Front Mol Neurosci
January 1, 2018;
Ketamine Modulates Zic5 Expression via the Notch Signaling Pathway in Neural Crest Induction.
Ketamine is a potent dissociative anesthetic and the most commonly used illicit drug. Many addicts are women at childbearing age. Although ketamine has been extensively studied as a clinical anesthetic, its effects on embryonic development are poorly understood. Here, we applied the Xenopus model to study the effects of ketamine on development. We found that exposure to ketamine from pre-gastrulation (stage 7) to early neural plate (stage 13.5) resulted in disruption of neural crest (NC) derivatives. Ketamine exposure did not affect mesoderm
development as indicated by the normal expression of Chordin
, and Fgf8
. However, ketamine treatment significantly inhibited Zic5
expression at early neural plate stage. Overexpression of Zic5
rescued ketamine-induced Slug
inhibition, suggesting the blockage of NC induction was mediated by Zic5
. Furthermore, we found Notch
signaling was altered by ketamine. Ketamine inhibited the expression of Notch
targeted genes including Hes5
.2b, and ESR1
and ketamine-treated embryos exhibited Notch
phenotypes. A 15 bp core binding element upstream of Zic5
was induced by Notch
signaling and caused transcriptional activation. These results demonstrated that Zic5
works as a downstream target gene of Notch
signaling in Xenopus NC induction. Our study provides a novel teratogenic mechanism whereby ketamine disrupts NC induction via targeting a Notch
Front Mol Neurosci
Notch signaling pathway
Disease Ontology terms:
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FIGURE 1. Ketamine exposure results in neurocristopathies. Upon ketamine exposure from stage 7 to stage 13.5. (A) Embryos exhibit shortened axis, faded trunk pigment (red arrowhead), and deficient eyes (blue arrowhead) at tailbud stage and severity of phenotypes are in a dose-dependent manner. (B) Sections of eyes in ketamine-treated embryos revealed a more rounding shape pigment, disorganized arrangement of cells (DAPI staining), reduced expression of β-tubulin III compared with the control group. Ketamine exposure also causes embryos to develop a mega-colon like phenotype (C), and cranial cartilage atrophy (D). GCL, ganglionic cell layer; INL, inner nuclear layer; ONL, outer nuclear layer; L, lens; Mk, Meckel’s cartilage; ch, ceratohyal cartilage; bh, basihyal cartilage; ba, branchial cartilage. Numbers in (A,D) indicate ketamine exposure concentration (mM) to embryos. Scale bar: (A–C) 100 μm, (D) 1 mm.
FIGURE 2. Ketamine blocks NC induction independent of mesoderm genesis. (A) The expression of the NC marker gene Slug was inhibited by ketamine in a dose-dependent manner. The numbers of embryos showing similar changes in gene expression and total embryos in each concentration group are indicated. (B) Early mesoderm development is not affected upon ketamine exposure. There were no significant differences in the expression pattern of mesoderm marker genes chordin, Xbra, Wnt8, and Fgf8 between control and ketamine-treated group prior to NC induction. (C) Quantitative analysis of gene expression patterns between control and ketamine group. Scale bar: 100 μm.
FIGURE 3. Ketamine targets Zic5 in NC induction. (A) In animal caps, Wnt8 (500 pg) and tBR (500 pg) induce expression of NC markers (lanes 1, 2). Upon ketamine exposure, the expression of Zic5 and Slug were inhibited (lanes 2, 3). The induced expression of Zic5 and Slug were reduced by ketamine treatment in animal cap. Gene expressions were normalized with H4. Data are shown as folds over the control animal caps. 15 animal caps were isolated for each group for single time experiments. The number represents Mean ± SEM, N = 3, ∗P < 0.05 by ANOVA. (B) In whole embryos, either ketamine exposure or injection (0.8 nmol/embryo) blocked Zic5 and Slug expression at stage 13 (when the NC is being induced) but not stage16 (at the onset of NC migration). Ketamine treatment did not affect most of the border genes from stage 12 to stage 16, but significantly blocked Zic5 and NC gene Slug at stage 13. For a single time experiment, 10 embryos were collected for each group for RT-PCR. Data are shown as folds over the control embryos. The number represents Mean ± SEM. N = 3, ∗P < 0.05 by ANOVA. (C) In situ hybridization suggests genes involved in NC induction, including Fgf8, Wnt8, Msx1, Pax3, and Zic1 were not affected by ketamine exposure. (D) Ketamine inhibited Zic5 expression comparing with MBS cultured control group. (E) The inhibition of Slug expression by ketamine was rescued by injection of Zic5 mRNA (500 pg) in both sides or unilaterally. Arrowhead indicate the unilaterally injected side. (F) Quantitative analysis of in situ hybridization results, left: MBS control group, right: ketamine exposure group. ∗P < 0.05 with t-test. Con, control; Ket, ketamine; Ket Inj, ketamine injection; St., stage; Zic5-Bi, zic5 bilateral injection; Zic5-Uni, zic5 unilateral injection. Scale bar: 100 μm.
FIGURE 4. Ketamine inhibits Zic5 through the Notch signaling pathway. (A) During NC induction, ketamine exposure down-regulated expression of Notch targeted genes including Hes5.2a, Hes5.2b, and ESR1. The number represents Mean ± SEM, N = 3, ∗P < 0.05 by Student’s t-test. (B) In the late neurula, MyoD transcription in early somite primordium became a little bit thin and slightly reduced the signal upon ketamine exposure. At tailbud stage, ketamine led to fewer somites and a shortened body axis. The somites are labeled with in situ hybridization of MyoD, and Six1. (C) Ketamine exposure increased the amount of ubiquitinated Notch protein in Jurkat cells. Upper part: ubiquitinated Notch proteins were immuno-precipitated (IP) with Notch-1 antibody, followed by anti-ubiquitin antibody (Ub-Ab) western blot staining. Lower part: Notch-1 loading control. (D) In Jurkat cells, ketamine exposure for 10 h reduced Notch protein level. The number represents Mean SEM, N = 3, P < 0.05 by Student’s t-test. (E) During NC induction, inhibiting Notch signaling by microinjection of 1 ng Delta-stu mRNA at 4-cell stage blocked Zic5 expression. Activation of Notch signaling by microinjection of 800 pg NICD mRNA into one dorsal cell at 4-cell stage induced ectopic Zic5 expression. Scale bar: 100 μm.
FIGURE 5. Identification of a novel NRE in Xenopus Zic5. (A) A ∼4 kb region (-4042 to -29) upstream of Zic5 contains a Zic5 promoter. Mutations of two putative canonical CSL binding sites within this 4 kb region did not affect notch’s activation of promoter activity. X’s represent the mutated site. (B) Deletion assay of Zic5 upstream regulatory sequence identified a 15 bp NRE locating between -200 and -186 bp in response to NICD activation. P-values relative to -186/-29 fragment are -200/-29 (P = 0.004); -286/-29 (P = 0.015); -533/-29 (P = 0.013); -1950/-29 (P = 0.004); -4042/-29 (P = 0.014). (C) Diagram of the constructs containing 15 bp CSL sequence and mutations in the promoter constructs. pXZic5-200 represents the wild-type and pXZic5-200M represents the 15 bp CBS mutant plasmid. (D) Mutation of the 15 bp CBS (pXZic5-200M) abolished the activation triggered by NICD in the reporter assay; P = 0.0017. (E) EMSA assay; NICD expression increased protein binding to the 15 bp NRE (see section “Materials and Methods”). The number represents Mean ± SEM, N = 3, ∗P < 0.05 with the post hoc Newman–Keuls test. RLU, relative luminant unit; Nuclear Ext, nuclear extraction; Zic5 UPS, fluorescent-labeling zic5 upstream Notch response element.