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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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Over-expression of skp2 blocks primary neurogenesis and reduces cell proliferation. (a-e) Embryos were injected with 1 ng wild-type (Wt) skp2 (a,d), 1 ng FBox skp2 (b,e), or 1 ng Wt MAFbx (c) mRNA, along with ßgal mRNA as a lineage tracer, in one blastomere at the two cell stage. Embryos were analyzed for expression of nßt mRNA (a-c) at stage 15. Dorsal views with injected side to the right. (d,e) Whole mount stage 15 embryos immunostained (red) to detect pH3 after injection of 1 ng Wt skp2 (d), or 1 ng FBox skp2 (e) mRNA, along with ßgal mRNA as a lineage tracer, in one blastomere at the two cell stage. Dorsal views with injected side to the right. (d',e') Detail of pH3-positive cells on the injected side relative to the uninjected side (boxed area in (d,e), dashed line is dorsal mid-line separating injected and uninjected halves). (f) Embryos were injected with 250 pg, 500 pg or 1 ng Wt skp2 mRNA (n = 41, 83, 87 embryos, respectively) in one blastomere at the two cell stage. Embryos were analyzed for expression of nßt mRNA at stage 15. Data shown in (f) are the percentages of embryos with no change, or moderate or substantial reduction of nßt positive cells on the injected side relative to the uninjected side for each injection (see Additional file 1 for photographs of representative embryos).
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Figure 3: Over-expression of skp2 blocks primary neurogenesis and reduces cell proliferation. (a-e) Embryos were injected with 1 ng wild-type (Wt) skp2 (a,d), 1 ng FBox skp2 (b,e), or 1 ng Wt MAFbx (c) mRNA, along with ßgal mRNA as a lineage tracer, in one blastomere at the two cell stage. Embryos were analyzed for expression of nßt mRNA (a-c) at stage 15. Dorsal views with injected side to the right. (d,e) Whole mount stage 15 embryos immunostained (red) to detect pH3 after injection of 1 ng Wt skp2 (d), or 1 ng FBox skp2 (e) mRNA, along with ßgal mRNA as a lineage tracer, in one blastomere at the two cell stage. Dorsal views with injected side to the right. (d',e') Detail of pH3-positive cells on the injected side relative to the uninjected side (boxed area in (d,e), dashed line is dorsal mid-line separating injected and uninjected halves). (f) Embryos were injected with 250 pg, 500 pg or 1 ng Wt skp2 mRNA (n = 41, 83, 87 embryos, respectively) in one blastomere at the two cell stage. Embryos were analyzed for expression of nßt mRNA at stage 15. Data shown in (f) are the percentages of embryos with no change, or moderate or substantial reduction of nßt positive cells on the injected side relative to the uninjected side for each injection (see Additional file 1 for photographs of representative embryos).

Mentions: While skp2 targets need to be phosphorylated prior to ubiquitination, the absolute amount of skp2 may also be important in determining target degradation; levels of skp2 protein itself are regulated in a cell cycle dependent manner [25,34] while its levels are elevated in a number of human tumours [3,35-54]. Therefore, we next determined the effect of over-expression of skp2 RNA on differentiation of primary neurons (Figure 3a–c,f). As controls, we used the mutant of skp2 that has had the F box Skp1 binding domain deleted (F box skp2), so is impaired in its ability to target substrates for destruction [21]. This F Box-deleted form of skp2 has been shown to be unable to target Xic1 for destruction in vitro [21]. As an additional control, we injected RNA encoding Xenopus MAFbx. MAFbx is a related ring finger E3 ligase that can ubquitinate MyoD in mammals [55] and, therefore, would not be expected to affect primary neurogenesis.


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)

Over-expression of skp2 blocks primary neurogenesis and reduces cell proliferation. (a-e) Embryos were injected with 1 ng wild-type (Wt) skp2 (a,d), 1 ng FBox skp2 (b,e), or 1 ng Wt MAFbx (c) mRNA, along with ßgal mRNA as a lineage tracer, in one blastomere at the two cell stage. Embryos were analyzed for expression of nßt mRNA (a-c) at stage 15. Dorsal views with injected side to the right. (d,e) Whole mount stage 15 embryos immunostained (red) to detect pH3 after injection of 1 ng Wt skp2 (d), or 1 ng FBox skp2 (e) mRNA, along with ßgal mRNA as a lineage tracer, in one blastomere at the two cell stage. Dorsal views with injected side to the right. (d',e') Detail of pH3-positive cells on the injected side relative to the uninjected side (boxed area in (d,e), dashed line is dorsal mid-line separating injected and uninjected halves). (f) Embryos were injected with 250 pg, 500 pg or 1 ng Wt skp2 mRNA (n = 41, 83, 87 embryos, respectively) in one blastomere at the two cell stage. Embryos were analyzed for expression of nßt mRNA at stage 15. Data shown in (f) are the percentages of embryos with no change, or moderate or substantial reduction of nßt positive cells on the injected side relative to the uninjected side for each injection (see Additional file 1 for photographs of representative embryos).
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Related In: Results  -  Collection

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Figure 3: Over-expression of skp2 blocks primary neurogenesis and reduces cell proliferation. (a-e) Embryos were injected with 1 ng wild-type (Wt) skp2 (a,d), 1 ng FBox skp2 (b,e), or 1 ng Wt MAFbx (c) mRNA, along with ßgal mRNA as a lineage tracer, in one blastomere at the two cell stage. Embryos were analyzed for expression of nßt mRNA (a-c) at stage 15. Dorsal views with injected side to the right. (d,e) Whole mount stage 15 embryos immunostained (red) to detect pH3 after injection of 1 ng Wt skp2 (d), or 1 ng FBox skp2 (e) mRNA, along with ßgal mRNA as a lineage tracer, in one blastomere at the two cell stage. Dorsal views with injected side to the right. (d',e') Detail of pH3-positive cells on the injected side relative to the uninjected side (boxed area in (d,e), dashed line is dorsal mid-line separating injected and uninjected halves). (f) Embryos were injected with 250 pg, 500 pg or 1 ng Wt skp2 mRNA (n = 41, 83, 87 embryos, respectively) in one blastomere at the two cell stage. Embryos were analyzed for expression of nßt mRNA at stage 15. Data shown in (f) are the percentages of embryos with no change, or moderate or substantial reduction of nßt positive cells on the injected side relative to the uninjected side for each injection (see Additional file 1 for photographs of representative embryos).
Mentions: While skp2 targets need to be phosphorylated prior to ubiquitination, the absolute amount of skp2 may also be important in determining target degradation; levels of skp2 protein itself are regulated in a cell cycle dependent manner [25,34] while its levels are elevated in a number of human tumours [3,35-54]. Therefore, we next determined the effect of over-expression of skp2 RNA on differentiation of primary neurons (Figure 3a–c,f). As controls, we used the mutant of skp2 that has had the F box Skp1 binding domain deleted (F box skp2), so is impaired in its ability to target substrates for destruction [21]. This F Box-deleted form of skp2 has been shown to be unable to target Xic1 for destruction in vitro [21]. As an additional control, we injected RNA encoding Xenopus MAFbx. MAFbx is a related ring finger E3 ligase that can ubquitinate MyoD in mammals [55] and, therefore, would not be expected to affect primary neurogenesis.

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