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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 expression is upregulated in Notch-deficient embryos. a The expression of dtx1 was analyzed in mibta52b homozygous mutants and wild-type siblings through in situ hybridization. The stages are shown in the bottom left corner. Embryos were produced by crossing the parents with heterozygous mutant genotype. Embryos at 8 hpf, 10 hpf, and 16 hpf stages were mixed genotypes containing heterozygous mibta52b mutation, homozygous mibta52b mutation, and wild-type mib, and they were grouped according to the substantial difference in dtx1 expression. The results indicate that approximately 75 % of wild-type and heterozygous mibta52b siblings had unaltered dtx1 expression, whereas approximately 25 % of homozygous mibta52b mutants had increased dtx1 expression. Embryos at 24 hpf and 48 hpf were grouped according the morphological defects that only appeared in mibta52b homozygous mutants. c. DAPT treatment was performed at different time points, and the embryos were harvested at different stages as indicated; the results show that DAPT treatment caused upregulation of dtx1 expression. b and d The results of in situ hybridization in a and c were quantitatively confirmed using qPCR analysis, respectively. *, P < 0.05
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Fig2: dtx1 expression is upregulated in Notch-deficient embryos. a The expression of dtx1 was analyzed in mibta52b homozygous mutants and wild-type siblings through in situ hybridization. The stages are shown in the bottom left corner. Embryos were produced by crossing the parents with heterozygous mutant genotype. Embryos at 8 hpf, 10 hpf, and 16 hpf stages were mixed genotypes containing heterozygous mibta52b mutation, homozygous mibta52b mutation, and wild-type mib, and they were grouped according to the substantial difference in dtx1 expression. The results indicate that approximately 75 % of wild-type and heterozygous mibta52b siblings had unaltered dtx1 expression, whereas approximately 25 % of homozygous mibta52b mutants had increased dtx1 expression. Embryos at 24 hpf and 48 hpf were grouped according the morphological defects that only appeared in mibta52b homozygous mutants. c. DAPT treatment was performed at different time points, and the embryos were harvested at different stages as indicated; the results show that DAPT treatment caused upregulation of dtx1 expression. b and d The results of in situ hybridization in a and c were quantitatively confirmed using qPCR analysis, respectively. *, P < 0.05

Mentions: A previous study showed that the constitutively active form of Notch upregulated Dtx1 expression in a mouse thymoma cell line [18]. To examine the response of dtx1 to Notch signaling in the developing nervous system, we analyzed its expression in mind bomb mutant embryos (mibta52b), which have a deficient Notch pathway [19] because of a missense mutation in the most C-terminal RING domain that bears ubiquitin ligase activity required for Delta ligand internalization and Notch activation [20]. Compared with wild-type embryos, dtx1 expression in the developing nervous system was considerably upregulated in mibta52b embryos (Fig. 2), suggesting that Delta–Notch signaling activation is essential for inhibiting dtx1 transcription. This result was further confirmed by the γ-secretase inhibitor, N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT), which has been shown to efficiently block Notch signaling at different time-points [21]. Embryos were treated with DAPT at the shield stage (6 hpf) and harvested at 75 % epiboly (8 hpf), at 8 hpf and harvested at the bud stage (10 hpf), 24 hpf, or 48 hpf. The embryos that were treated with DAPT at different time-points exhibited an upregulation of dtx1 expression to a level similar to that observed in the mibta52b mutant (Fig. 2b,d), which demonstrated that Notch signaling was essential for the inhibition of dtx1 expression. The converse regulatory mechanism between the thymoma cell line and developing nervous system suggested that the regulation of Dtx1/dtx1 expression by Notch signaling is highly dependent on the cell type.Fig. 2


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 expression is upregulated in Notch-deficient embryos. a The expression of dtx1 was analyzed in mibta52b homozygous mutants and wild-type siblings through in situ hybridization. The stages are shown in the bottom left corner. Embryos were produced by crossing the parents with heterozygous mutant genotype. Embryos at 8 hpf, 10 hpf, and 16 hpf stages were mixed genotypes containing heterozygous mibta52b mutation, homozygous mibta52b mutation, and wild-type mib, and they were grouped according to the substantial difference in dtx1 expression. The results indicate that approximately 75 % of wild-type and heterozygous mibta52b siblings had unaltered dtx1 expression, whereas approximately 25 % of homozygous mibta52b mutants had increased dtx1 expression. Embryos at 24 hpf and 48 hpf were grouped according the morphological defects that only appeared in mibta52b homozygous mutants. c. DAPT treatment was performed at different time points, and the embryos were harvested at different stages as indicated; the results show that DAPT treatment caused upregulation of dtx1 expression. b and d The results of in situ hybridization in a and c were quantitatively confirmed using qPCR analysis, respectively. *, P < 0.05
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Fig2: dtx1 expression is upregulated in Notch-deficient embryos. a The expression of dtx1 was analyzed in mibta52b homozygous mutants and wild-type siblings through in situ hybridization. The stages are shown in the bottom left corner. Embryos were produced by crossing the parents with heterozygous mutant genotype. Embryos at 8 hpf, 10 hpf, and 16 hpf stages were mixed genotypes containing heterozygous mibta52b mutation, homozygous mibta52b mutation, and wild-type mib, and they were grouped according to the substantial difference in dtx1 expression. The results indicate that approximately 75 % of wild-type and heterozygous mibta52b siblings had unaltered dtx1 expression, whereas approximately 25 % of homozygous mibta52b mutants had increased dtx1 expression. Embryos at 24 hpf and 48 hpf were grouped according the morphological defects that only appeared in mibta52b homozygous mutants. c. DAPT treatment was performed at different time points, and the embryos were harvested at different stages as indicated; the results show that DAPT treatment caused upregulation of dtx1 expression. b and d The results of in situ hybridization in a and c were quantitatively confirmed using qPCR analysis, respectively. *, P < 0.05
Mentions: A previous study showed that the constitutively active form of Notch upregulated Dtx1 expression in a mouse thymoma cell line [18]. To examine the response of dtx1 to Notch signaling in the developing nervous system, we analyzed its expression in mind bomb mutant embryos (mibta52b), which have a deficient Notch pathway [19] because of a missense mutation in the most C-terminal RING domain that bears ubiquitin ligase activity required for Delta ligand internalization and Notch activation [20]. Compared with wild-type embryos, dtx1 expression in the developing nervous system was considerably upregulated in mibta52b embryos (Fig. 2), suggesting that Delta–Notch signaling activation is essential for inhibiting dtx1 transcription. This result was further confirmed by the γ-secretase inhibitor, N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT), which has been shown to efficiently block Notch signaling at different time-points [21]. Embryos were treated with DAPT at the shield stage (6 hpf) and harvested at 75 % epiboly (8 hpf), at 8 hpf and harvested at the bud stage (10 hpf), 24 hpf, or 48 hpf. The embryos that were treated with DAPT at different time-points exhibited an upregulation of dtx1 expression to a level similar to that observed in the mibta52b mutant (Fig. 2b,d), which demonstrated that Notch signaling was essential for the inhibition of dtx1 expression. The converse regulatory mechanism between the thymoma cell line and developing nervous system suggested that the regulation of Dtx1/dtx1 expression by Notch signaling is highly dependent on the cell type.Fig. 2

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