-month-old adult zebrafish and total RNA was obtained using standard TRIzol (Life Technologies) RNA extraction, with the exception of the homogenization method. Nerves were pooled into a total of 500 μl TRIzol and then thoroughly homogenized following these steps in succession: vortexing for 30 s, disruption with a plastic-tipped electric homogenizer for 1 min, and passaged through a syringe and successively smaller needles (22.5 and 27 gauge), 10 times each.
To make cDNA, 1 μg of total RNA was reverse transcribed using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems) using random hexamers as per manufacturer instructions. RT-PCR for dock1 was performed on adult PLLn cDNA. The following primers were used: F: 5’-CGGAGTGGCCGTCTACAACTATG-3′ (bordering exons 1 and 2) and R: 5’-CAAGCCGGAAACACACCCTTC-3′ (bordering exons 3 and 4). Milli-Q water was used as a substrate for a control RT-PCR.
Full-length zebrafish dock1 was cloned into pCS2+ using Gibson Assembly. dock1 was amplified in two pieces from zebrafish cDNA using a Phusion mastermix (NEB) and the following primers: part 1: F: 5’-TCTTTTTGCAGGATCCCATAGAGAAGCGAGAAAAAGTGTG-3′ and R: 5’-CTCCATGATGATCTGCACGTG-3′ and part 2: F: 5’-TCAGCGACATACTGGAGGTGC-3′ and R: 5’-TAATACGACTCACTATAGTTGAGGTGTCAGCTGCTTTTCCG-3′. Gibson Assembly was then performed using an in-house Gibson reaction mixture (gifted by the Solnica-Krezel lab, Washington University in St. Louis). Briefly, the fragments were gel extracted and purified using the QIAquick Gel Purification Kit (Qiagen). 30 ng of pCS2+, linearized with Clal and Xbal, were combined with 5-fold excess of the dock1 PCR fragments and 15 μl of the Gibson Assembly enzyme-reagent mixture. The mixture was incubated at 50 °C for 1 h and then 10 μl were transformed into DH5 alpha cells and plated on ampicillin plates. Subsequent colonies were grown, miniprepped with a Qiagen Kit, and Sanger sequenced. Synthetic mRNA for injection was generated by linearizing dock1 in pCS2+ with Not1 and then transcribing with the mMESSAGE mMACHINE SP6 ULTRA Kit (Ambion). Approximately 120 pg of dock1 mRNA in 2 nl was injected into 1-cell stage embryos generated from a dock1stl145 heterozygous in-cross. In situ hybridization for mbp was performed and scoring of expression was performed blinded to genotype.
Whole mount in situ hybridization and qualitative scoring
Whole mount in situ hybridization (WISH) was performed as described [32, 33] on larvae treated with 0.003% phenylthiourea from 24 h post fertilization (hpf) to inhibit pigmentation until fixation in 4% paraformaldehyde. The previously characterized riboprobes used in this study were: sox10 , krox20 , and mbp . All phenotypes were scored with the scorer blinded to genotype. The PLLn was scored for strength of staining: “strong” = strong and consistent expression along the entirety of the PLLn; “reduced” = consistent but reduced mpb expression along PLLn; and “strongly reduced” = patches of mbp expression or no expression, similar to scoring as performed previously . “Strong” mbp expression was assigned a value of 3, “reduced,” a value of 2, and “strongly reduced,” a value of 1 to code each phenotype as a number for a Chi-squared anaylsis.
Transmission Electron microscopy and quantifications
TEM was performed on 3 dpf, 5 dpf, and 21 dpf cross-sections of the PLLn according to standard protocols [38, 39]. Larvae were cut between body segments 5 and 6 and juvenile 21 dpf fish were cut immediately posterior to the heart. A Jeol JEM-1400 (Jeol USA) electron microscope and AMT V601 digital camera were used to image samples. Quantification of percent myelinated axons, sorted axons, total axon number, and number of Schwann cell nuclei was performed on the entire cross section of the PLLn. The scorer was blinded to genotype, and quantification was performed manually as described previously .
Lifeact microinjections and live imaging
One-cell stage zebrafish embryos were injected with ~ 15–20 ng of sox10:Lifeact-RFP (a gift from the Lyons lab, University of Edinburgh) and 25 ng of transposase mRNA. 1 dpf larvae were then screened for expression of sox10:Lifeact-RFP in Schwann cells at 24 hpf. For live-imaging, larvae were anesthetized in Tricaine and embedded in 0.8% agarose on a 35 mm glass bottom dish filled with 0.2% Tricaine and covered with a 22 × 22 mm2 coverslip on top of vacuum grease . The larvae were then imaged with a Zeiss LSM 880 confocal microscope at 20× for 3 h at 3 min intervals. Still images were captured with a Zeiss LSM 880 II Airyscan FAST confocal microscope at 40xW with a 1.8 zoom. To examine blood vesssls, 4 dpf larvae with Tg(kdlr:mcherry) were imaged at 13.5× with a Nikon SMZ18 fluorescent dissecting microscope.
Eos Photoconversion and quantification of Schwann cell number
Tg(foxd3:gfp);dock1stl145/+ fish were crossed to Tg(sox10(4.9):nls-eos);dock1stl145/+ fish and offspring were screened for both transgenes at 1 dpf. At 2 dpf, larvae were placed in 0.8% low-melt agarose and mounted for imaging as described above. Before counting, larvae were individually exposed to 30 s of UV light using the DAPI filter with the 20× objective of a Zeiss LSM 880 confocal microscope. The number of GFP and RFP positive cells along the PLLn spanning ~ 8 body segments were the counted manually in ImageJ. The observer was blinded to genotype.
Neuromast labeling and quantification
3 dpf larvae derived from a dock1stl145 heterozygous in-cross were incubated with 50 μl of DASPEI (40 mg/ 100 mL in distilled water) in 4 mL of egg water for 15 min at room temperature. The DASPEI solution was removed and replaced with fresh egg water. The number of neuromasts along the PLLn were counted under a fluorescent dissecting microscope using a GFP filter.
Immunohistochemistry for acetylated tubulin was performed as described in  with mouse anti-acetylated alpha-tubulin used at a dilution of 1:1000 (Sigma). Larvae were fixed at 4 dpf and were derived from a dock1stl145 heterozygous in-cross. Heavy myosin within somites was detected with chicken MF 20 antibody at a dilution of 1:20 (Developmental Studies Hybridoma Bank). MF 20 was deposited to the DSHB by Fischman, D.A. (DSHB Hybridoma Product MF 20). For MF 20 staining, embryos were fixed at 1 dpf in 4% paraformaldehyde for 1 h and washed twice with 1X PBS for 10 min. Samples were then blocked with 0.05% Triton in PBS and 10% goat serum and then incubated with MF 20 in block overnight at 4 °C. After incubation, larvae were washed twice with PBS and then incubated secondary antibody in PBS for 2 h at room temperature. Primary antibodies were detected IgG2b with secondary antibody conjugated to either Alexa 568 or 488 (Invitrogen) at a 1:2000 dilution. Immunostained larvae were imaged with a Nikon SMZ18 fluorescent dissecting microscope.
GraphPad Prism 7 was utilized to perform statistical tests. Unpaired t-tests with Welch’s correction were used to test significance of all TEM, neuromast number, and Schwann cell number data. A Chi-squared analysis was utilized to determine significance for all WISH data. Phenotypes of “strong,” “reduced,” and “strongly reduced” were assigned a number of 3, 2, or 1, respectively, in order to compare phenotypes with a Chi-squared analysis. An unpaired t-test with Welch’s correction showed no significant difference between wild-type and heterozygous animals; therefore, for TEM, WISH, neuromast, and Schwann cell number data, wild-type and heterozygous animals were combined as controls.
Mutations in dock1 result in decreased myelin basic protein expression in the peripheral nervous system
Schwann cell myelination is significantly reduced in d o ck1 stl145 mutants at early stages
Neither Schwann cell migration nor number are affected in dock1 stl145 mutants
Because migration is not affected in dock1stl145 mutants, we examined whether decreased myelination in dock1stl145 mutant nerves was the result of fewer Schwann cells. To do this, we generated and analyzed 2 dpf double transgenic tg(foxd3:gfp);tg(sox10:nls-eos);dock1stl145 larvae. The sox10:nls-eos transgene enabled manual counting of Schwann cell nuclei along the PLLn, while the foxd3:gfp transgene provided a co-label to ensure Schwann cell identity. Counting the number of double positive cells at 2 dpf showed that dock1stl145/+ and wild-type siblings do not exhibit a significant difference in cell number (p = 0.2218) and were thus combined as the control group. No significant difference in the number of Schwann cells between mutants and control siblings was observed (p = 0.1243), suggesting that a reduction in Schwann cell number is not a contributing factor to decreased myelination of the PNS in dock1stl145 mutants (Fig. 4 l-n). Overall, these experiments demonstrate that SCP migration and number are not overtly affected in dock1stl145 mutants.
Defects in Schwann cell development are first observed during radial sorting and myelination initiation
A critical component of Schwann cell development is the remodeling of the cytoskeleton to promote shape changes to facilitate proper myelination of the PNS [3, 43]. Although some of the intracellular components involved in cytoskeletal rearrangements have been identified, such as Rac1, the full complement of proteins involved in this process has not been comprehensively defined. Through a forward genetic screen in zebrafish, we identified an early stop codon in the Rac1 binding domain of dock1, an atypical GEF, that causes decreased mbp expression in the PNS at 3 dpf and 5 dpf. Rescue experiments and complementation tests with two newly engineered alleles of dock1 confirmed that mutations in dock1 result in decreased mbp expression.
TEM analysis showed fewer myelinated axons in mutants at 3 dpf, 5 dpf, and 21 dpf, whereas axon number is not significantly affected at any stage assessed. However, we did note that several unmyelinated axons in some mutant nerves were abnormally large in diameter and had many mitochondria (data not shown). We demonstrated that reduced mbp expression and the reduction of myelinated axons in mutants is not caused by absence or loss of Schwann cell number. While two other members of the Dock1-family of atypical GEFs, Dock7 and Dock8, affect SCP migration in mammals [20, 21], this does not appear to be the case of Dock1 in zebrafish. This is not entirely unexpected, since loss of Rac1 in mouse Schwann cells did not affect Schwann cell migration [5, 6]. Although overt defects in migration were not detected using live-imaging, these experiments enabled visualization of F-actin localization, which showed that F-actin is localized at the back of migrating Schwann cells. This live-imaging data with Lifeact supports previously reported data from 3D culture of Schwann cells showing that migrating Schwann cells in vivo move in an amoeboid-like fashion , as contractions seem to occur at the back of the cell. In the future, it will be interesting to generate dock7 and dock8 zebrafish genetic mutants and observe how migration and F-actin localization is affected in SCPs.
Although a significant reduction in the percent myelinated axons is observed at 5 dpf, Schwann cells in dock1stl145 mutants do have the capability to myelinate axons, suggesting that dock1 is involved in the timing of myelination onset. It is also possible that other Dock1 family members compensate for dock1 loss of function in our mutants. Further experiments are needed to determine if Dock1 functions in a Schwann cell-autonomous or non-cell-autonomous manner. Consistent with data showing that dock1 mutant Schwann cells are delayed in radial sorting and myelination, expression of krox20, a transcription factor essential for expression of myelin genes, is decreased in dock1stl145 mutant nerves. Importantly, Schwann cell number is not affected in dock1stl145 mutants and overall PLLn development is not affected in dock1stl145 mutants compared to controls, as determined by acetylated tubulin staining and counting neuromast number. Combined, these data demonstrate that Schwann cell radial sorting and myelination are delayed in dock1 mutants.
The cell autonomy of Dock1 function and the upstream signals that trigger Dock1 activation remain to be elucidated. Dock1 has a DHR1 domain that can bind phosphatidylinositols  located in the cell membrane, making Dock1 is an attractive candidate to serve as a link between cell surfaces receptor and the cytoskeleton. Additionally, Dock1 could be a representative of a class of drug targets for diseases affecting peripheral myelin, especially because GEFs may contribute to myelination disease states in human patients [4, 46, 47, 48]. Although RhoGTPases are critical for cytoskeletal rearrangements, their ubiquity in many cell types limits their ability to serve as useful therapeutic drug targets. Alternatively, GEFs, particularly atypical GEFs like Dock1, could open a door to indirectly affect RhoGTPases in a more cell-specific manner and thus influence the cell shape changes that promote proper Schwann cell development and myelination.
In this study, we demonstrate that mutations in an atypical GEF, dock1, result in defects in Schwann cell radial sorting and myelination. Schwann cells are slower to extend processes into axon bundles and subsequently myelinate fewer axons. Schwann cell number and migration are not affected in these mutants; however, Schwann cells in dock1 mutants fail to robustly express markers such as mbp and krox20 in early development, suggesting that dock1 aids in the temporal regulation of Schwann cell radial sorting and development. Moreover, Dock1 may represent a link between extracellular signals and the intracellular cytoskeletal rearrangements necessary for radial sorting and myelination.
We thank members of the Monk, Solnica-Krezel, and Kaufman laboratories for assistance with the screen and helpful discussions; the Washington University Center for Cellular Imaging; the Washington University Zebrafish Consortium; S. Kucenas (University of Virginia) for the Tg(sox10(4.9):nls-eos) line; D. Lyons for the sox10:lifeact-RFP plasmid; C. Shiau (University of North Carolina) for the Tg(kdlr:mcherry) line; M. Mokallad (Washington University) for the acetylated alpha-tubulin antibody, and the Solnica-Krezel laboratory (Washington University) for the MF 20 antibody.
A.L.H., B.L.H, S.D.A., and K.R.M. performed the genetic screen and identified the stl145 mutant. R.L.C. and K.R.M designed research, and R.L.C. performed research. R.L.C. and K.R.M analyzed data, and R.L.C. and K.R.M. wrote the paper. All authors edited and approved of the manuscript.
R.L.C. is supported by the National Science Foundation Graduate Research Fellowship (DGE-1745038). This work was also supported by the Philip and Sima Needleman Student Fellowship in Regenerative Medicine (A.L.H.) and by the National Institute of Neurological Disorders and Stroke (NINDS) Grants F31 NS096814 (A.L.H), F31 NS094004 (to B.L.H.), and F31 NS087801 (to S.D.A.), and by a National Institute of Child Health and Human Development Grant R01 HD80601 (to K.R.M.). K.R.M. is a Harry Weaver Neuroscience Scholar of the NMSS.
Availability of data and materials
Data, reagents, and zebrafish lines available on request from the authors.
Ethics approval and consent to participate
Zebrafish were reared in accordance with the Washington University IRB and animal protocols and were raised in the Washington University Zebrafish Consortium (http://zebrafish.wustl.edu/husbandry.htm).
Consent for publication
The authors have no competing interests.
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