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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 induces the expression of markers for glial precursors and mature glial cells. a Expression of slc1a3a, mag, and gfap was induced in embryos that overexpressed dtx1full. An injection with dtx1ΔIII or dtx1 morpholino downregulated slc1a3a, mag, and gfap expression, and this effect could be rescued by a concomitant injection of dtx1full cRNA. The embryo stages are shown in the bottom left corner of each panel. b qPCR analysis and cell count confirmed the results obtained through in situ hybridization shown in A. *, P < 0.05; **, P < 0.01; ***, P < 0.001
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Fig6: Dtx1 induces the expression of markers for glial precursors and mature glial cells. a Expression of slc1a3a, mag, and gfap was induced in embryos that overexpressed dtx1full. An injection with dtx1ΔIII or dtx1 morpholino downregulated slc1a3a, mag, and gfap expression, and this effect could be rescued by a concomitant injection of dtx1full cRNA. The embryo stages are shown in the bottom left corner of each panel. b qPCR analysis and cell count confirmed the results obtained through in situ hybridization shown in A. *, P < 0.05; **, P < 0.01; ***, P < 0.001

Mentions: Gliogenesis proceeds after neurogenesis. Recent in vitro studies have shown that murine DTX1 is capable of promoting gliogenic specification in neural crest cell cultures [31, 32] and is essential for oligodendrocyte development in primary glial cultures [12, 33]. We accordingly analyzed the role of zebrafish Dtx1 during gliogenesis. Overexpression of dtx1full cRNA upregulates the expression of the early glial marker slc1a3a [Glast in mammals, for glial progenitors [23, 34, 35]], increasing the expression 2.5-fold, according to qPCR analysis (Fig. 6). This suggests that Dtx1 is effective in inducing gliogenesis. By contrast, slc1a3a was downregulated in embryos injected with dtx1ΔIII or dtx1 morpholinos (Fig. 6), and this finding was also confirmed by qPCR analysis showing 2-fold and 1.9-fold decreases (Fig. 6). The effect of dtx1 morpholinos could be restored by coinjection with dtx1full cRNA (Fig. 6). To test whether the effects of slc1a3a were caused by irregular glial differentiation, we evaluated the expression of the mature glial cell marker myelin-associated glycoprotein (mag, for myelinated glial cells) and glial fibrillary acidic protein (gfap, for radial glia and astrocytes) [36]. Overexpression of dtx1 caused increased mag expression (2.0-fold increases according to qPCR results; Fig. 6), whereas injecting dtx1ΔIII or the dtx1 morpholinos caused decreased mag expression (Fig. 6), as confirmed using qPCR (Fig. 6). Overall, these results show that Dtx1 is necessary for inducing glial differentiation.Fig. 6


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 induces the expression of markers for glial precursors and mature glial cells. a Expression of slc1a3a, mag, and gfap was induced in embryos that overexpressed dtx1full. An injection with dtx1ΔIII or dtx1 morpholino downregulated slc1a3a, mag, and gfap expression, and this effect could be rescued by a concomitant injection of dtx1full cRNA. The embryo stages are shown in the bottom left corner of each panel. b qPCR analysis and cell count confirmed the results obtained through in situ hybridization shown in A. *, 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

Fig6: Dtx1 induces the expression of markers for glial precursors and mature glial cells. a Expression of slc1a3a, mag, and gfap was induced in embryos that overexpressed dtx1full. An injection with dtx1ΔIII or dtx1 morpholino downregulated slc1a3a, mag, and gfap expression, and this effect could be rescued by a concomitant injection of dtx1full cRNA. The embryo stages are shown in the bottom left corner of each panel. b qPCR analysis and cell count confirmed the results obtained through in situ hybridization shown in A. *, P < 0.05; **, P < 0.01; ***, P < 0.001
Mentions: Gliogenesis proceeds after neurogenesis. Recent in vitro studies have shown that murine DTX1 is capable of promoting gliogenic specification in neural crest cell cultures [31, 32] and is essential for oligodendrocyte development in primary glial cultures [12, 33]. We accordingly analyzed the role of zebrafish Dtx1 during gliogenesis. Overexpression of dtx1full cRNA upregulates the expression of the early glial marker slc1a3a [Glast in mammals, for glial progenitors [23, 34, 35]], increasing the expression 2.5-fold, according to qPCR analysis (Fig. 6). This suggests that Dtx1 is effective in inducing gliogenesis. By contrast, slc1a3a was downregulated in embryos injected with dtx1ΔIII or dtx1 morpholinos (Fig. 6), and this finding was also confirmed by qPCR analysis showing 2-fold and 1.9-fold decreases (Fig. 6). The effect of dtx1 morpholinos could be restored by coinjection with dtx1full cRNA (Fig. 6). To test whether the effects of slc1a3a were caused by irregular glial differentiation, we evaluated the expression of the mature glial cell marker myelin-associated glycoprotein (mag, for myelinated glial cells) and glial fibrillary acidic protein (gfap, for radial glia and astrocytes) [36]. Overexpression of dtx1 caused increased mag expression (2.0-fold increases according to qPCR results; Fig. 6), whereas injecting dtx1ΔIII or the dtx1 morpholinos caused decreased mag expression (Fig. 6), as confirmed using qPCR (Fig. 6). Overall, these results show that Dtx1 is necessary for inducing glial differentiation.Fig. 6

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