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Deltex1 is inhibited by the Notch-Hairy/E(Spl) signaling pathway and induces neuronal and glial differentiation.

Cheng YC, Huang YC, Yeh TH, Shih HY, Lin CY, Lin SJ, Chiu CC, Huang CW, Jiang YJ - Neural Dev (2015)

Bottom Line: Examination of the expression of her2 and her8a in embryos with altered Dtx1 expression showed that Dxt1-induced neuronal differentiation did not require a regulatory effect on the Notch-Hairy/E(Spl) pathway.Our results demonstrated that Dtx1 is regulated by Notch-Hairy/E(Spl) signaling and is a major factor specifically regulating neural differentiation.Thus, our results provide new insights into the mediation of neural development by the Notch signaling pathway.

View Article: PubMed Central - PubMed

Affiliation: Graduate Institute of Biomedical Sciences, College of Medicine, Chang Gung University, 259 Wen-Hwa 1 Road, Taoyuan, 33383, Taiwan. yccheng@mail.cgu.edu.tw.

ABSTRACT

Background: Notch signaling has been conserved throughout evolution and plays a fundamental role in various neural developmental processes and the pathogenesis of several human cancers and genetic disorders. However, how Notch signaling regulates various cellular processes remains unclear. Although Deltex proteins have been identified as cytoplasmic downstream elements of the Notch signaling pathway, few studies have been reported on their physiological role.

Results: We isolated zebrafish deltex1 (dtx1) and showed that this gene is primarily transcribed in the developing nervous system, and its spatiotemporal expression pattern suggests a role in neural differentiation. The transcription of dtx1 was suppressed by the direct binding of the Notch downstream transcription factors Her2 and Her8a. Overexpressing the complete coding sequence of Dtx1 was necessary for inducing neuronal and glial differentiation. By contrast, disrupting Dtx1 expression by using a Dtx1 construct without the RING finger domain reduced neuronal and glial differentiation. This effect was phenocopied by the knockdown of endogenous Dtx1 expression by using morpholinos, demonstrating the essential function of the RING finger domain and confirming the knockdown specificity. Cell proliferation and apoptosis were unaltered in Dtx1-overexpressed and -deficient zebrafish embryos. Examination of the expression of her2 and her8a in embryos with altered Dtx1 expression showed that Dxt1-induced neuronal differentiation did not require a regulatory effect on the Notch-Hairy/E(Spl) pathway. However, both Dtx1 and Notch activation induced glial differentiation, and Dtx1 and Notch activation negatively inhibited each other in a reciprocal manner, which achieves a proper balance for the expression of Dtx1 and Notch to facilitate glial differentiation. We further confirmed that the Dtx1-Notch-Hairy/E(Spl) cascade was sufficient to induce neuronal and glial differentiation by concomitant injection of an active form of Notch with dtx1, which rescued the neuronogenic and gliogenic defects caused by the activation of Notch signaling.

Conclusions: Our results demonstrated that Dtx1 is regulated by Notch-Hairy/E(Spl) signaling and is a major factor specifically regulating neural differentiation. Thus, our results provide new insights into the mediation of neural development by the Notch signaling pathway.

No MeSH data available.


Related in: MedlinePlus

Dtx1 regulates Notch signaling in a temporal manner. aIn situ hybridization and qPCR results show that expression of her2 and her8a was not affected by the alteration of dtx1 expression at 10 hpf. In contrast, the expression of her2 and her8a was downregulated by dtx1full cRNA and upregulated by dtx1ΔIII or dtx1 morpholino after 16 hpf. The embryo stages are shown in the bottom left corner of each panel. b Injection of NICD inhibits neurog1 expression and induces slc1a3a and mag expression. This effect of NICD could be abrogated by co-injection with dtx1full cRNA at all stage examined. The result of in situ hybridization was confirmed by qPCR quantification showing in a and b c Western blot analysis with anti-Myc antibody revealed that injection of dtx1full cRNA caused ubiquitination of NICD-myc, indicated by multiple bands in samples at 16 hpf (bracket). Asterisks indicating non-specific bands
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Fig7: Dtx1 regulates Notch signaling in a temporal manner. aIn situ hybridization and qPCR results show that expression of her2 and her8a was not affected by the alteration of dtx1 expression at 10 hpf. In contrast, the expression of her2 and her8a was downregulated by dtx1full cRNA and upregulated by dtx1ΔIII or dtx1 morpholino after 16 hpf. The embryo stages are shown in the bottom left corner of each panel. b Injection of NICD inhibits neurog1 expression and induces slc1a3a and mag expression. This effect of NICD could be abrogated by co-injection with dtx1full cRNA at all stage examined. The result of in situ hybridization was confirmed by qPCR quantification showing in a and b c Western blot analysis with anti-Myc antibody revealed that injection of dtx1full cRNA caused ubiquitination of NICD-myc, indicated by multiple bands in samples at 16 hpf (bracket). Asterisks indicating non-specific bands

Mentions: Dtx1 has been identified as a positive and negative mediator for Notch signaling [9, 12, 37]. To examine whether Dtx1 reciprocally regulates Notch signaling in neuronal differentiation, we examined the expression of her2 and her8a in embryos with dtx1 gain- and loss-of function at different developmental stages. Overexpressing dtx1full cRNA did not alter the expression of her2 and her8a at 10 hpf, and injection of dtx1ΔIII or morpholino also had no effect on her2 and her8a expression at 10 hpf (Fig. 7a), indicating that Dtx1 does not reciprocally regulate Notch and HES/Her expression at this stage. Because Dtx1 is regulated by Her2 and Her8a and induces neuronal differentiation at this stage, this result also suggested that Dtx1-mediated regulation of neuronal differentiation does not require a reciprocal regulatory effect on Notch. On the contrary, overexpression of dtx1full inhibited her2 and her8a expression from 16 hpf, and injection of dtx1ΔIII or dtx1 morpholino resulted in upregulation of her2 and her8a after 16 hpf (Fig. 7a), suggesting that Dtx1 inhibits Notch signaling and confirmed that the RING domain is essential for inhibiting Notch signaling in these later stages.Fig. 7


Deltex1 is inhibited by the Notch-Hairy/E(Spl) signaling pathway and induces neuronal and glial differentiation.

Cheng YC, Huang YC, Yeh TH, Shih HY, Lin CY, Lin SJ, Chiu CC, Huang CW, Jiang YJ - Neural Dev (2015)

Dtx1 regulates Notch signaling in a temporal manner. aIn situ hybridization and qPCR results show that expression of her2 and her8a was not affected by the alteration of dtx1 expression at 10 hpf. In contrast, the expression of her2 and her8a was downregulated by dtx1full cRNA and upregulated by dtx1ΔIII or dtx1 morpholino after 16 hpf. The embryo stages are shown in the bottom left corner of each panel. b Injection of NICD inhibits neurog1 expression and induces slc1a3a and mag expression. This effect of NICD could be abrogated by co-injection with dtx1full cRNA at all stage examined. The result of in situ hybridization was confirmed by qPCR quantification showing in a and b c Western blot analysis with anti-Myc antibody revealed that injection of dtx1full cRNA caused ubiquitination of NICD-myc, indicated by multiple bands in samples at 16 hpf (bracket). Asterisks indicating non-specific bands
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4696291&req=5

Fig7: Dtx1 regulates Notch signaling in a temporal manner. aIn situ hybridization and qPCR results show that expression of her2 and her8a was not affected by the alteration of dtx1 expression at 10 hpf. In contrast, the expression of her2 and her8a was downregulated by dtx1full cRNA and upregulated by dtx1ΔIII or dtx1 morpholino after 16 hpf. The embryo stages are shown in the bottom left corner of each panel. b Injection of NICD inhibits neurog1 expression and induces slc1a3a and mag expression. This effect of NICD could be abrogated by co-injection with dtx1full cRNA at all stage examined. The result of in situ hybridization was confirmed by qPCR quantification showing in a and b c Western blot analysis with anti-Myc antibody revealed that injection of dtx1full cRNA caused ubiquitination of NICD-myc, indicated by multiple bands in samples at 16 hpf (bracket). Asterisks indicating non-specific bands
Mentions: Dtx1 has been identified as a positive and negative mediator for Notch signaling [9, 12, 37]. To examine whether Dtx1 reciprocally regulates Notch signaling in neuronal differentiation, we examined the expression of her2 and her8a in embryos with dtx1 gain- and loss-of function at different developmental stages. Overexpressing dtx1full cRNA did not alter the expression of her2 and her8a at 10 hpf, and injection of dtx1ΔIII or morpholino also had no effect on her2 and her8a expression at 10 hpf (Fig. 7a), indicating that Dtx1 does not reciprocally regulate Notch and HES/Her expression at this stage. Because Dtx1 is regulated by Her2 and Her8a and induces neuronal differentiation at this stage, this result also suggested that Dtx1-mediated regulation of neuronal differentiation does not require a reciprocal regulatory effect on Notch. On the contrary, overexpression of dtx1full inhibited her2 and her8a expression from 16 hpf, and injection of dtx1ΔIII or dtx1 morpholino resulted in upregulation of her2 and her8a after 16 hpf (Fig. 7a), suggesting that Dtx1 inhibits Notch signaling and confirmed that the RING domain is essential for inhibiting Notch signaling in these later stages.Fig. 7

Bottom Line: Examination of the expression of her2 and her8a in embryos with altered Dtx1 expression showed that Dxt1-induced neuronal differentiation did not require a regulatory effect on the Notch-Hairy/E(Spl) pathway.Our results demonstrated that Dtx1 is regulated by Notch-Hairy/E(Spl) signaling and is a major factor specifically regulating neural differentiation.Thus, our results provide new insights into the mediation of neural development by the Notch signaling pathway.

View Article: PubMed Central - PubMed

Affiliation: Graduate Institute of Biomedical Sciences, College of Medicine, Chang Gung University, 259 Wen-Hwa 1 Road, Taoyuan, 33383, Taiwan. yccheng@mail.cgu.edu.tw.

ABSTRACT

Background: Notch signaling has been conserved throughout evolution and plays a fundamental role in various neural developmental processes and the pathogenesis of several human cancers and genetic disorders. However, how Notch signaling regulates various cellular processes remains unclear. Although Deltex proteins have been identified as cytoplasmic downstream elements of the Notch signaling pathway, few studies have been reported on their physiological role.

Results: We isolated zebrafish deltex1 (dtx1) and showed that this gene is primarily transcribed in the developing nervous system, and its spatiotemporal expression pattern suggests a role in neural differentiation. The transcription of dtx1 was suppressed by the direct binding of the Notch downstream transcription factors Her2 and Her8a. Overexpressing the complete coding sequence of Dtx1 was necessary for inducing neuronal and glial differentiation. By contrast, disrupting Dtx1 expression by using a Dtx1 construct without the RING finger domain reduced neuronal and glial differentiation. This effect was phenocopied by the knockdown of endogenous Dtx1 expression by using morpholinos, demonstrating the essential function of the RING finger domain and confirming the knockdown specificity. Cell proliferation and apoptosis were unaltered in Dtx1-overexpressed and -deficient zebrafish embryos. Examination of the expression of her2 and her8a in embryos with altered Dtx1 expression showed that Dxt1-induced neuronal differentiation did not require a regulatory effect on the Notch-Hairy/E(Spl) pathway. However, both Dtx1 and Notch activation induced glial differentiation, and Dtx1 and Notch activation negatively inhibited each other in a reciprocal manner, which achieves a proper balance for the expression of Dtx1 and Notch to facilitate glial differentiation. We further confirmed that the Dtx1-Notch-Hairy/E(Spl) cascade was sufficient to induce neuronal and glial differentiation by concomitant injection of an active form of Notch with dtx1, which rescued the neuronogenic and gliogenic defects caused by the activation of Notch signaling.

Conclusions: Our results demonstrated that Dtx1 is regulated by Notch-Hairy/E(Spl) signaling and is a major factor specifically regulating neural differentiation. Thus, our results provide new insights into the mediation of neural development by the Notch signaling pathway.

No MeSH data available.


Related in: MedlinePlus