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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

Disruption of Dtx1 expression by using dtx1ΔIII or dtx1 morpholino reduces neuronal differentiation. a The injection of dtx1ΔIII or dtx1 morpholino caused an identical phenotype, which downregulated neurog1 (a) and HuC/D expression (b) The phenotypes caused by the morpholino injection could be rescued by a concomitant injection of dtx1full cRNA. The embryo stages are shown in the bottom left corner of each panel. c qPCR analysis confirmed the results obtained through in situ hybridization in a d Counting the HuC/D-positive cells confirmed the results obtained in b *, P < 0.05; **, P < 0.01; ***, P < 0.001
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Fig5: Disruption of Dtx1 expression by using dtx1ΔIII or dtx1 morpholino reduces neuronal differentiation. a The injection of dtx1ΔIII or dtx1 morpholino caused an identical phenotype, which downregulated neurog1 (a) and HuC/D expression (b) The phenotypes caused by the morpholino injection could be rescued by a concomitant injection of dtx1full cRNA. The embryo stages are shown in the bottom left corner of each panel. c qPCR analysis confirmed the results obtained through in situ hybridization in a d Counting the HuC/D-positive cells confirmed the results obtained in b *, P < 0.05; **, P < 0.01; ***, P < 0.001

Mentions: Studies have shown that the RING finger motif is essential for Deltex function during Drosophila wing formation and rat oligodendrocyte development [11, 30]. Accordingly, we created a deletion zebrafish dtx1 cDNA construct lacking the RING domain (dtx1ΔIII) to gain insights into the structural requirements for Dtx1 function. We found that neurogenin1 was downregulated in the dtx1ΔIII-injected embryos at the bud stage and 24-hpf embryos (Fig. 5). HuC/D expression analysis also revealed downregulation in dtx1ΔIII-injected embryos (Fig. 5). This result suggested that dtx1ΔIII was effective in disrupting neuronal differentiation.Fig. 5


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)

Disruption of Dtx1 expression by using dtx1ΔIII or dtx1 morpholino reduces neuronal differentiation. a The injection of dtx1ΔIII or dtx1 morpholino caused an identical phenotype, which downregulated neurog1 (a) and HuC/D expression (b) The phenotypes caused by the morpholino injection could be rescued by a concomitant injection of dtx1full cRNA. The embryo stages are shown in the bottom left corner of each panel. c qPCR analysis confirmed the results obtained through in situ hybridization in a d Counting the HuC/D-positive cells confirmed the results obtained in b *, P < 0.05; **, P < 0.01; ***, P < 0.001
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig5: Disruption of Dtx1 expression by using dtx1ΔIII or dtx1 morpholino reduces neuronal differentiation. a The injection of dtx1ΔIII or dtx1 morpholino caused an identical phenotype, which downregulated neurog1 (a) and HuC/D expression (b) The phenotypes caused by the morpholino injection could be rescued by a concomitant injection of dtx1full cRNA. The embryo stages are shown in the bottom left corner of each panel. c qPCR analysis confirmed the results obtained through in situ hybridization in a d Counting the HuC/D-positive cells confirmed the results obtained in b *, P < 0.05; **, P < 0.01; ***, P < 0.001
Mentions: Studies have shown that the RING finger motif is essential for Deltex function during Drosophila wing formation and rat oligodendrocyte development [11, 30]. Accordingly, we created a deletion zebrafish dtx1 cDNA construct lacking the RING domain (dtx1ΔIII) to gain insights into the structural requirements for Dtx1 function. We found that neurogenin1 was downregulated in the dtx1ΔIII-injected embryos at the bud stage and 24-hpf embryos (Fig. 5). HuC/D expression analysis also revealed downregulation in dtx1ΔIII-injected embryos (Fig. 5). This result suggested that dtx1ΔIII was effective in disrupting neuronal differentiation.Fig. 5

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