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Disruption of microtubule integrity initiates mitosis during CNS repair.

Bossing T, Barros CS, Fischer B, Russell S, Shepherd D - Dev. Cell (2012)

Bottom Line: A pilot screen analyzing transcriptomes of single cells during repair pointed to downregulation of the microtubule-stabilizing GTPase mitochondrial Rho (Miro) and upregulation of the Jun transcription factor Jun-related antigen (Jra).Conversely, loss of Jra renders midline cells unable to replace damaged siblings.The conservation of the identified molecules suggests that similar mechanisms may operate in vertebrates.

View Article: PubMed Central - PubMed

Affiliation: School of Biological Sciences, Bangor University, Deiniol Road, Bangor LL57 2UW, UK. t.bossing@bangor.ac.uk

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Related in: MedlinePlus

Disruption of Microtubules Triggers Cell DivisionsGenotypes listed at top of panel. Ventral views, anterior up. Bar, 6 μm.(A) In stage 13 embryos, a minority of midline cells (green) divide (arrows, red, pH3).(B) Depletion of Miro by expression of UAS::MiroRNAi in all tissues results in increased cell divisions in midline cells only.(C–E) Expression of GFP (green, C) does not increase mitosis (arrows, red, pH3) in stage 13 embryos but midline expression of α-tubulin (green, D) and, to a lesser degree, β-tubulin (green, E), triggers extra mitosis.(F) Number of dividing midline cells in all trunk segments of early stage 13 embryos. ANOVA, bars represent SEM. CD8::GFP, sim::GAL4/ UAS::CD8-GFP (n = 39)/ sim::GAL4/ +. α-tubulin, UAS::GFP-α−tubulin/ sim::GAL4; UAS::GFP-α-tubulin/ sim::GAL4 (n = 35). β-tubulin, UAS::GFP-β−tubulin/ sim::GAL4; UAS::GFP-β-tubulin/ sim::GAL4 (n = 36). GAL4V2h (n = 10). Gal4V2h/+; UAS::MiroRNATRIP02775/+ (n = 16). Gal4V2h/UAS::MiroRNAKK106683 (n = 23). Error bars: SEM.(G) In water-injected stage 10 embryos, midline cells (green) do not divide (red) and show a cortical accumulation of β-tubulin (blue).(H and I) Partial depolymerization of microtubules in stage 10 embryos injected with colcemid (n = 14) or vinblastine (n = 27) drives midline cells into division. Insets show higher magnification of boxed area and β-tubulin only.(I) Midline precursor 1 (MP1) in wild-type gives rise to two MP1-neurons (arrowheads).(J) Midline expression of α-tubulin results in extra MP1-neurons (arrowheads) generated by one precursor.See also Figures S2 and S3.
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fig2: Disruption of Microtubules Triggers Cell DivisionsGenotypes listed at top of panel. Ventral views, anterior up. Bar, 6 μm.(A) In stage 13 embryos, a minority of midline cells (green) divide (arrows, red, pH3).(B) Depletion of Miro by expression of UAS::MiroRNAi in all tissues results in increased cell divisions in midline cells only.(C–E) Expression of GFP (green, C) does not increase mitosis (arrows, red, pH3) in stage 13 embryos but midline expression of α-tubulin (green, D) and, to a lesser degree, β-tubulin (green, E), triggers extra mitosis.(F) Number of dividing midline cells in all trunk segments of early stage 13 embryos. ANOVA, bars represent SEM. CD8::GFP, sim::GAL4/ UAS::CD8-GFP (n = 39)/ sim::GAL4/ +. α-tubulin, UAS::GFP-α−tubulin/ sim::GAL4; UAS::GFP-α-tubulin/ sim::GAL4 (n = 35). β-tubulin, UAS::GFP-β−tubulin/ sim::GAL4; UAS::GFP-β-tubulin/ sim::GAL4 (n = 36). GAL4V2h (n = 10). Gal4V2h/+; UAS::MiroRNATRIP02775/+ (n = 16). Gal4V2h/UAS::MiroRNAKK106683 (n = 23). Error bars: SEM.(G) In water-injected stage 10 embryos, midline cells (green) do not divide (red) and show a cortical accumulation of β-tubulin (blue).(H and I) Partial depolymerization of microtubules in stage 10 embryos injected with colcemid (n = 14) or vinblastine (n = 27) drives midline cells into division. Insets show higher magnification of boxed area and β-tubulin only.(I) Midline precursor 1 (MP1) in wild-type gives rise to two MP1-neurons (arrowheads).(J) Midline expression of α-tubulin results in extra MP1-neurons (arrowheads) generated by one precursor.See also Figures S2 and S3.

Mentions: In our pilot study, Miro was the most downregulated transcript (T.B. et al., unpublished data). Miro, encodes a widely conserved atypical GTPase, regulating mitochondrial transport and microtubule stabilization (Guo et al., 2005). If downregulation of Miro permits midline cells to enter into divisions, we would expect more progeny per midline precursor in Miro mutants. Single labeled midline precursors in MiroSD32 mutants do not, however, divide more often than wild-type precursors (data not shown). This is likely due to a maternal contribution of Miro RNA (www.flyexpress.net) and we therefore lowered the amount of Miro RNA by ubiquitously expressing UAS::MiroRNAiTRIPiJF02775 or UAS::MiroRNAiKKi106683 with a maternal GAL4 driver (GAL4V2h). Strikingly, embryos from mothers expressing either of the UAS::MiroRNAi constructs show an increase in midline cell division at the end of germband shortening (Figures 2A, 2B, 2F, and 3C–3F). Compared to control embryos (GAL4V2h) with an average of 4.8 mitotic cells in all 10 trunk neuromeres, the number of midline cell divisions in embryos depleted of Miro increases to an average of 15.6 cells (UAS::MiroRNAiTRIPJF02775) or 11.1 cells (UAS::MiroRNAiKK106683; Figure 2F). If Miro downregulation is essential for damage-induced division, midline targeted upregulation may block these divisions. Indeed, in contrast to wild-type embryos, ablations in early stage 11 embryos with midline Miro expression can result in undamaged cells which survive but neither divide nor differentiate (16%, n = 13; Figure S4). Yet, most ablations result in repair (46%) or death (38%). The low percentage of embryos with no repair may indicate that the expression of Miro in most surviving siblings is not high enough to prevent repair.


Disruption of microtubule integrity initiates mitosis during CNS repair.

Bossing T, Barros CS, Fischer B, Russell S, Shepherd D - Dev. Cell (2012)

Disruption of Microtubules Triggers Cell DivisionsGenotypes listed at top of panel. Ventral views, anterior up. Bar, 6 μm.(A) In stage 13 embryos, a minority of midline cells (green) divide (arrows, red, pH3).(B) Depletion of Miro by expression of UAS::MiroRNAi in all tissues results in increased cell divisions in midline cells only.(C–E) Expression of GFP (green, C) does not increase mitosis (arrows, red, pH3) in stage 13 embryos but midline expression of α-tubulin (green, D) and, to a lesser degree, β-tubulin (green, E), triggers extra mitosis.(F) Number of dividing midline cells in all trunk segments of early stage 13 embryos. ANOVA, bars represent SEM. CD8::GFP, sim::GAL4/ UAS::CD8-GFP (n = 39)/ sim::GAL4/ +. α-tubulin, UAS::GFP-α−tubulin/ sim::GAL4; UAS::GFP-α-tubulin/ sim::GAL4 (n = 35). β-tubulin, UAS::GFP-β−tubulin/ sim::GAL4; UAS::GFP-β-tubulin/ sim::GAL4 (n = 36). GAL4V2h (n = 10). Gal4V2h/+; UAS::MiroRNATRIP02775/+ (n = 16). Gal4V2h/UAS::MiroRNAKK106683 (n = 23). Error bars: SEM.(G) In water-injected stage 10 embryos, midline cells (green) do not divide (red) and show a cortical accumulation of β-tubulin (blue).(H and I) Partial depolymerization of microtubules in stage 10 embryos injected with colcemid (n = 14) or vinblastine (n = 27) drives midline cells into division. Insets show higher magnification of boxed area and β-tubulin only.(I) Midline precursor 1 (MP1) in wild-type gives rise to two MP1-neurons (arrowheads).(J) Midline expression of α-tubulin results in extra MP1-neurons (arrowheads) generated by one precursor.See also Figures S2 and S3.
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fig2: Disruption of Microtubules Triggers Cell DivisionsGenotypes listed at top of panel. Ventral views, anterior up. Bar, 6 μm.(A) In stage 13 embryos, a minority of midline cells (green) divide (arrows, red, pH3).(B) Depletion of Miro by expression of UAS::MiroRNAi in all tissues results in increased cell divisions in midline cells only.(C–E) Expression of GFP (green, C) does not increase mitosis (arrows, red, pH3) in stage 13 embryos but midline expression of α-tubulin (green, D) and, to a lesser degree, β-tubulin (green, E), triggers extra mitosis.(F) Number of dividing midline cells in all trunk segments of early stage 13 embryos. ANOVA, bars represent SEM. CD8::GFP, sim::GAL4/ UAS::CD8-GFP (n = 39)/ sim::GAL4/ +. α-tubulin, UAS::GFP-α−tubulin/ sim::GAL4; UAS::GFP-α-tubulin/ sim::GAL4 (n = 35). β-tubulin, UAS::GFP-β−tubulin/ sim::GAL4; UAS::GFP-β-tubulin/ sim::GAL4 (n = 36). GAL4V2h (n = 10). Gal4V2h/+; UAS::MiroRNATRIP02775/+ (n = 16). Gal4V2h/UAS::MiroRNAKK106683 (n = 23). Error bars: SEM.(G) In water-injected stage 10 embryos, midline cells (green) do not divide (red) and show a cortical accumulation of β-tubulin (blue).(H and I) Partial depolymerization of microtubules in stage 10 embryos injected with colcemid (n = 14) or vinblastine (n = 27) drives midline cells into division. Insets show higher magnification of boxed area and β-tubulin only.(I) Midline precursor 1 (MP1) in wild-type gives rise to two MP1-neurons (arrowheads).(J) Midline expression of α-tubulin results in extra MP1-neurons (arrowheads) generated by one precursor.See also Figures S2 and S3.
Mentions: In our pilot study, Miro was the most downregulated transcript (T.B. et al., unpublished data). Miro, encodes a widely conserved atypical GTPase, regulating mitochondrial transport and microtubule stabilization (Guo et al., 2005). If downregulation of Miro permits midline cells to enter into divisions, we would expect more progeny per midline precursor in Miro mutants. Single labeled midline precursors in MiroSD32 mutants do not, however, divide more often than wild-type precursors (data not shown). This is likely due to a maternal contribution of Miro RNA (www.flyexpress.net) and we therefore lowered the amount of Miro RNA by ubiquitously expressing UAS::MiroRNAiTRIPiJF02775 or UAS::MiroRNAiKKi106683 with a maternal GAL4 driver (GAL4V2h). Strikingly, embryos from mothers expressing either of the UAS::MiroRNAi constructs show an increase in midline cell division at the end of germband shortening (Figures 2A, 2B, 2F, and 3C–3F). Compared to control embryos (GAL4V2h) with an average of 4.8 mitotic cells in all 10 trunk neuromeres, the number of midline cell divisions in embryos depleted of Miro increases to an average of 15.6 cells (UAS::MiroRNAiTRIPJF02775) or 11.1 cells (UAS::MiroRNAiKK106683; Figure 2F). If Miro downregulation is essential for damage-induced division, midline targeted upregulation may block these divisions. Indeed, in contrast to wild-type embryos, ablations in early stage 11 embryos with midline Miro expression can result in undamaged cells which survive but neither divide nor differentiate (16%, n = 13; Figure S4). Yet, most ablations result in repair (46%) or death (38%). The low percentage of embryos with no repair may indicate that the expression of Miro in most surviving siblings is not high enough to prevent repair.

Bottom Line: A pilot screen analyzing transcriptomes of single cells during repair pointed to downregulation of the microtubule-stabilizing GTPase mitochondrial Rho (Miro) and upregulation of the Jun transcription factor Jun-related antigen (Jra).Conversely, loss of Jra renders midline cells unable to replace damaged siblings.The conservation of the identified molecules suggests that similar mechanisms may operate in vertebrates.

View Article: PubMed Central - PubMed

Affiliation: School of Biological Sciences, Bangor University, Deiniol Road, Bangor LL57 2UW, UK. t.bossing@bangor.ac.uk

Show MeSH
Related in: MedlinePlus