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SNX17 regulates Notch pathway and pancreas development through the retromer-dependent recycling of Jag1.

Yin W, Liu D, Liu N, Xu L, Li S, Lin S, Shu X, Pei D - Cell Regen (Lond) (2012)

Bottom Line: SNX17 is a sorting nexin family protein implicated in vesicular trafficking and we find it is specifically required in the ligand-expressing cells for Notch signaling.Mechanistically, SNX17 regulates the protein level of Jag1a on plasma membrane by binding to Jag1a and facilitating the retromer-dependent recycling of the ligand.In zebrafish, inhibition of this SNX17-mediated Notch signaling pathway results in defects in neurogenesis as well as pancreas development.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Regenerative Biology, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, 510530 China.

ABSTRACT

Background: Notch is one of the most important signaling pathways involved in cell fate determination. Activation of the Notch pathway requires the binding of a membrane-bound ligand to the Notch receptor in the adjacent cell which induces proteolytic cleavages and the activation of the receptor. A unique feature of the Notch signaling is that processes such as modification, endocytosis or recycling of the ligand have been reported to play critical roles during Notch signaling, however, the underlying molecular mechanism appears context-dependent and often controversial.

Results: Here we identified SNX17 as a novel regulator of the Notch pathway. SNX17 is a sorting nexin family protein implicated in vesicular trafficking and we find it is specifically required in the ligand-expressing cells for Notch signaling. Mechanistically, SNX17 regulates the protein level of Jag1a on plasma membrane by binding to Jag1a and facilitating the retromer-dependent recycling of the ligand. In zebrafish, inhibition of this SNX17-mediated Notch signaling pathway results in defects in neurogenesis as well as pancreas development.

Conclusions: Our results reveal that SNX17, by acting as a cargo-specific adaptor, promotes the retromer dependent recycling of Jag1a and Notch signaling and this pathway is involved in cell fate determination during zebrafish neurogenesis and pancreas development.

No MeSH data available.


SNX17 regulates Notch signaling in zebrafish. (A) Expression pattern of SNX17 at day 2 as detected by in situ hybridization. SNX17 was enriched in the liver and pancreas (arrows) at this stage. (B) Efficiencies of splice-blocking MOs in inducing the alternative splicing of SNX17 mRNA as determined by RT-PCR. (C) Knockdown of SNX17 did not disrupt liver development. lfabp is a marker for hepatocytes. (D, E) The expression of the endocrine β-cell marker insulin (ins) was mildly increased while the exocrine marker trypsin (trp) was down-regulated in SNX17 morphants. The trp defect was rescued by co-injection of mRNA encoding human SNX17. (F) Knockdown of SNX17 or mib enhanced the expression of pro-neuronal marker huC. Injection of mRNA encoding the NICD rescued the SNX17-knockdown induced neurogenic defect. (G) Real-time RT-PCR analysis of huC and her/hes family endogenous Notch target genes. β-actin is the reference and data represent mean ± SD from three independent assays.
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Fig1: SNX17 regulates Notch signaling in zebrafish. (A) Expression pattern of SNX17 at day 2 as detected by in situ hybridization. SNX17 was enriched in the liver and pancreas (arrows) at this stage. (B) Efficiencies of splice-blocking MOs in inducing the alternative splicing of SNX17 mRNA as determined by RT-PCR. (C) Knockdown of SNX17 did not disrupt liver development. lfabp is a marker for hepatocytes. (D, E) The expression of the endocrine β-cell marker insulin (ins) was mildly increased while the exocrine marker trypsin (trp) was down-regulated in SNX17 morphants. The trp defect was rescued by co-injection of mRNA encoding human SNX17. (F) Knockdown of SNX17 or mib enhanced the expression of pro-neuronal marker huC. Injection of mRNA encoding the NICD rescued the SNX17-knockdown induced neurogenic defect. (G) Real-time RT-PCR analysis of huC and her/hes family endogenous Notch target genes. β-actin is the reference and data represent mean ± SD from three independent assays.

Mentions: Previous studies in various cultured cells have reported that SNX17 is involved in the endocytosis of LDLR [20] and P-Selectin [21], the recycling of LRP [22], the scavenger receptor FEEL-1 [23] and integrins [24, 25] to plasma membrane, as well as the escape of the lysosomal degradation of human papillomavirus L2 capsid protein during virus infection [26], however, the physiological function of SNX17 remains unknown. We used the zebrafish model to investigate the in vivo function of SNX17. We first examined the embryonic expression pattern of SNX17 by in situ hybridization and found it was not spatially restricted during gastrulation and early somitogenesis stages, but became enriched in the pancreas, liver, eye and neuronal tissues starting from day 2 (Figure 1A). We performed morpholino (MO) mediated knockdown of SNX17 and determined the downstream effects of this treatment. MOs targeting the exon 3/intron 3 junction (MO1) or the exon 2/intron 2 junction (MO2) of SNX17 gene were synthesized and both of them induced alternative splicing of SNX17 mRNA effectively (Figure 1B). The reading frames in these abnormally spliced RNAs are shifted and their protein products are non-functional. We treated embryos with these MOs and found that the expression of hepatocyte marker lfabp (liver fatty acid binding protein 1a) was normal in the morphants (Figure 1C). Interestingly, the expression of insulin (ins, a marker for endocrine β-cells) was mildly increased while that of trypsin (trp, a marker for exocrine pancreas) was severely reduced in SNX17 morphants (Figure 1D, E). Furthermore, the trp defect can be rescued by the over-expression of human SNX17, indicating this defect is SNX17 specific and not induced by the off-target effect of MOs. The pancreatic defect in SNX17 morphants is similar to that induced by inhibition of Notch, either in the mib mutant or after DAPT treatment, which promotes the endocrine pancreas at the expense of exocrine pancreas [27]. In addition, Notch is well known to regulate neurogenesis during zebrafish embryogenesis [28], so we tested whether or not SNX17 was required for neurogenesis. As shown in Figure 1F, similar to that in mib morphants, the expression of pro-neuronal marker huC was enhanced when SNX17 was inhibited. This defect can be reversed by the injection of mRNA encoding the NICD. These data suggest that SNX17 is essential for Notch activation in vivo. We then determined the expression levels of several Notch target genes by real-time RT-PCR to further characterize the Notch signaling defect in SNX17 morphants. We found the expression levels of her45, 12 and hes5 were significantly reduced in MO1 as well as mib-MO injected embryos. On the other hand, the expression of huC was increased in both morphants, which was consistent with the previous in situ hybridization result (Figure 1G). Taken together, these data clearly demonstrate that SNX17 is required for Notch signaling in vivo.Figure 1


SNX17 regulates Notch pathway and pancreas development through the retromer-dependent recycling of Jag1.

Yin W, Liu D, Liu N, Xu L, Li S, Lin S, Shu X, Pei D - Cell Regen (Lond) (2012)

SNX17 regulates Notch signaling in zebrafish. (A) Expression pattern of SNX17 at day 2 as detected by in situ hybridization. SNX17 was enriched in the liver and pancreas (arrows) at this stage. (B) Efficiencies of splice-blocking MOs in inducing the alternative splicing of SNX17 mRNA as determined by RT-PCR. (C) Knockdown of SNX17 did not disrupt liver development. lfabp is a marker for hepatocytes. (D, E) The expression of the endocrine β-cell marker insulin (ins) was mildly increased while the exocrine marker trypsin (trp) was down-regulated in SNX17 morphants. The trp defect was rescued by co-injection of mRNA encoding human SNX17. (F) Knockdown of SNX17 or mib enhanced the expression of pro-neuronal marker huC. Injection of mRNA encoding the NICD rescued the SNX17-knockdown induced neurogenic defect. (G) Real-time RT-PCR analysis of huC and her/hes family endogenous Notch target genes. β-actin is the reference and data represent mean ± SD from three independent assays.
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Related In: Results  -  Collection

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Fig1: SNX17 regulates Notch signaling in zebrafish. (A) Expression pattern of SNX17 at day 2 as detected by in situ hybridization. SNX17 was enriched in the liver and pancreas (arrows) at this stage. (B) Efficiencies of splice-blocking MOs in inducing the alternative splicing of SNX17 mRNA as determined by RT-PCR. (C) Knockdown of SNX17 did not disrupt liver development. lfabp is a marker for hepatocytes. (D, E) The expression of the endocrine β-cell marker insulin (ins) was mildly increased while the exocrine marker trypsin (trp) was down-regulated in SNX17 morphants. The trp defect was rescued by co-injection of mRNA encoding human SNX17. (F) Knockdown of SNX17 or mib enhanced the expression of pro-neuronal marker huC. Injection of mRNA encoding the NICD rescued the SNX17-knockdown induced neurogenic defect. (G) Real-time RT-PCR analysis of huC and her/hes family endogenous Notch target genes. β-actin is the reference and data represent mean ± SD from three independent assays.
Mentions: Previous studies in various cultured cells have reported that SNX17 is involved in the endocytosis of LDLR [20] and P-Selectin [21], the recycling of LRP [22], the scavenger receptor FEEL-1 [23] and integrins [24, 25] to plasma membrane, as well as the escape of the lysosomal degradation of human papillomavirus L2 capsid protein during virus infection [26], however, the physiological function of SNX17 remains unknown. We used the zebrafish model to investigate the in vivo function of SNX17. We first examined the embryonic expression pattern of SNX17 by in situ hybridization and found it was not spatially restricted during gastrulation and early somitogenesis stages, but became enriched in the pancreas, liver, eye and neuronal tissues starting from day 2 (Figure 1A). We performed morpholino (MO) mediated knockdown of SNX17 and determined the downstream effects of this treatment. MOs targeting the exon 3/intron 3 junction (MO1) or the exon 2/intron 2 junction (MO2) of SNX17 gene were synthesized and both of them induced alternative splicing of SNX17 mRNA effectively (Figure 1B). The reading frames in these abnormally spliced RNAs are shifted and their protein products are non-functional. We treated embryos with these MOs and found that the expression of hepatocyte marker lfabp (liver fatty acid binding protein 1a) was normal in the morphants (Figure 1C). Interestingly, the expression of insulin (ins, a marker for endocrine β-cells) was mildly increased while that of trypsin (trp, a marker for exocrine pancreas) was severely reduced in SNX17 morphants (Figure 1D, E). Furthermore, the trp defect can be rescued by the over-expression of human SNX17, indicating this defect is SNX17 specific and not induced by the off-target effect of MOs. The pancreatic defect in SNX17 morphants is similar to that induced by inhibition of Notch, either in the mib mutant or after DAPT treatment, which promotes the endocrine pancreas at the expense of exocrine pancreas [27]. In addition, Notch is well known to regulate neurogenesis during zebrafish embryogenesis [28], so we tested whether or not SNX17 was required for neurogenesis. As shown in Figure 1F, similar to that in mib morphants, the expression of pro-neuronal marker huC was enhanced when SNX17 was inhibited. This defect can be reversed by the injection of mRNA encoding the NICD. These data suggest that SNX17 is essential for Notch activation in vivo. We then determined the expression levels of several Notch target genes by real-time RT-PCR to further characterize the Notch signaling defect in SNX17 morphants. We found the expression levels of her45, 12 and hes5 were significantly reduced in MO1 as well as mib-MO injected embryos. On the other hand, the expression of huC was increased in both morphants, which was consistent with the previous in situ hybridization result (Figure 1G). Taken together, these data clearly demonstrate that SNX17 is required for Notch signaling in vivo.Figure 1

Bottom Line: SNX17 is a sorting nexin family protein implicated in vesicular trafficking and we find it is specifically required in the ligand-expressing cells for Notch signaling.Mechanistically, SNX17 regulates the protein level of Jag1a on plasma membrane by binding to Jag1a and facilitating the retromer-dependent recycling of the ligand.In zebrafish, inhibition of this SNX17-mediated Notch signaling pathway results in defects in neurogenesis as well as pancreas development.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Regenerative Biology, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, 510530 China.

ABSTRACT

Background: Notch is one of the most important signaling pathways involved in cell fate determination. Activation of the Notch pathway requires the binding of a membrane-bound ligand to the Notch receptor in the adjacent cell which induces proteolytic cleavages and the activation of the receptor. A unique feature of the Notch signaling is that processes such as modification, endocytosis or recycling of the ligand have been reported to play critical roles during Notch signaling, however, the underlying molecular mechanism appears context-dependent and often controversial.

Results: Here we identified SNX17 as a novel regulator of the Notch pathway. SNX17 is a sorting nexin family protein implicated in vesicular trafficking and we find it is specifically required in the ligand-expressing cells for Notch signaling. Mechanistically, SNX17 regulates the protein level of Jag1a on plasma membrane by binding to Jag1a and facilitating the retromer-dependent recycling of the ligand. In zebrafish, inhibition of this SNX17-mediated Notch signaling pathway results in defects in neurogenesis as well as pancreas development.

Conclusions: Our results reveal that SNX17, by acting as a cargo-specific adaptor, promotes the retromer dependent recycling of Jag1a and Notch signaling and this pathway is involved in cell fate determination during zebrafish neurogenesis and pancreas development.

No MeSH data available.