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. 2011 Dec;25(12):4184–4197. doi: 10.1096/fj.11-186239

Disc1 regulates both β-catenin-mediated and noncanonical Wnt signaling during vertebrate embryogenesis

Gianluca De Rienzo *, Joshua A Bishop †,‡, Yingwei Mao §, Luyuan Pan ‖, Taylur P Ma ‖, Cecilia B Moens ‖, Li-Huei Tsai †,, Hazel Sive *,**,1
PMCID: PMC3236629  PMID: 21859895

Abstract

Disc1 is a schizophrenia risk gene that engages multiple signaling pathways during neurogenesis and brain development. Using the zebrafish as a tool, we analyze the function of zebrafish Disc1 (zDisc1) at the earliest stages of brain and body development. We define a “tool” as a biological system that gives insight into mechanisms underlying a human disorder, although the system does not phenocopy the disorder. A zDisc1 peptide binds to GSK3β, and zDisc1 directs early brain development and neurogenesis, by promoting β-catenin-mediated Wnt signaling and inhibiting GSK3β activity. zDisc1 loss-of-function embryos additionally display a convergence and extension phenotype, demonstrated by abnormal movement of dorsolateral cells during gastrulation, through changes in gene expression, and later through formation of abnormal, U-shaped muscle segments, and a truncated tail. These phenotypes are caused by alterations in the noncanonical Wnt pathway, via Daam and Rho signaling. The convergence and extension phenotype can be rescued by a dominant negative GSK3β construct, suggesting that zDisc1 inhibits GSK3β activity during noncanonical Wnt signaling. This is the first demonstration that Disc1 modulates the noncanonical Wnt pathway and suggests a previously unconsidered mechanism by which Disc1 may contribute to the etiology of neuropsychiatric disorders.—De Rienzo, G., Bishop, J. A., Mao, Y., Pan, L., Ma, T. P., Moens, C. B., Tsai, L. H., Sive, H. Disc1 regulates both β-catenin-mediated and noncanonical Wnt signaling during vertebrate embryogenesis.

Keywords: Disc1, zebrafish, brain, neurons, GSK3β, pcp, mutant

Disrupted in schizophrenia 1 (Disc1) is a gene associated with schizophrenia, identified as a (1:11) (q42.1; q14.3) chromosomal translocation, with subsequent studies indicating that multiple mutations of human Disc1 (hDisc1) may be linked to mental health disorders (1). The molecular biology of Disc1 is complicated. Mouse Disc1 (mDisc1) regulates neuronal migration, partly through modulating microtubules, possibly though interaction with LIS1 and NDEL1, which regulate microtubule assembly (2). Consistently, mDisc1 interacts with kinesin-1 and the adaptor protein Grb2, and may regulate transport along axonal microtubules (3). Recently, mDisc1 has been associated with the basal body of cilia (4).

mDisc1 binds to the transcription factor, FEZ1 and expression of the FEZ1-binding domain of mDisc1 inhibits neurite outgrowth (5), while the phosphodiesterase 4 family of enzymes are implicated in schizophrenia, and also interact with mDisc1 (6). mDisc1 loss of function in adult-born hippocampal dentate granule cells increases dendritic outgrowth and ectopic dendrite formation (7). Girdin and mDisc1 interact (8), and after either Girdin or mDisc1 loss of function in adult-born neurons of the dentate gyrus, dendritic number increases and abnormal migration is observed (8). Migration defects in cortical neurons are also caused by loss of function in Dixdc1, which interacts with mDisc1and NDEL (9).

Disc1 directly affects the Wnt-signaling pathway. mDisc1 binds to GSK3β, a negative regulator of β-catenin-mediated Wnt signaling, through a 15-aa N-terminal region (10). In dentate gyrus neurons, mDisc1 inhibits GSK3β and thereby activates downstream β-catenin function, while inhibition of mDisc1 reduces lymphoid enhancer factor/T cell factor (TCF/LEF) mediated transcription in culture (10). Dixdc1 can also increase TCF/LEF-mediated transcription; however, it does so indirectly, through interaction with mDisc1 (9).

Several reports suggest that localization or levels of Wnt signaling components in cortex and hippocampus are abnormal in patients with schizophrenia. Wnt-1 immunoreactivity (11), intraneuronal staining of β-catenin (12), and expression of the secreted Wnt antagonist Dickkopf-3 (13) are decreased relative to controls in postmortem brains of patients with schizophrenia. GSK3β levels and phosphorylation (on Ser-9) may be abnormal among individuals with schizophrenia (14), perhaps related to the interface of GSK3β and mood stabilizers such as lithium (15).

Wnt can also signal through the noncanonical pathway, which does not involve β-catenin. This pathway is responsible for morphogenetic changes, such as cell sheet lengthening or bending (16), and neuronal migration (17). Noncanonical Wnt signaling acts through 2 branches, one involving the formin-domain protein Daam1, the GTPase Rho, and the kinase Rock, while the other involves the GTPase Rac (18).

In the present study, we use the zebrafish as a tool (19) to analyze the mechanisms by which zDisc1 functions during embryogenesis. We define a “tool” as a biological system that gives insight into mechanisms underlying a human disorder, although the system does not phenocopy the disorder. We demonstrate that zDisc1 modulates brain development through the β-catenin-mediated Wnt pathway and show, for the first time, that zDisc1 regulates the noncanonical Wnt pathway. The data implicate an additional major pathway by which Disc1 may affect neuropsychiatric disorders.

MATERIALS AND METHODS

Fish lines and maintenance

Embryos were obtained from natural spawnings. Developmental stages are reported as hours postfertilization (hpf) at 28°C. Disc1fh291 mutants were isolated from a library of 8600 ENU-mutagenized F1 fish (20). The Tg(TOP:GFP)w25 line was kindly provided by Dr. Randall Moon (University of Washington School of Medicine, Seattle, WA, USA), and the Tg(CM-isl1:GFP) line was kindly provided by Dr. Anand Chandrasekhar (University of Missouri, Columbia, MO, USA).

Morpholino oligo injections

Antisense morpholino oligonucleotides (MOs; Gene Tools, LLC, Philomath, OR, USA) were as follows: Disc1MO, 5′-TCGCAGTTTTGTCTTACCTGTCCTC-3′; CMO, 5′-CCTCTTACCTCAGTTACAATTTATA-3′; p53MO, 5′-GCGCCATTGCTTTGCAAGAATTG-3′. MO (1 nl) was injected into a single cell of a 1–2 cell embryo, using 3.5 ng for CMO, 3–3.5 ng for Disc1MO. The p53MO was used at 1.5-fold greater mass than the mass of experimental or control MO in all experiments.

Microscopy

Methods for brightfield microscopy have been described previously (21). Confocal imaging was performed on a Zeiss LSM710 (Carl Zeiss, Oberkochen, Germany) as described previously (21).

Brain ventricle imaging

Brain ventricle imaging was described previously (22).

Transgenic analysis

Transgenic analysis was generally performed in transient transgenics (23). Each construct (1.0 nl of 30 ng/ml), together with 0.2 U/ml of I-SceI meganuclease (New England BioLabs, Beverly, MA, USA), was injected into 1-cell fertilized embryos. The miR124 promoter is specifically expressed in the developing central nervous system (24), the Ntl promoter (a kind gift from Jim Smith, Gurdon Institute, Cambridge, UK) in the marginal zone and later in notochord (25). miR124:Disc1-IresGFP and miR124:DN-GSK3β-IresGFP were obtained by PCR of full-length zDisc1 cDNA or the Xenopus DN-GSK3β (a kind gift from David Kimelman, University of Washington, Seattle, WA, USA) and cloning into miR124:IresGFP (unpublished results). Ntl:DN-GSK3β-IresGFP was obtained from Ntl:CFP (25), DN-GSK3β from pCS2DN-GSK3β clone, and IresGFP-pA from p3E-Ires-GFP-pA from the Tol2Kit.

In situ hybridization

In situ hybridization for single- or dual-color labeling is described elsewhere (26).

Immunohistochemistry

Whole-mount immunostaining used mouse anti-acetylated α-tubulin (1:1000; Sigma, St. Louis, MO, USA), and mouse anti-Znp1 (1:200; Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA, USA). Goat anti-mouse Alexa Fluor 488 (1:500; Molecular Probes, Eugene, OR, USA) was used as a secondary antibody alone or in combination with phalloidin Texas Red (1:100; Molecular Probes).

Genotyping

Genomic DNA was extracted as reported in Westerfield et al. (27) and amplified with the following primers: GT134, 5′-ACTCATCAAAGTCTTCAAATAAACACCAAT-3′; GT135, 5′-GCCCTGACGCTGATTCAGAT-3′. The resulting 189-bp fragment was digested with MfeI, which cuts only mutant DNA.

RT-PCR and qRT-PCR

Total embryo RNA was extracted using Trizol (Invitrogen, Carlsbad, CA, USA), followed by chloroform extraction and isopropanol precipitation. cDNA synthesis was performed with Super Script III Reverse Transcriptase (Invitrogen) and random hexamers.

Primers for RT-PCR were the following: Disc1D1up, 5′-GCAGCCCAACGGAGGATGAC-3′; Disc1D1rev, 5′-TTTTGGGGCACTGCTGGTTTC-3′.

qRT-PCR was performed using an ABI Prism 7900 (Applied Biosystems, Inc., Foster City, CA, USA). Fluorescence detection chemistry utilized SYBR green dye master mix (Roche, Indianapolis, IN, USA). qRT-PCR primer sequences were as follows: GFPup, 5′-CCCTCGTGACCACCCTGACCTAC-3′; GFPrev, 5′-CCTTGATGCCGTTCTTCTGCTTGTC-3′. The relative amount of product was calculated using ΔCT and normalized to β-actin.

Pharmacological treatments

GSK3β was inhibited with 2,4-dibenzyl-5-oxothiadiazolidine-3-thione (Chiron 99021), generously provided by Dr. Stephen Haggarty (Center for Human Genetic Research, Massachusetts General Hospital, Boston, MA, USA). Embryos were soaked in 400 μM Chiron 99021 in embryo medium, from 30% epiboly to 8 somites at 28°C, prior to dechorionation and fixation. Controls were treated with DMSO (1%). Dexamethasone (DEX) was added to a final concentration of 10 μg/ml at 50% epiboly.

RNA injections

PCR fragments were cloned into pGEM-T Easy Vector (Promega, Madison, WI, USA), and subcloned into the EcoRI site of pCS2+. 6xHisTag fusion protein constructs were obtained by PCR and cloned into pCS2+. The following colleagues generously provided constructs: Dr. Nobue Itasaki (National Institute for Medical Research, London, UK), GR-LEFΔN-βCTA; Dr. David Kimelman (University of Washington), DN-GSK3β; Dr. Steven Wilson (University College London, London, UK), NDsh; Dr. Raymond Habas (Temple University, Philadelphia, PA, USA), Daam1A12; Dr. Xi He (Harvard Medical School, Cambridge, MA, USA), N-Daam1; Dr. Rudi Winklbauer (University of Toronto, Toronto, ON, Canada), RhoAV14; Dr. Karen Symes (Boston University, Boston, MA, USA), RacV12, and Dr. Jeremy Green (Dana-Farber Cancer Institute, Boston, MA, USA) for membrane-targeted CAAX-eGFP. Amounts of mRNA injected: hDisc1, 200 pg; GR-LEFΔN-βCTA, 50 pg; DN-GSK3β, 20 pg; NDsh, 30 pg; DaamA12, 100 pg; NDaam, 100 pg; RhoV14, 0.2 pg; RacV12, 5 pg.

Western blot analysis

Methods for Western blot analysis have been described previously (21).

Luciferase assay

Luciferase assay was performed as described previously (10).

Surface plasmon resonance (SPR) analysis of zDisctide Binding to GSK3β

An S CM5 chip was loaded into a Biacore T100 SPR and conditioned with pulses of 50 mM NaOH and glycine (GE Life Sciences, Pittsburgh, PA, USA) at 30 μl/min for 30 s. Anti-GST antibody was immobilized using standard amine coupling conditions in PBS buffer at ∼10,000 RU on Flowcell 1 and Flowcell 2 (FC1/2). Recombinant GST (BPS Bioscience, San Diego, CA, USA; 10 μg/ml in capture buffer) was captured at ∼1000 RU on FC1 as a reference for non-specific binding. GST tagged GSK3β (BPS Bioscience; 10 μg/ml in capture buffer) was captured to a level of ∼1000 RU on FC2. The GSK3β binding peptide L803-mts (Tocris Bioscience, Ellisville, MO, USA) was used as a positive control. L803-mts and zDisctide (GenScript, Piscataway, NJ, USA) solutions were generated at 50 μM in TBS sample buffer with 2% DMSO (v/v), and diluted to 1.5625 μM. Samples were loaded into a Biacore T100 reagent rack and flowrate was set at 30 μl/min for 60 s followed by 60 s of disassociation. Each injection was run in duplicate, and binding analysis was done on Scrubber 2.0 (http://www.cores.utah.edu/interaction/scrubber.html).

Statistical analysis

Tests of significance were performed using Anova single factor. Values are reported as means ± sd.

Convergence and extension assay

Convergence and extension assays were performed as described previously (28).

RESULTS

Zebrafish Disc1 is required for normal embryogenesis and neurogenesis

The putative zebrafish Disc1 protein has limited overall sequence conservation with human Disc1 (29); however, conserved domains are found throughout the protein (Supplemental Fig. S1). zDisc1 is expressed maternally and zygotically, with strong expression in the developing nervous system (Supplemental Fig. S2). To assay zDisc1 function during zebrafish embryogenesis, we analyzed a zDisc1 mutant and also used an antisense approach to inhibit zDisc1 function.

In collaboration with the zebrafish TILLING Consortium, the Disc1fh291 mutant was defined. The mutant is caused by an early stop codon, in exon 2, downstream from the first nuclear localization site at Lys115 (NLS1, Fig. 1A). A second mutant, Disc1fh292, was difficult to recover and has not been further studied. An antisense morpholino-modified oligonucleotide (Disc1MO) targeted the intron 1/exon 2 boundary, soon after NLS1, and putatively resulting in inclusion of intron 1, to add 14 aa to exon 1 until the first stop codon.

Figure 1.

Figure 1.

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Loss of zDisc1 function results in abnormal brain development and impaired axonogenesis. A) a) Schematic of zDisc1 gene. Bar indicates position of zDisc1 morpholino (Disc1MO); star indicates stop codon in Disc1fh291 line. b) Schematic of zDisc1 protein. c) Predicted protein truncation in Disc1fh291 embryos. d) Predicted protein truncation in Disc1 morphants. B) a) Genomic PCR for Disc1fh291 locus followed by MfeI digestion performed on single embryo. b) RT-PCR showing decrease in correctly spliced zDisc1 mRNA after injection of Disc1MO. Actin is loading control. C) Morphology of Disc1 mutant and morphants at 24 hpf. a–a″) Wild-type embryos. b–b″) fh291/fh291 embryos. c–c″) fh291/fh291 coinjected with hDisc1 RNA. d–d″) Embryos injected with Disc1MO. Brain ventricle visualization is described in Materials and Methods. a–d) Lateral view of head. a′–d′) Lateral view of whole embryo. a″–d″) Dorsal view of head. f, forebrain; m, midbrain; h, hindbrain; asterisks, otic vesicles. D) Disc1fh291 morphological analysis at 3 and 5 dpf. a) Wild-type. b) fh291/fh291 showing abnormal pigmentation (bracket). c) Wild-type. d) fh291/fh291 showing bent tail. E) Analysis of neurons. fh291/fh291 embryos injected with CMO or Disc1MO immunostained for acetylated tubulin at 36 hpf. a, b) CMO-injected embryos (100% normal, n=10). c, d) fh291/fh291 embryos (0% normal; n=10). e, f) Disc1MO injected embryos (0% normal; n=10). a, c, e) Lateral view of head neurons. b, d, f) Dorsal view of hindbrain neurons. ac, anterior commissure; poc, postoptic commissure; dvdt, dorsoventral diencephalic tract; tpc, tract of posterior commissure; sot, supraoptic tract; r, rhombomeres. F) Analysis of muscle segments. Embryos were immunostained in two independent experiments with phalloidin, which marks actin filaments at 30 hpf. Lateral view of muscle segments, anterior to left. Dotted lines: muscle segment shape. a) CMO or wild-type. b) Disc1fh291 mutant. c) Disc1MO (CMO or wild-type: 100% normal, n=14; Disc1fh291 mutant: 13% normal, n=15; Disc1MO: 0% normal, n=15). G) Experimental design. H) Effects of zDisc1 expression in the CNS, analyzed by brain ventricle injection. Embryos assayed at 24 hpf in 2 independent experiments. a–a″) Embryos injected with Disc1MO (0% normal, n=40). b–b″) F0 Tg(miR124:IresGFP) embryos + Disc1MO (0% normal, n=79). c–c″) F0 Tg(miR124:Disc1-IresGFP) embryos + Disc1MO (3 ng/embryo; 53% normal, n=106). a–c) Lateral view of head. a′–c′) Lateral view of whole embryo. a″–c″) Dorsal view of head. I) Effects of zDisc1 expression of zDisc1 in the CNS, assayed at 36 hpf in 2 independent experiments, by immunostaining for acetylated tubulin. Dorsal view. a) Disc1MO-injected embryos (0% normal, n=10). b) Tg(miR124:IresGFP) embryos + Disc1MO (0% normal, n=10). c) Tg(miR124:Disc1-IresGFP) embryo + Disc1MO (80% normal, n=10).

After outcrossing Disc1fh291 to wild-type (AB strain) fish for 5 generations, a true-breeding line was obtained, and PCR genotyping identified wild-type, heterozygous, and homozygous mutant embryos (Fig. 1Ba). Injection of the Disc1MO led to a 4.5-fold decrease in levels of normal zDisc1 RNA (Fig. 1Bb). While heterozygote Disc1fh291 fish showed no abnormal phenotype, homozygous mutant embryos assayed at 24 hpf (late somitogenesis) showed abnormal brain morphology, with small ventricles as seen after injection with Rhodamine dextran (22), and an abnormally bent tail (Fig. 1Ca–b″). Specificity of the phenotype for Disc1 was confirmed by rescue with injected human Disc1 (hDisc1) RNA (Fig. 1Cc, c″). A similar phenotype was observed after injection of Disc1MO into the 1-cell embryo (Fig. 1Cd, d″). Cell death due to off-target effects of MOs was decreased by coinjection of a p53 MO (30). In both Disc1fh291 mutants and Disc1MO embryos, muscle segments were abnormally shaped and the tail was bent. At 5 d postfertilization, the Disc1fh291 mutant embryo remained bent, with abnormal pigmentation and an uninflated swim bladder (Fig. 1D).

Axon tracts in both Disc1fh291 mutants and zDisc1 morphants were abnormal (Fig. 1D). At 36 hpf, the forebrain axon scaffold, labeled with anti-acetylated tubulin, was disorganized, with the postoptic commissure, anterior commissure, and supraoptic tract missing or strongly reduced (Fig. 1Ec, d, arrows and asterisks). Hindbrain axon tracts (Fig. 1De) were severely disorganized. Subsequent development of the brain is abnormal, with reduced forebrain and hindbrain axon tracts, and abnormal motor neuron differentiation (Supplemental Fig. S3).

After treating fixed embryos at 24 hpf with phalloidin, which stains actin, it was clear that Disc1fh291 mutants and zDisc1 morphants showed abnormal, U-shaped muscle segments (Fig. 2F). Together with the curved body shape, this morphology suggested a convergence and extension phenotype (31). In further analyses, zDisc1 mutant embryos showed a 1.5-fold increase in apoptosis at 24 hpf, relative to controls (late somitogenesis), and levels of cell proliferation remained unchanged (not shown).

Figure 2.

Figure 2.

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zDisc1 binds GSK3β and hDisc1 rescues the zDisc1 loss of function phenotype. A) The zDisctide peptide (134–149 aa) binds GSK3β. SPR sensorgrams for GSK3β binding with control L803 (a) and zDisctide1 (b). B) zDisc1 and hDisc1 mutants, and alignment of putative GSK3b binding domains. Asterisk (*) indicates identical; two dots (:), strongly similar; one dot (.), more weakly similar. Red box, GSK3β binding domain; blue box, N-terminal 6x-His tag; light blue box, N-terminal-flag tag. C) a) Western blot using a monoclonal anti-His antibody on total protein extracted from 30 hpf Disc1MO embryos, coinjected with hDisc1 (lane 1), Δ1hDisc1 (lane 2) or Δ2hDisc1 RNA (lane 3). b) Western blot using a monoclonal antiflag antibody on total protein extracted from 30 hpf CMO (lane 1) and Disc1MO (lane 2) embryos coinjected with zDisc1 RNA. D) Phenotypic rescue. Embryos were injected with CMO or Disc1MO, together with mRNA encoding hDisc1 (200 pg), zDisc1* (100 pg), or GFP (200 pg), and assayed at 24 hpf in 3 independent experiments. a–a″) CMO + GFP (100% normal, n=60). b–b″) CMO + zDisc1 or zDisc1* (80% normal, n=20). c–c″) CMO + hDisc1 or Δ1hDisc1 or Δ2hDisc1 (97% normal, n=58). d–d″) Disc1MO + GFP (0% normal, n=18). e–e″) Disc1MO + zDisc1 (84% normal, n=25). f–f″) Disc1MO + hDisc1 (69% normal, n=23). g–g″) Disc1MO + zDisc1* (20% normal, n=30). (h, h″) Disc1MO + Δ1hDisc1 (64% normal, n=25). i–i″) Disc1MO + Δ2hDisc1 (0% normal, n=18). a–i) Lateral views of whole embryo. a′–i′) Lateral views of head. a″–i″) Dorsal views of head after brain ventricle injection. f, forebrain; m, midbrain; h, hindbrain. E) Muscle segments after coinjection of CMO or Disc1MO and GFP, hDisc1, Δ1hDisc1 or Δ2hDisc1 mRNAs. Embryos were immunostained with phalloidin, which marks actin filaments, at 30 hpf in 2 independent experiments. Lateral view of muscle segments, anterior to left. Dotted lines: muscle segment shape. a) CMO + hDisc1, Δ1hDisc1, or Δ2hDisc1 (90–100% normal, n=25). b) Disc1MO + GFP (7% normal, n=15). c) Disc1MO + hDISC1 (87% normal, n=15). d) Disc1MO + Δ1hDisc1 (80% normal, n=15). e) Disc1MO + Δ2hDisc1 (0% normal, n=13).

Since defects outside the brain after zDisc1 loss of function could have influenced neural development, we asked whether zDisc1 expressed from the central nervous sytem-specific miR124 promoter, in transient (F0) transgenics, could overcome the Disc1 morphant phenotype (Fig. 1G). Tg(miR124:Disc1) restored normal brain morphology (Fig. 1Ha–c″) and axonal development to zDisc1 morphants (Fig. 1Ia–c), indicating that zDisc1 acts in the neuroectoderm. Tail defects were not corrected. These data show that zDisc1 is required for normal embryogenesis and functions within the developing brain to regulate brain morphology and axonogenesis and elsewhere to regulate somite and tail formation.

Function of Disc1 is conserved between humans and zebrafish, and requires a GSK3β binding domain

By sequence analysis and genome comparison, zDisc1, mDisc1, and hDisc1 are orthologs (Supplemental Fig. S1). We also applied a more rigorous definition of orthology, by asking whether zDisc1 and hDisc have interchangeable function. One key domain in mDisc1 is the 15-aa GSK3β binding region identified by Mao et al. (10), of which hDisc1 shares 13/15 residues, while zDisc1 has 7/15 residues identical to both mDisc1 and hDisc1, with conservative substitutions in 3 additional residues (Fig. 2A, B). To determine whether this zebrafish domain binds GSK3β, SPR was used (Fig. 2A) and demonstrated a robust binding interaction between zDisctide and GSK3β. L803-mts, a known GSK3β inhibitor (32), was used as a positive control. L803-mts produced a KD value in the low micromolar range (69 μM), while zDisctide gave a KD value of 392 nM, a nearly 200-fold change in dissociation values. A second characteristic of SPR data is the percentage theoretical Rmax (%Rmax), a ratio of the observed and theoretical Rmax values. True binding interactions have %Rmax near 100. L803-mts gave a %Rmax value of 91, while zDisctide gave a comparable value of 88. These data illustrate that zDisctide can interact directly with GSK3β at levels analogous to a known GSK3β binder, and equivalent to the analogous mDisc1 peptide (10).

We then assayed interchangeability of zDisc1 and hDisc1 in their ability to restore normal development to zDisc1 loss of function embryos. In addition, several mutations were tested, including zDisc1*, with 5 point mutations in the GSK3β binding domain; Δ1hDisc1, extending from aa 166 and including the GSK3β binding domain; and Δ2hDisc1, extending from aa 226 and lacking the GSK3β binding domain (Fig. 2B). Protein levels were equivalent for all constructs (Fig. 2C). Neither wild-type nor mutant Disc1 proteins produced an overexpression phenotype when coinjected with control morpholino (Fig. 2Da–c″). When wild-type zDisc1 or hDisc1 mRNA was coinjected with the Disc1MO, normal brain morphology was restored (Fig. 2Dd–f″). Expression of zDisc1* mRNA did not restore a normal phenotype (Fig. 2Dg, g″), Δ1hDisc1 led to partial rescue (Fig. 2Dh, h″), while Δ2hDisc1 failed to rescue either brain or muscle segment defects seen in Disc1MO morphants (Fig. 2Di, i″). Consistently, muscle segments were normal V-shaped after coinjection of the Disc1MO with zDisc1 or hDisc1mRNAs (Fig. 2Ea–f), but abnormal after zDisc1* and Δ2hDisc1mRNA injection. Δ1hDisc1 mRNA prevented abnormal muscle segment shape (Fig. 2De), suggesting residual function.

These data demonstrate that zebrafish, mouse, and human Disc1 proteins are orthologs, with evolutionarily related sequence identity and functional interchangeability. Orthology is further supported since a zDisc1 peptide homologous to a region of hDisc1 and mDisc1 binds GSK3β tightly, and this region is required for normal zebrafish forebrain development and muscle segment morphology. Thus, the pathways in which Disc1 proteins of fish and mammals act are likely to be conserved.

β-Catenin and dominant negative GSK3β rescue forebrain and axonal defects after zDisc1 loss of function

The requirement for the GSK3β binding region suggested that zDisc1 modulates downstream signaling factors, including β-catenin, a key player in one branch of the Wnt signaling pathway (10, 33). We first tested this by asking whether zDisc1 could substitute for mDisc1 in activating a reporter containing by TCF/LEF binding sites, and responsive to β-catenin activity (Fig. 3A). TCF/LEF signaling was activated in mouse P19 cells by expression of Wnt3a, and shRNAs against mDisc1 decreased luciferase reporter activity (12). Cotransfection of zDisc1 cDNA with mDisc1 shRNAs increased TCF/LEF-mediated luciferase activity in controls, and restored activity after mDisc1 shRNA cotransfection (Fig. 3A). These data suggest that zDisc1 can activate β-catenin-mediated gene expression.

Figure 3.

Figure 3.

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zDisc1 acts through the β-catenin pathway. A) LEF/TCF-mediated luciferase activity is rescued by zDisc1 expression after inhibition of mDisc1. n = 3, P = 2.4E-06, mean ± se. B) Experimental prediction using a DEX-inducible β-catenin protein. C) Experimental design. D) Effects of inducible β-catenin. Embryos were injected with CMO (3.5 ng) or Disc1MO (3.5 ng), together with 20 pg of GR-LEF-βcat mRNA. DEX was added at early gastrula (50% epiboly); embryos were assayed at late somitogenesis (24 hpf). Two to 4 independent experiments were performed. a–b″) Embryos injected with CMO + GR-LEF-βcat without DEX (a–a″; 100% normal, n=53) or with DEX (b–b″; 29% normal, n=35). c–d″) Disc1MO + GR-LEF-βcat without DEX (c–c″; 0% normal, n=44) or with DEX (d–d″; 57% normal, n=40). a–d) Lateral views of heads, anterior to left. a′–d′) Dorsal views of heads after ventricle injection. a″–d″) Lateral view of whole embryo, anterior to left. f, forebrain ventricle; m, midbrain ventricle; h, hindbrain ventricle. Dark stripe in forebrain (arrowhead) of zDisc1 morphants was caused by accumulation of debris (unpublished results). E) Effects of GR-LEF-βca. Axons stained with acetylated tubulin at 36 hpf. a, b) CMO + GR-LEF-βcat without DEX. c, d) Disc1MO + GR-LEF-βcat without DEX. e, f) Disc1MO GR-LEF-βcat with DEX. a, c, e) Forebrain, lateral view, anterior to left. b, d, f) Hindbrain, dorsal view, anterior to top. Arrows: sot, supraoptic tract; arrowheads: r, rhombomeric neurons. F) Experimental prediction. G) Effects of DN-GSK3β. Embryos injected with CMO (3.5 ng) or Disc1MO (3.5 ng), together with mRNA encoding GFP (20 pg) or DN-GSK3β (20 pg), and assayed at 24 hpf in 3 independent experiments. a) CMO + GFP (90% normal, n=30). b) CMO + DN-GSK3β (17% normal, n=60). c) Disc1MO + GFP (0% normal, n=34). d) Disc1MO + DN-GSK3β (69% normal, n=34). a–d) Dorsal views of head after ventricle injection. e–n) Analysis of neurons. Embryos coinjected with CMO plus GFP or DN-GSK3β and Disc1MO plus GFP or DN-GSK3β were immunostained for acetylated tubulin at 36 hpf in 2 separate experiments. e, f) CMO + GFP (100% normal, n=15). g, h) CMO + DN-GSK3β (13% normal, n=15). i, j) Disc1MO + GFP (0% normal, n=10). k, l) Disc1MO plus DN-GSK3β (85% normal, n=20). e, g, i, k) Lateral view of head. f, h, j, l) Dorsal view of hindbrain. sot, supraoptic tract; r, rhombomeres.

We further hypothesized that activation of β-catenin in zDisc1 morphants would overcome the zDisc1 loss of function phenotype (Fig. 3B). We tested this using a fusion protein, GR-LEFΔNβCTA (34), comprising a stable activation domain of β-catenin, fused to the DNA binding domain of the coactivator, Lef1, and to the hormone binding domain of the glucocorticoid receptor (GR), allowing conditional activation by DEX (ref. 35 and Fig. 3C). Consistent with posteriorization caused by β-catenin-mediated Wnt signaling (36), activation of the GR construct in controls resulted in loss of forebrain and eyes (Fig. 3Da–b″). However, this construct prevented the Disc1MO phenotype in 57% of morphants where normal brain morphology and eye development was observed (Fig. 3Dc–d″). Inducible β-catenin also restored formation of the supraoptic axon tracts (Fig. 3Ea, c, e, arrows,) and rhombomeric axons (Fig. 3Db, d, f, arrowheads) to zDisc1 morphants, but did not rescue tail or muscle segment defects, suggesting that other pathways contribute to these phenotypes (Fig. 3Dc, d″).

Since GSK3β inhibits β-catenin function, we asked whether a dominant negative GSK3β (DN-GSK3β; ref. 36) could prevent the zDisc1 loss of function phenotype (Fig. 3F). While expression of DN-GSK3β posteriorized the head (ref. 36 and Fig. 3Gb), normal brain morphology (Fig. 3Gc, d) and axonal outgrowth (Fig. 3H) were seen in Disc1MO embryos, demonstrating that this construct counteracts loss of zDisc1 function.

Consistently, zDisc1 loss of function decreased expression of a β-catenin-responsive reporter in the Tg(TOP:GFP)w25 line (37), and expression was restored by a GSKβ inhibitor (Supplemental Fig. S4). zDisc1 and Wnt8b (considered a canonical Wnt) synergize in loss of function assays (not shown), and changes in gastrula stage gene expression seen after Wnt8 expression were also seen after zDisc1 loss of function (Supplemental Fig. S5A–D). Loss of canonical Wnt expression leads to anterior expansion, in many systems (36), and we observed an expansion of telencephalic (emx1) and a decrease in diencephalic (nr2f2) gene expression after Disc1 loss of function (Supplemental Fig. S5E–H).

These data suggest that zDisc1 acts through β-catenin and GSK3β to direct normal brain morphology and correct axonal outgrowth. Forced expression of activated β-catenin did not rescue tail formation or somite shape (not shown) in zDisc1 loss of function embryos, indicating that β-catenin is not involved in these aspects of zDisc1 function.

zDisc1 regulates convergence and extension

The curved body and abnormal muscle segments seen after zDisc loss of function suggested that convergence and extension was abnormal. We tested this further in several ways. First, in control embryos, labeled dorsolateral cells converge dorsally and extend along the head-to-tail axis during gastrulation (Fig. 4Aa–c), whereas this movement is defective in zDisc1 morphants (Fig. 4Ad–f). Second, inhibition of zDisc1 led to a significantly shortened body axis by the end of gastrulation (bud stage; Fig. 4Ba–c). Third, axial mesodermal width increased, indicated by flh expression as assayed at bud stage (Fig. 4Bd, e), while at the 12 somite stage, as measured by myoD expression, somite width increased and somites were compressed (Fig. 4Ca–d), while by 17-somites, the axial mesoderm remained S-shaped (Fig. 4Ce, f). These data mirror that seen in convergence and extension mutants, and indicate that this process is abnormal after zDisc1 loss of function.

Figure 4.

Figure 4.

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zDisc1 is required for convergence and extension. A) Disc1 loss of function inhibits cell movement during gastrulation. Images of live embryos injected with CMO (a–c) or Disc1MO (d–f) and injected in a single blastomere with fluorescein dextran at the 32-cell stage. Embryos analyzed further had dorsolateral staining at shield. a, d) 50% epiboly. b, e) 70% epiboly. c, f) Bud. V, ventral; D, dorsal. B) Body length of CMO (a, d) and Disc1MO-injected (b, e) embryos at bud stage. a, b) Lateral view. c) Quantification of angle between head and tail at bud stage of CMO vs. Disc1MO injected embryos (average of 30 embryos/treatment). d, e) flh expression in CMO (d) or Disc1MO-injected (e) embryos. C) Embryos injected with CMO (a, c, e) or Disc1MO (b, d, f), views of embryos hybridized for myoD1 RNA expression (c–f) at 12 somites. a, b) Dorsal view. c, d) Lateral view. e, f) Live embryos at 17 somites.

Noncanonical Wnt pathway mediators are able to rescue somite and tail zDisc1 loss-of-function effects

Since the noncanonical Wnt pathway regulates convergence and extension (30), we asked whether modulators of this pathway could rescue muscle segment and tail phenotypes after zDisc1 loss of function. N-terminal truncated Disheveled lacking the DIX domain (NDsh) primarily transduces noncanonical Wnt signals (38, 39), and while NDsh RNA had little effect on control embryos (Fig. 5Ab–b″), when coinjected with the Disc1MO, normal muscle segment morphology was seen (Fig. 5Aj–j″). RNA encoding an activated Daam1 protein (Daam1A12; ref. 40) did not perturb control embryos (Fig. 5Ac–c″), but prevented muscle segment and tail abnormalities in Disc1MO embryos (Fig. 5Ak–k″). GSK3β has not previously been reported to interface with embryonic noncanonical Wnt signaling, but injection of RNA encoding DN-GSK3β resulted in normal muscle segment and tail morphology (Fig. 5Al–l″). Use of a dominant negative Daam1 (N-Daam1) construct showed that suppression of the zDisc1 loss-of-function phenotype by DN-GSK3β required Daam1 function (Fig. 5Am–n″). Consistent with involvement of the Daam-1 branch of noncanonical signaling, activated RhoA (RhoAV14) prevented the zDisc1 loss of function muscle segment and tail phenotype (Fig. 5Ao–o″). In contrast, an activated form of Rac (RacV12), acting in the other branch of the noncanonical pathway, did not restore normal phenotypes to zDisc1 morphants (Fig. 5Ap–p″).

Figure 5.

Figure 5.

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Noncanonical Wnt pathway modulators rescue zDisc1 loss of function defects. A), Embryos injected with CMO (a–h′) or Disc1MO (i–p′), together with mRNA encoding GFP, NDsh, DaamA12, DN-GSK3β, NDaam1, Ndaam1 and DN-GSK3β, RhoAV14, or RacV12 as indicated, and assayed at 24 hpf in 3 independent experiments. a–p) Lateral view of whole embryo, head to left. a′–p′) Lateral view of muscle segments. a″–p″) Muscle segments immunostained with phalloidin, at 30 hpf. Lateral view, anterior to the left. Dotted lines: muscle segment shape. B) Percentage of normal (blue) vs. abnormal (red) embryos in Aa–h. C) Percentage of normal (blue) vs. abnormal (red) embryos in Ai-p. D) Expression of flh at bud stage (10 hpf) in embryos injected with CMO (a–f) or Disc1MO (g–l), together with mRNA encoding GFP, NDsh, DaamA12, RhoAV14, RacV12, or DN-GSK3β, as indicated. Dorsal view with anterior to top. E) Percentage of normal (blue) vs. abnormal (red) embryos as reported in Da–f. F) Percentage of normal (blue) vs. abnormal (red) embryos in Dg–j.

An additional measure of convergence and extension is expression of flh RNA in the axial mesoderm. This is wider than normal in zDisc1 loss of function embryos (Figs. 4 and 5Da, g), and was thinner and equivalent to controls, after coinjection of the Disc1MO with NDsh, DaamA12 or RhoAV14, DN-GSK3β RNAs (Fig. 5Dh–l). In contrast (and consistent with Fig. 5Ap–p″), RacV12 failed to normalize flh expression (Fig. 5Dk). Daam1A12 expression failed to rescue axonal defects caused by zDisc1 loss of function (data not shown), indicating that the noncanonical Wnt pathway does not regulate axonogenesis.

These data indicate that zDisc1 modulates convergence and extension through the Daam1/Rho branch of the noncanonical Wnt pathway. zDisc1 may be a negative modulator of GSK3β, and this protein has recently been implicated in noncanonical Wnt signaling using tissue culture analyses (41); however, in this case GSK3β activated Wnt coreceptor function.

DN-GSK3β expressed in the CNS rescues neuronal defects, while DN-GSK3β expressed in the mesendoderm rescues somite defects in zDisc1 morphants

Since ubiquitous expression of DN-GSK3β rescued all defects caused by zDisc1 loss of function, we asked whether the DN-GSK3β could act specifically in neurectoderm or mesendoderm (Fig. 6). DN-GSK3β expression was driven by the central nervous specific promoter miR124 in transient transgenics (Fig. 6A and ref. 25). When a control morpholino was injected into the F0 Tg(miR124:DN-GSK3β) transgenics, embryos appeared smaller (Fig. 6Bc, d), although head truncation observed after RNA injection and earlier expression of the protein was not seen (Fig. 3H). After injection of the Disc1MO, Tg(miR124:DN-GSK3β) transgenics showed normal brain morphology (Fig. 6Be–h), forebrain and hindbrain axon tracts (Fig. 6Ce–h). These data indicate that both GSK3β and zDisc1 act in the neuroectoderm, consistent with data shown in Fig. 1H. Muscle segment shape was also normal in zDisc1 morphants expressing the DN-GSK3β in the CNS (Fig. 6Dc, d). This was not due to ectopic expression (not shown) but is likely due to contribution of the neurectoderm in regulation of convergence and extension (39).

Figure 6.

Figure 6.

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A–D) Expression of DN-GSK3β in the central nervous system prevents zDisc1 loss of function defects. A) Experimental design. B) Effects of DN-GSK3β in the CNS. a–b) F0 Tg(miR124:IresGFP) + CMO (88% normal, n=31). c–d) F0 Tg(miR124:DN-GSK3β-IresGFP) + CMO (70% normal, n=30). e–f) F0 Tg(miR124:IresGFP) + Disc1MO (0% normal, n=32). g–h) F0 Tg(miR124:DN-GSK3β-IresGFP) + Disc1MO (82% normal, n=32). a, c, e, g) Lateral view of whole embryo. a′, c′, e′, g′) Lateral view of muscle segments. b, d, f, h) Dorsal view of head after ventricle injection. C) Effects of DN-GSK3β in CNS on hindbrain axons. Embryos assayed at 36 hpf in 2 independent experiments, and immunostained for acetylated tubulin. a, b) F0 Tg(miR124:IresGFP) + CMO (100% normal, n=10). c, d) F0 Tg(miR124:DN-GSK3β-IresGFP) + CMO (100% normal, n=10). e, f) F0 Tg(miR124:IresGFP) + Disc1MO (0% normal, n=10). g, h) F0 Tg(miR124:DN-GSK3β-IresGFP) + Disc1MO (90% normal, n=10). a, c, e, g) Lateral view of head neurons. b, d, f, h) Dorsal view of hindbrain neurons. D) Effects of DN-GSK3β in CNS on muscle segments. Embryos immunostained for phalloidin at 30 hpf in 2 independent experiments. a) F0 Tg(miR124:IresGFP) + CMO (100% normal, n=10). b) F0 Tg(miR124:DN-GSK3β-IresGFP) + CMO (100% normal, n=10). c) F0 Tg(miR124:IresGFP) + Disc1MO (0% normal, n=10). d) F0 Tg(miR124:DN-GSK3β-IresGFP) + Disc1MO (80% normal, n=10). Lateral view of somites, anterior to the left. Dotted lines: muscle segment shape. E–H) Expression of DN-GSK3β in mesendoderm prevents muscle segment defects after zDisc1 loss of function. E) Experimental design. F) Effects of DN-GSK3β in the mesendoderm. a–b) F0 Tg(Ntl:IresGFP) + CMO (85% normal, n=34). c–d) F0 Tg(Ntl:DN-GSK3β-IresGFP) + CMO (75% normal, n=32). e–f) F0 Tg(miR124:IresGFP) + Disc1MO (0% normal, n=30). g–h) F0 Tg(miR124:DN-GSK3β-IresGFP) + Disc1MO (84% normal, n=32). a, c, e, g) Lateral view of whole embryo. a′, c′, e′, g′) Lateral view of the muscle segments. b, d, f, h) Dorsal view of head. G) Effects of DN-GSK3β in mesendoderm on muscle segments. Embryos were immunostained for phalloidin at 30 hpf in 2 independent experiments. a) F0 Tg(Ntl:IresGFP) + CMO (100% normal, n=10). b) F0 Tg(Ntl:DN-GSK3β-IresGFP) + CMO (100% normal, n=10). c) F0 Tg(Ntl:IresGFP) + Disc1MO (0% normal, n=12). d) F0 Tg(Ntl:DN-GSK3β-IresGFP) + Disc1MO (83% normal, n=12). Lateral view of muscle segments, anterior to left. Dotted lines: muscle segment shape. H) Effects of DN-GSK3β in mesendoderm on hindbrain axons. Embryos assayed at 36 hpf in 2 independent experiments, and immunostained for acetylated tubulin. a, b) F0 Tg(Ntl:IresGFP) embryos + CMO (100% normal, n=11). c, d) F0 Tg(Ntl:DN-GSK3β-IresGFP) + CMO (100% normal, n=9). e, f) F0 Tg(Ntl:IresGFP) + Disc1MO (0% normal, n=11). (g, h) F0 Tg(Ntl:DN-GSK3β-IresGFP) + Disc1MO (2% normal, n=12). a, c, e, g) Lateral view of head neurons. b, d, f, h) Dorsal view of hindbrain neurons. I) Schematic of Disc1interaction with the β-catenin-mediated and noncanonical Wnt pathways. ac, anterior commissure; poc, postoptic commissure; sot, supraoptic tract; tpc, tract of posterior commissure; r, rhombomeres.

DN-GSK3β expression was also driven in the germ ring and notochord by the no-tail (Ntl) promoter (ref. 25 and Fig. 6E). F0 transient transgenics expressing Ntl:DN-GSK3β injected with the Disc1MO, displayed straight tails and normal muscle segment shape (Fig. 6F, G), suggesting that zDisc1 and GSK3β act together in the mesendoderm to regulate convergence and extension. Expression of DN-GSK3β in the mesendoderm did not rescue axonal defects characteristic of zDisc1 loss of function (Fig. 6H).

Thus, the entire spectrum of defects observed after loss of zDisc1 function is rescued by suppression of GSK3β activity. These include brain and axon defects, dependent on β-catenin, as well as abnormal somite and tail morphology, dependent on Daam1 and Rho, and suggest a dual pathway by which zDisc1 modulates both β-catenin and noncanonical Wnt pathways, during early embryogenesis (Fig. 6I).

DISCUSSION

Although the hDisc1 gene is believed to play a pivotal role in development of schizophrenia and other mental health disorders, the mechanisms underlying this activity remain unclear. This study shows that zDisc1 function is required for brain and body development, acting through β-catenin-mediated Wnt signaling and, for the first time, demonstrates that Disc1 is a positive modulator of the noncanonical Wnt pathway. Conservation of zebrafish, mouse and human Disc1 protein domains, and interchangeability of these proteins, suggests that Disc1 engages similar pathways in teleosts and mammals.

In zebrafish, Disc1 modulates β-catenin-mediated Wnt signaling during the initial stages of brain development, since normal brain development can be restored after loss of zDisc1 function by expression of activated β-catenin and inhibition of GSK3β. These data are consistent with changes in neuronal outgrowth observed in midgestation mouse embryos, which require mDisc1 and β-catenin activity (10), although the mDisc1 phenotype has not been described at earlier times during mouse gestation, comparable to the timepoints examined in this study.

β-Catenin-mediated Wnt signaling is required for posterior development in many animal phyla (42), and we therefore predicted that zDisc1 loss of function would anteriorize the embryo. Loss of diencephalic gene expression during somitogenesis, and some expansion of telencephalic gene expression does occur after loss of Disc1 function, as predicted. However, the phenotype is complex, and very similar to that observed after inhibition of Wnt8 function (43), likely due to multiple targets involved, and the different roles for Wnt signaling at different times of development.

While mDisc1, acting through β-catenin, is necessary for neuronal proliferation in midgestation mouse embryos (10), it is not required in the zebrafish early embryonic neurectoderm. Unlike the mouse, we do find an increase in apoptosis after zDisc1 loss of function, reflecting either species differences, or, possibly differential temporal responsiveness to zDisc1.

Part of the zDisc1 phenotype results from abnormal convergence and extension during gastrulation. These defects are due to modulation of noncanonical Wnt signaling, by the Daam1/Rho branch of this pathway. In contrast, Disc 1 regulates mouse glutamatergic synaptic spine formation through Rac1 (44). Noncanonical Wnt signaling modulates neuronal migration in zebrafish and mammals (45, 46), suggesting that Disc1 may direct normal neural circuit formation.

Since zDisc1 and hDisc1 proteins lacking the GSK3β binding site (10) could rescue neither convergence and extension defects nor brain and neural phenotypes, this site regulates all embryonic aspects of zDisc1 function. Recent data suggest that β-catenin-mediated and noncanonical Wnt signaling pathways share common components. Both Wnt5a, considered a noncanonical Wnt, and Wnt3a, considered a β-catenin-mediated Wnt, compete for the same Fzd receptor (40). In both embryos and tissue culture, the LRP5/6 coreceptor is generally used for the β-catenin-mediated Wnt pathway, while in tissue culture, Ror1 and Ror2 coreceptors are used for the noncanonical pathway. In both cases, membrane-bound GSK3β phosphorylates and activates the coreceptors (40). Subsequent inhibition of cytosolic Gskβ leads to dephosphorylation of β-catenin and activation of Wnt/β-catenin signaling (47). Our data suggest that in the noncanonical pathway, GSK3β inhibits downstream signaling, and we hypothesize that this effect is also mediated by cytosolic GSK3β.

Two previous studies analyzed aspects of zDisc1 function, different to those addressed here. Wood et al. (48) demonstrated that zDisc1 is involved in development of oligodendrocytes, analyzed at larval stages, however, it was not clear when zDisc1 affected this lineage or what signaling pathways are involved. zDisc1 has been associated with neural crest development (49); however, the antisense oligos used to alter gene function would have led to expression of truncated zDisc1 proteins, rather than loss of protein expression, as we achieved in this study. We have shown that zDisc1 protein truncation leads to a dominant interfering phenotype that is milder, and distinct from loss of function, and does not act through GSK3β modulation (data not shown and ref. 50), and a similar effect is observed in mice (51).

Disc1 interfaces with many signaling pathways, and the networks regulated by Disc1 in different regions of the nervous system, and at different times of development, are not yet clear. Our data newly adds the noncanonical Wnt pathway to the milieu of Disc1 functionalities, which should now be considered as one of the pathways that may be abnormal in neuropsychiatric disorders, which may be affected by Disc1 function.

Supplementary Material

Supplemental Data
supp_25_12_4184__index.html (889B, html)

Acknowledgments

The authors thank the members of the H.S. laboratory for discussion, and Olivier Paugois for expert fish husbandry. The authors are grateful to Ed Scolnick, Stephen Haggarty, Pamela Sklar, and other members of the Stanley Center for input. The authors thank the following colleagues for generous gifts of reagents, as noted in Materials and Methods: Anand Chandrasekhar (University of Missouri, Columbia, MO, USA), Jeremy Green (Dana-Farber Cancer Institute, Boston, MA, USA), Raymond Habas (Temple University, Philadelphia, PA, USA), Stephen Haggarty (Center for Human Genetic Research, Massachusetts General Hospital, Boston, MA, USA), Xi He (Harvard Medical School, Cambridge, MA, USA), David Kimelman (University of Washington, Seattle, WA, USA), Jim Smith (Gurdon Institute, Cambridge, UK), Randall Moon (University of Washington School of Medicine, Seattle, WA, USA), Steven Wilson (University College London, London, UK), Rudi Winklbauer (University of Toronto, Toronto, ON, Canada), and Karen Symes (Boston University, Boston, MA, USA).

This work was supported by the Stanley Medical Research Institute, via a grant to the Stanley Center for Psychiatric Research at the Broad Institute. The Disc1fh291 and Disc1fh292 mutants were identified with the support of U.S. National Institutes of Health (NIH) grant R01 HG002995 to C.B.M. This work was partially supported by NIH grant RO1 MH091115 to L.-H.T.

Footnotes

This article includes supplemental data. Please visit http://www.fasebj.org to obtain this information.

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