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FASEB J. 2011 Dec;25(12):4184-97. doi: 10.1096/fj.11-186239. Epub 2011 Aug 22.

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

De Rienzo G, Bishop JA, Mao Y, Pan L, Ma TP, Moens CB, Tsai LH, Sive H.

Source

Whitehead institute for Biomedical Research, 9 Cambridge Center, Cambridge, Massachusetts 02142, USA.

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.

PMID:: 21859895; [PubMed - indexed for MEDLINE]
PMCID:: PMC3236629

Free PMC Article

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Figure 1.

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 Disc1^fh291 line. b) Schematic of zDisc1 protein. c) Predicted protein truncation in Disc1^fh291 embryos. d) Predicted protein truncation in Disc1 morphants. B) a) Genomic PCR for Disc1^fh291 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) Disc1^fh291 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) Disc1^fh291 mutant. c) Disc1MO (CMO or wild-type: 100% normal, n=14; Disc1^fh291 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).

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

FASEB J. 2011 December;25(12):4184-4197.

Figure 3.

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.

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

FASEB J. 2011 December;25(12):4184-4197.

Figure 2.

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).

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

FASEB J. 2011 December;25(12):4184-4197.

Figure 6.

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.

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

FASEB J. 2011 December;25(12):4184-4197.