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The E3 ubiquitin ligase skp2 regulates neural differentiation independent from the cell cycle.

Boix-Perales H, Horan I, Wise H, Lin HR, Chuang LC, Yew PR, Philpott A - Neural Dev (2007)

Bottom Line: Xenopus skp2 shows a dynamic expression pattern in early embryonic neural tissue and depletion of skp2 results in generation of extra primary neurons.We conclude that the SCFskp2 complex has functions in the control of neuronal differentiation additional to its role in cell cycle regulation.Thus, it is well placed to be a co-ordinating factor regulating both cell proliferation and cell differentiation directly.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Oncology, University of Cambridge, Hutchison/MRC Research Centre, Addenbrookes Hospital, Hills Road, Cambridge CB2 0XZ, UK. hectorboix@googlemail.com

ABSTRACT

Background: The SCFskp2 complex is an E3 ubiquitin ligase that is known to target a number of cell cycle regulators, including cyclin-dependent kinase inhibitors, for proteolysis. While its role in regulation of cell division has been well documented, additional functions in differentiation, including in the nervous system, have not been investigated.

Results: Using Xenopus as a model system, here we demonstrate that skp2 has an additional role in regulation of differentiation of primary neurons, the first neurons to differentiate in the neural plate. Xenopus skp2 shows a dynamic expression pattern in early embryonic neural tissue and depletion of skp2 results in generation of extra primary neurons. In contrast, over-expression of skp2 inhibits neurogenesis in a manner dependent on its ability to act as part of the SCFskp2 complex. Moreover, inhibition of neurogenesis by skp2 occurs upstream of the proneural gene encoding NeuroD and prior to cell cycle exit. We have previously demonstrated that the Xenopus cyclin dependent kinase inhibitor Xic1 is essential for primary neurogenesis at an early stage, and before these cells exit the cell cycle. We show that SCFskp2 degrades Xic1 in embryos and this contributes to the ability of skp2 to regulate neurogenesis.

Conclusion: We conclude that the SCFskp2 complex has functions in the control of neuronal differentiation additional to its role in cell cycle regulation. Thus, it is well placed to be a co-ordinating factor regulating both cell proliferation and cell differentiation directly.

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Loss of skp2 protein promotes primary neurogenesis. (a) Embryos were injected with wild-type (Wt) skp2, skp2 1–2 alone or in combination with skp2 Mo or Con Mo as indicated. Protein (30 μg) from a stage 15 embryo was western blotted to determine skp2 levels; tubulin was used as a loading control. (b) Western blot for endogenous skp2 protein levels on stage 15 embryos that were injected with 20 ng, 30 ng, or 40 ng skp2 Mo or 40 ng Con Mo at the one cell stage, arrow to skp2 protein band. In vitro translated (IVT) skp2 protein is run in lane 6, and alpha-tubulin was used as a loading control. (c) The percentages of embryos with mild increase, no change, or moderate or substantial reduction of nßt positive cells on the injected side relative to the uninjected side for 20 ng, 30 ng, and 40 ng skp2 Mo, or 30 ng and 40 ng Con Mo (see Additional file 1 for photographs of representative embryos). (d,e) Embryos were injected with 30 ng skp2 Mo (d) or 30 ng Con Mo (e) in one blastomere at the two cell stage, along with ßgal mRNA as a lineage tracer, and analyzed for nßt mRNA expression at stage 15 ((d) arrow to show expansion of primary neurons). The view is dorsal with injected side to the right. (f,g) In situ hybridisation sections, which are transverse across the centre of the embryo, with injected side to the right (f) Section of an early neurula embryo injected with 30 ng skp2 Mo, indicating nßt upregulation by skp2 protein depletion. (g) Section of a mid neurula embryo injected with 30 ng Con Mo showing no difference in nßt distribution. Arrows (f, g) denoting staining of nßt in primary neurons. (h,i) Whole mount stage 15 embryos immunostained (red) to detect pH3 after injection of 30 ng skp2 Mo (h) or 30 ng Con Mo (i) in one blastomere at the two cell stage. ßgal mRNA was co-injected and X-Gal staining (blue) was performed to reveal injected side. Dorsal views with injected side to the right. (h',i') Detail of pH3 cells on the injected side relative to the uninjected side of representative embryos (boxed area in (h,i), dashed line is dorsal mid-line separating injected and uninjected halves).
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Figure 2: Loss of skp2 protein promotes primary neurogenesis. (a) Embryos were injected with wild-type (Wt) skp2, skp2 1–2 alone or in combination with skp2 Mo or Con Mo as indicated. Protein (30 μg) from a stage 15 embryo was western blotted to determine skp2 levels; tubulin was used as a loading control. (b) Western blot for endogenous skp2 protein levels on stage 15 embryos that were injected with 20 ng, 30 ng, or 40 ng skp2 Mo or 40 ng Con Mo at the one cell stage, arrow to skp2 protein band. In vitro translated (IVT) skp2 protein is run in lane 6, and alpha-tubulin was used as a loading control. (c) The percentages of embryos with mild increase, no change, or moderate or substantial reduction of nßt positive cells on the injected side relative to the uninjected side for 20 ng, 30 ng, and 40 ng skp2 Mo, or 30 ng and 40 ng Con Mo (see Additional file 1 for photographs of representative embryos). (d,e) Embryos were injected with 30 ng skp2 Mo (d) or 30 ng Con Mo (e) in one blastomere at the two cell stage, along with ßgal mRNA as a lineage tracer, and analyzed for nßt mRNA expression at stage 15 ((d) arrow to show expansion of primary neurons). The view is dorsal with injected side to the right. (f,g) In situ hybridisation sections, which are transverse across the centre of the embryo, with injected side to the right (f) Section of an early neurula embryo injected with 30 ng skp2 Mo, indicating nßt upregulation by skp2 protein depletion. (g) Section of a mid neurula embryo injected with 30 ng Con Mo showing no difference in nßt distribution. Arrows (f, g) denoting staining of nßt in primary neurons. (h,i) Whole mount stage 15 embryos immunostained (red) to detect pH3 after injection of 30 ng skp2 Mo (h) or 30 ng Con Mo (i) in one blastomere at the two cell stage. ßgal mRNA was co-injected and X-Gal staining (blue) was performed to reveal injected side. Dorsal views with injected side to the right. (h',i') Detail of pH3 cells on the injected side relative to the uninjected side of representative embryos (boxed area in (h,i), dashed line is dorsal mid-line separating injected and uninjected halves).

Mentions: To determine whether skp2 may play a role in regulation of primary neurogenesis, we designed a skp2 morpholino (skp2 Mo) to inhibit skp2 protein expression, along with a matched control morpholino (Con Mo) that has five base changes, so would be unable to bind to skp2 message. As expected, skp2 Mo but not Con Mo was able to prevent translation of co-injected skp2 message (Figure 2a), although skp2Mo is unable to target a modified skp2 that is missing the first six base-pairs after the initiator ATG (skp2 1–2). We then injected increasing amounts of skp2 Mo into two of two cells and performed western blot analysis to look at the level of endogenous skp2 protein (Figure 2b). While endogenous skp2 is expressed at low levels, probably as it is an unstable protein [28], it is nevertheless clearly detected in both uninjected embryos and those injected with 40 ng Con Mo (Figure 2b, lanes 4 and 5). In contrast, 20 ng of skp2 Mo substantially inhibited expression of skp2 compared to uninjected or Con Mo injected embryos (Figure 2b, lane 1), and at 30 ng and 40 ng of skp2 Mo, skp2 protein was undetectable (Figure 2b, lanes 2 and 3). Thus, skp2 Mo specifically blocks translation of skp2 message, resulting in an absence of detectable skp2 protein, while a matched control morpholino has no effect.


The E3 ubiquitin ligase skp2 regulates neural differentiation independent from the cell cycle.

Boix-Perales H, Horan I, Wise H, Lin HR, Chuang LC, Yew PR, Philpott A - Neural Dev (2007)

Loss of skp2 protein promotes primary neurogenesis. (a) Embryos were injected with wild-type (Wt) skp2, skp2 1–2 alone or in combination with skp2 Mo or Con Mo as indicated. Protein (30 μg) from a stage 15 embryo was western blotted to determine skp2 levels; tubulin was used as a loading control. (b) Western blot for endogenous skp2 protein levels on stage 15 embryos that were injected with 20 ng, 30 ng, or 40 ng skp2 Mo or 40 ng Con Mo at the one cell stage, arrow to skp2 protein band. In vitro translated (IVT) skp2 protein is run in lane 6, and alpha-tubulin was used as a loading control. (c) The percentages of embryos with mild increase, no change, or moderate or substantial reduction of nßt positive cells on the injected side relative to the uninjected side for 20 ng, 30 ng, and 40 ng skp2 Mo, or 30 ng and 40 ng Con Mo (see Additional file 1 for photographs of representative embryos). (d,e) Embryos were injected with 30 ng skp2 Mo (d) or 30 ng Con Mo (e) in one blastomere at the two cell stage, along with ßgal mRNA as a lineage tracer, and analyzed for nßt mRNA expression at stage 15 ((d) arrow to show expansion of primary neurons). The view is dorsal with injected side to the right. (f,g) In situ hybridisation sections, which are transverse across the centre of the embryo, with injected side to the right (f) Section of an early neurula embryo injected with 30 ng skp2 Mo, indicating nßt upregulation by skp2 protein depletion. (g) Section of a mid neurula embryo injected with 30 ng Con Mo showing no difference in nßt distribution. Arrows (f, g) denoting staining of nßt in primary neurons. (h,i) Whole mount stage 15 embryos immunostained (red) to detect pH3 after injection of 30 ng skp2 Mo (h) or 30 ng Con Mo (i) in one blastomere at the two cell stage. ßgal mRNA was co-injected and X-Gal staining (blue) was performed to reveal injected side. Dorsal views with injected side to the right. (h',i') Detail of pH3 cells on the injected side relative to the uninjected side of representative embryos (boxed area in (h,i), dashed line is dorsal mid-line separating injected and uninjected halves).
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Figure 2: Loss of skp2 protein promotes primary neurogenesis. (a) Embryos were injected with wild-type (Wt) skp2, skp2 1–2 alone or in combination with skp2 Mo or Con Mo as indicated. Protein (30 μg) from a stage 15 embryo was western blotted to determine skp2 levels; tubulin was used as a loading control. (b) Western blot for endogenous skp2 protein levels on stage 15 embryos that were injected with 20 ng, 30 ng, or 40 ng skp2 Mo or 40 ng Con Mo at the one cell stage, arrow to skp2 protein band. In vitro translated (IVT) skp2 protein is run in lane 6, and alpha-tubulin was used as a loading control. (c) The percentages of embryos with mild increase, no change, or moderate or substantial reduction of nßt positive cells on the injected side relative to the uninjected side for 20 ng, 30 ng, and 40 ng skp2 Mo, or 30 ng and 40 ng Con Mo (see Additional file 1 for photographs of representative embryos). (d,e) Embryos were injected with 30 ng skp2 Mo (d) or 30 ng Con Mo (e) in one blastomere at the two cell stage, along with ßgal mRNA as a lineage tracer, and analyzed for nßt mRNA expression at stage 15 ((d) arrow to show expansion of primary neurons). The view is dorsal with injected side to the right. (f,g) In situ hybridisation sections, which are transverse across the centre of the embryo, with injected side to the right (f) Section of an early neurula embryo injected with 30 ng skp2 Mo, indicating nßt upregulation by skp2 protein depletion. (g) Section of a mid neurula embryo injected with 30 ng Con Mo showing no difference in nßt distribution. Arrows (f, g) denoting staining of nßt in primary neurons. (h,i) Whole mount stage 15 embryos immunostained (red) to detect pH3 after injection of 30 ng skp2 Mo (h) or 30 ng Con Mo (i) in one blastomere at the two cell stage. ßgal mRNA was co-injected and X-Gal staining (blue) was performed to reveal injected side. Dorsal views with injected side to the right. (h',i') Detail of pH3 cells on the injected side relative to the uninjected side of representative embryos (boxed area in (h,i), dashed line is dorsal mid-line separating injected and uninjected halves).
Mentions: To determine whether skp2 may play a role in regulation of primary neurogenesis, we designed a skp2 morpholino (skp2 Mo) to inhibit skp2 protein expression, along with a matched control morpholino (Con Mo) that has five base changes, so would be unable to bind to skp2 message. As expected, skp2 Mo but not Con Mo was able to prevent translation of co-injected skp2 message (Figure 2a), although skp2Mo is unable to target a modified skp2 that is missing the first six base-pairs after the initiator ATG (skp2 1–2). We then injected increasing amounts of skp2 Mo into two of two cells and performed western blot analysis to look at the level of endogenous skp2 protein (Figure 2b). While endogenous skp2 is expressed at low levels, probably as it is an unstable protein [28], it is nevertheless clearly detected in both uninjected embryos and those injected with 40 ng Con Mo (Figure 2b, lanes 4 and 5). In contrast, 20 ng of skp2 Mo substantially inhibited expression of skp2 compared to uninjected or Con Mo injected embryos (Figure 2b, lane 1), and at 30 ng and 40 ng of skp2 Mo, skp2 protein was undetectable (Figure 2b, lanes 2 and 3). Thus, skp2 Mo specifically blocks translation of skp2 message, resulting in an absence of detectable skp2 protein, while a matched control morpholino has no effect.

Bottom Line: Xenopus skp2 shows a dynamic expression pattern in early embryonic neural tissue and depletion of skp2 results in generation of extra primary neurons.We conclude that the SCFskp2 complex has functions in the control of neuronal differentiation additional to its role in cell cycle regulation.Thus, it is well placed to be a co-ordinating factor regulating both cell proliferation and cell differentiation directly.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Oncology, University of Cambridge, Hutchison/MRC Research Centre, Addenbrookes Hospital, Hills Road, Cambridge CB2 0XZ, UK. hectorboix@googlemail.com

ABSTRACT

Background: The SCFskp2 complex is an E3 ubiquitin ligase that is known to target a number of cell cycle regulators, including cyclin-dependent kinase inhibitors, for proteolysis. While its role in regulation of cell division has been well documented, additional functions in differentiation, including in the nervous system, have not been investigated.

Results: Using Xenopus as a model system, here we demonstrate that skp2 has an additional role in regulation of differentiation of primary neurons, the first neurons to differentiate in the neural plate. Xenopus skp2 shows a dynamic expression pattern in early embryonic neural tissue and depletion of skp2 results in generation of extra primary neurons. In contrast, over-expression of skp2 inhibits neurogenesis in a manner dependent on its ability to act as part of the SCFskp2 complex. Moreover, inhibition of neurogenesis by skp2 occurs upstream of the proneural gene encoding NeuroD and prior to cell cycle exit. We have previously demonstrated that the Xenopus cyclin dependent kinase inhibitor Xic1 is essential for primary neurogenesis at an early stage, and before these cells exit the cell cycle. We show that SCFskp2 degrades Xic1 in embryos and this contributes to the ability of skp2 to regulate neurogenesis.

Conclusion: We conclude that the SCFskp2 complex has functions in the control of neuronal differentiation additional to its role in cell cycle regulation. Thus, it is well placed to be a co-ordinating factor regulating both cell proliferation and cell differentiation directly.

Show MeSH