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A spindle-independent cleavage pathway controls germ cell formation in Drosophila.

Cinalli RM, Lehmann R - Nat. Cell Biol. (2013)

Bottom Line: The primordial germ cells (PGCs) are the first cells to form during Drosophila melanogaster embryogenesis.In addition to using core regulators of cleavage, including the small GTPase RhoA (Drosophila rho1) and the Rho-associated kinase, ROCK (Drosophila drok), we show that this pathway requires Germ cell-less (GCL), a conserved BTB-domain protein not previously implicated in cleavage mechanics.This alternative form of cell formation suggests that organisms have evolved multiple molecular strategies for regulating the cytoskeleton during cleavage.

View Article: PubMed Central - PubMed

Affiliation: HHMI and Kimmel Center for Biology and Medicine at the Skirball Institute, Department of Cell Biology, New York University School of Medicine, New York, New York 10016, USA.

ABSTRACT
The primordial germ cells (PGCs) are the first cells to form during Drosophila melanogaster embryogenesis. Whereas the process of somatic cell formation has been studied in detail, the mechanics of PGC formation are poorly understood. Here, using four-dimensional multi-photon imaging combined with genetic and pharmacological manipulations, we find that PGC formation requires an anaphase spindle-independent cleavage pathway. In addition to using core regulators of cleavage, including the small GTPase RhoA (Drosophila rho1) and the Rho-associated kinase, ROCK (Drosophila drok), we show that this pathway requires Germ cell-less (GCL), a conserved BTB-domain protein not previously implicated in cleavage mechanics. This alternative form of cell formation suggests that organisms have evolved multiple molecular strategies for regulating the cytoskeleton during cleavage.

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A spindle-independent cleavage pathway directs bud furrow cleavage(a) Micrographs of vehicle- and C3 peptide-injected embryos (Vasa (green) and F-actin (red)). Arrows mark the PGCs in vehicle-injected embryos. Total number of embryos injected and scored in (a): vehicle-injected = 15, C3 peptide-injected = 12. (b) Time–lapse micrographs and below quantification of Anillin-GFP at the BF in vehicle- and C3 peptide-injected embryos (see Supplementary Video 3 and 4). Arrowheads mark the BF. Quantification shows the mean of 4 embryos, with 3 buds measured in each embryo. Error bars: S.D. (c) Micrographs of vehicle- and Y27682-injected embryos (Vasa (green) and F-actin (red)). Arrows mark the PGCs in vehicle-injected embryos. Total number of embryos injected and scored in c: vehicle-injected = 21, Y27682-injected = 26. (d) MIP micrographs of single PGCs from vehicle- and colcemid-injected embryos (Anillin (green), F-actin (red) and DNA (blue)). Total number of embryos injected and scored in d: vehicle-injected = 10, colcemid-injected = 18. (e) Time-lapse micrographs (single optical sections) of a bud from a colcemid-injected Anillin-GFP expressing embryo showing complete constriction of BF (see Supplementary Video 5). Arrowhead marks the BF. Note the absence of the AF in the time series. Total number of embryos observed in e = 4. Scale bars = 5 μm
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Figure 2: A spindle-independent cleavage pathway directs bud furrow cleavage(a) Micrographs of vehicle- and C3 peptide-injected embryos (Vasa (green) and F-actin (red)). Arrows mark the PGCs in vehicle-injected embryos. Total number of embryos injected and scored in (a): vehicle-injected = 15, C3 peptide-injected = 12. (b) Time–lapse micrographs and below quantification of Anillin-GFP at the BF in vehicle- and C3 peptide-injected embryos (see Supplementary Video 3 and 4). Arrowheads mark the BF. Quantification shows the mean of 4 embryos, with 3 buds measured in each embryo. Error bars: S.D. (c) Micrographs of vehicle- and Y27682-injected embryos (Vasa (green) and F-actin (red)). Arrows mark the PGCs in vehicle-injected embryos. Total number of embryos injected and scored in c: vehicle-injected = 21, Y27682-injected = 26. (d) MIP micrographs of single PGCs from vehicle- and colcemid-injected embryos (Anillin (green), F-actin (red) and DNA (blue)). Total number of embryos injected and scored in d: vehicle-injected = 10, colcemid-injected = 18. (e) Time-lapse micrographs (single optical sections) of a bud from a colcemid-injected Anillin-GFP expressing embryo showing complete constriction of BF (see Supplementary Video 5). Arrowhead marks the BF. Note the absence of the AF in the time series. Total number of embryos observed in e = 4. Scale bars = 5 μm

Mentions: What are the molecular mechanisms that control paired furrow activity during PGC formation? The small GTPase RhoA (Drosophila Rho) is a major regulator of cellular contractility and functions upstream of anillin and diaphanous during cytokinesis24,25. To determine whether PGC formation also requires RhoA activity, we injected the RhoA inhibitor, C3 peptide26,27, into embryos shortly after bud formation. Injection of the C3 peptide, but not vehicle, blocked PGC formation (# embryos with PGCs, vehicle-injected = 15/15, C3-injected = 0/12) (Fig. 2a). In Drosophila S2 cells, RhoA targets Anillin to the cleavage furrow during cytokinesis25. Therefore, we asked whether targeting of Anillin-GFP to the BF was dependent on RhoA activity. Using our live imaging assay, we monitored Anillin-GFP at the BF following RhoA inhibition. In contrast to vehicle-controls, C3 peptide-injected embryos exhibited a 2.5-fold reduction in Anillin-GFP at the BF shortly after injection (Fig. 2b, Supplementary Video S3 and S4). These data demonstrate that PGC formation requires RhoA and suggest that a common RhoA signaling cascade regulates Anillin localization during both PGC formation and cytokinesis.


A spindle-independent cleavage pathway controls germ cell formation in Drosophila.

Cinalli RM, Lehmann R - Nat. Cell Biol. (2013)

A spindle-independent cleavage pathway directs bud furrow cleavage(a) Micrographs of vehicle- and C3 peptide-injected embryos (Vasa (green) and F-actin (red)). Arrows mark the PGCs in vehicle-injected embryos. Total number of embryos injected and scored in (a): vehicle-injected = 15, C3 peptide-injected = 12. (b) Time–lapse micrographs and below quantification of Anillin-GFP at the BF in vehicle- and C3 peptide-injected embryos (see Supplementary Video 3 and 4). Arrowheads mark the BF. Quantification shows the mean of 4 embryos, with 3 buds measured in each embryo. Error bars: S.D. (c) Micrographs of vehicle- and Y27682-injected embryos (Vasa (green) and F-actin (red)). Arrows mark the PGCs in vehicle-injected embryos. Total number of embryos injected and scored in c: vehicle-injected = 21, Y27682-injected = 26. (d) MIP micrographs of single PGCs from vehicle- and colcemid-injected embryos (Anillin (green), F-actin (red) and DNA (blue)). Total number of embryos injected and scored in d: vehicle-injected = 10, colcemid-injected = 18. (e) Time-lapse micrographs (single optical sections) of a bud from a colcemid-injected Anillin-GFP expressing embryo showing complete constriction of BF (see Supplementary Video 5). Arrowhead marks the BF. Note the absence of the AF in the time series. Total number of embryos observed in e = 4. Scale bars = 5 μm
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Related In: Results  -  Collection

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Figure 2: A spindle-independent cleavage pathway directs bud furrow cleavage(a) Micrographs of vehicle- and C3 peptide-injected embryos (Vasa (green) and F-actin (red)). Arrows mark the PGCs in vehicle-injected embryos. Total number of embryos injected and scored in (a): vehicle-injected = 15, C3 peptide-injected = 12. (b) Time–lapse micrographs and below quantification of Anillin-GFP at the BF in vehicle- and C3 peptide-injected embryos (see Supplementary Video 3 and 4). Arrowheads mark the BF. Quantification shows the mean of 4 embryos, with 3 buds measured in each embryo. Error bars: S.D. (c) Micrographs of vehicle- and Y27682-injected embryos (Vasa (green) and F-actin (red)). Arrows mark the PGCs in vehicle-injected embryos. Total number of embryos injected and scored in c: vehicle-injected = 21, Y27682-injected = 26. (d) MIP micrographs of single PGCs from vehicle- and colcemid-injected embryos (Anillin (green), F-actin (red) and DNA (blue)). Total number of embryos injected and scored in d: vehicle-injected = 10, colcemid-injected = 18. (e) Time-lapse micrographs (single optical sections) of a bud from a colcemid-injected Anillin-GFP expressing embryo showing complete constriction of BF (see Supplementary Video 5). Arrowhead marks the BF. Note the absence of the AF in the time series. Total number of embryos observed in e = 4. Scale bars = 5 μm
Mentions: What are the molecular mechanisms that control paired furrow activity during PGC formation? The small GTPase RhoA (Drosophila Rho) is a major regulator of cellular contractility and functions upstream of anillin and diaphanous during cytokinesis24,25. To determine whether PGC formation also requires RhoA activity, we injected the RhoA inhibitor, C3 peptide26,27, into embryos shortly after bud formation. Injection of the C3 peptide, but not vehicle, blocked PGC formation (# embryos with PGCs, vehicle-injected = 15/15, C3-injected = 0/12) (Fig. 2a). In Drosophila S2 cells, RhoA targets Anillin to the cleavage furrow during cytokinesis25. Therefore, we asked whether targeting of Anillin-GFP to the BF was dependent on RhoA activity. Using our live imaging assay, we monitored Anillin-GFP at the BF following RhoA inhibition. In contrast to vehicle-controls, C3 peptide-injected embryos exhibited a 2.5-fold reduction in Anillin-GFP at the BF shortly after injection (Fig. 2b, Supplementary Video S3 and S4). These data demonstrate that PGC formation requires RhoA and suggest that a common RhoA signaling cascade regulates Anillin localization during both PGC formation and cytokinesis.

Bottom Line: The primordial germ cells (PGCs) are the first cells to form during Drosophila melanogaster embryogenesis.In addition to using core regulators of cleavage, including the small GTPase RhoA (Drosophila rho1) and the Rho-associated kinase, ROCK (Drosophila drok), we show that this pathway requires Germ cell-less (GCL), a conserved BTB-domain protein not previously implicated in cleavage mechanics.This alternative form of cell formation suggests that organisms have evolved multiple molecular strategies for regulating the cytoskeleton during cleavage.

View Article: PubMed Central - PubMed

Affiliation: HHMI and Kimmel Center for Biology and Medicine at the Skirball Institute, Department of Cell Biology, New York University School of Medicine, New York, New York 10016, USA.

ABSTRACT
The primordial germ cells (PGCs) are the first cells to form during Drosophila melanogaster embryogenesis. Whereas the process of somatic cell formation has been studied in detail, the mechanics of PGC formation are poorly understood. Here, using four-dimensional multi-photon imaging combined with genetic and pharmacological manipulations, we find that PGC formation requires an anaphase spindle-independent cleavage pathway. In addition to using core regulators of cleavage, including the small GTPase RhoA (Drosophila rho1) and the Rho-associated kinase, ROCK (Drosophila drok), we show that this pathway requires Germ cell-less (GCL), a conserved BTB-domain protein not previously implicated in cleavage mechanics. This alternative form of cell formation suggests that organisms have evolved multiple molecular strategies for regulating the cytoskeleton during cleavage.

Show MeSH
Related in: MedlinePlus