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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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Anillin-GFP and Myosin-GFP localize to paired cleavage furrows during Drosophila PGC formation(a) Diagram of 4D-imaging strategy used to capture PGC formation at the posterior of the Drosophila embryo. The embryo is positioned with its dorsal surface closest to the cover slip. 40 Z-slices (red line) spaced 1 μm apart along the dorsal-ventral axis were acquired per time point at the apex of the posterior pole. (b) Micrographs of time-lapse maximum intensity projections (MIP) of paired furrows during PGC formation revealed with Anillin-GFP (see Supplementary Video 1). (c) Time-lapse micrographs (single optical sections) of a single bud during PGC formation revealed by Myosin-GFP and Vasa-KO (see Supplementary Video 2). (b, c) Arrows and arrowheads mark the anaphase furrow (AF) and bud furrow (BF) respectively. (d and e) Kymographs showing the localization and quantification of (d) Anillin-GFP and (e) Myosin-GFP at a single paired furrow during PGC formation. We consistently observed biphasic enrichment of Myosin-GFP at the BF suggesting regulation by the cell cycle as previously reported31. Data shows the mean of quantifications done in (d) = 4 embryos and (e) = 4 embryos, with 3 buds measured in each embryo. Error bars: S.D. (f) Graphical description of PGC formation showing the remodeling of one bud into two cells. Scale bars = 5 μm
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Figure 1: Anillin-GFP and Myosin-GFP localize to paired cleavage furrows during Drosophila PGC formation(a) Diagram of 4D-imaging strategy used to capture PGC formation at the posterior of the Drosophila embryo. The embryo is positioned with its dorsal surface closest to the cover slip. 40 Z-slices (red line) spaced 1 μm apart along the dorsal-ventral axis were acquired per time point at the apex of the posterior pole. (b) Micrographs of time-lapse maximum intensity projections (MIP) of paired furrows during PGC formation revealed with Anillin-GFP (see Supplementary Video 1). (c) Time-lapse micrographs (single optical sections) of a single bud during PGC formation revealed by Myosin-GFP and Vasa-KO (see Supplementary Video 2). (b, c) Arrows and arrowheads mark the anaphase furrow (AF) and bud furrow (BF) respectively. (d and e) Kymographs showing the localization and quantification of (d) Anillin-GFP and (e) Myosin-GFP at a single paired furrow during PGC formation. We consistently observed biphasic enrichment of Myosin-GFP at the BF suggesting regulation by the cell cycle as previously reported31. Data shows the mean of quantifications done in (d) = 4 embryos and (e) = 4 embryos, with 3 buds measured in each embryo. Error bars: S.D. (f) Graphical description of PGC formation showing the remodeling of one bud into two cells. Scale bars = 5 μm

Mentions: To determine the mechanism of PGC formation, we began our studies by analyzing the events leading to PGC formation. When nuclei reach the embryonic cortex at the tenth nuclear cycle, they induce membrane and cytoplasmic protrusions, called ‘buds’4,20 (Supplementary Fig. S1a). Although the majority of these buds collapse shortly after their nuclei enter mitosis, the small fraction of buds that form within the germ plasm are reorganized into PGCs. To capture the transformation of buds into cells, we developed a 4D-imaging assay (Fig. 1a) that revealed and quantified the localization of green fluorescent protein (GFP), fused to either of two known cleavage furrow components, Myosin-II regulatory light chain-GFP (MRLC, Drosophila Sqh; now called ‘Myosin-GFP’) and Anillin-GFP (Drosophila Scraps), along with a kusabira orange fused germ plasm marker, Vasa-KO21-23 (Fig. 1b and c). We found that both Anillin-GFP and Myosin-GFP were enriched at the neck of posterior buds (hereafter termed the ‘bud furrow’, BF)(Fig. 1d and e, Supplementary Fig. 1e). When nuclei within these buds entered mitosis, the BF constricted beneath the chromosomes, in a plane parallel to the mitotic spindle. During anaphase, a second cleavage furrow (hereafter termed the ‘anaphase furrow’, AF) assembled orthogonally to both the mitotic spindle and BF (Fig. 1b and c, Supplementary Fig. S1b and f, Supplementary Video S1 and S2). Although the AF ingressed asymmetrically, it divided the bud into two daughter cells in a manner similar to a cytokinetic furrow (Fig. 1c, Supplementary Fig. S1f). In contrast, BF cleavage separated the bud from the embryo, asymmetrically partitioning the germ plasm, marked by Vasa-KO, into the PGCs (Fig. 1c and Supplementary Video S2). Following their constriction, these paired furrows (AF-BF) resolved into a tripartite midbody-like structure that attached the newly formed cells to the embryonic cortex (Supplementary Fig. S1c and d). We conclude that the constriction of two orthogonally paired furrows remodels one bud into two PGCs (Fig. 1f).


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

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

Anillin-GFP and Myosin-GFP localize to paired cleavage furrows during Drosophila PGC formation(a) Diagram of 4D-imaging strategy used to capture PGC formation at the posterior of the Drosophila embryo. The embryo is positioned with its dorsal surface closest to the cover slip. 40 Z-slices (red line) spaced 1 μm apart along the dorsal-ventral axis were acquired per time point at the apex of the posterior pole. (b) Micrographs of time-lapse maximum intensity projections (MIP) of paired furrows during PGC formation revealed with Anillin-GFP (see Supplementary Video 1). (c) Time-lapse micrographs (single optical sections) of a single bud during PGC formation revealed by Myosin-GFP and Vasa-KO (see Supplementary Video 2). (b, c) Arrows and arrowheads mark the anaphase furrow (AF) and bud furrow (BF) respectively. (d and e) Kymographs showing the localization and quantification of (d) Anillin-GFP and (e) Myosin-GFP at a single paired furrow during PGC formation. We consistently observed biphasic enrichment of Myosin-GFP at the BF suggesting regulation by the cell cycle as previously reported31. Data shows the mean of quantifications done in (d) = 4 embryos and (e) = 4 embryos, with 3 buds measured in each embryo. Error bars: S.D. (f) Graphical description of PGC formation showing the remodeling of one bud into two cells. Scale bars = 5 μm
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Figure 1: Anillin-GFP and Myosin-GFP localize to paired cleavage furrows during Drosophila PGC formation(a) Diagram of 4D-imaging strategy used to capture PGC formation at the posterior of the Drosophila embryo. The embryo is positioned with its dorsal surface closest to the cover slip. 40 Z-slices (red line) spaced 1 μm apart along the dorsal-ventral axis were acquired per time point at the apex of the posterior pole. (b) Micrographs of time-lapse maximum intensity projections (MIP) of paired furrows during PGC formation revealed with Anillin-GFP (see Supplementary Video 1). (c) Time-lapse micrographs (single optical sections) of a single bud during PGC formation revealed by Myosin-GFP and Vasa-KO (see Supplementary Video 2). (b, c) Arrows and arrowheads mark the anaphase furrow (AF) and bud furrow (BF) respectively. (d and e) Kymographs showing the localization and quantification of (d) Anillin-GFP and (e) Myosin-GFP at a single paired furrow during PGC formation. We consistently observed biphasic enrichment of Myosin-GFP at the BF suggesting regulation by the cell cycle as previously reported31. Data shows the mean of quantifications done in (d) = 4 embryos and (e) = 4 embryos, with 3 buds measured in each embryo. Error bars: S.D. (f) Graphical description of PGC formation showing the remodeling of one bud into two cells. Scale bars = 5 μm
Mentions: To determine the mechanism of PGC formation, we began our studies by analyzing the events leading to PGC formation. When nuclei reach the embryonic cortex at the tenth nuclear cycle, they induce membrane and cytoplasmic protrusions, called ‘buds’4,20 (Supplementary Fig. S1a). Although the majority of these buds collapse shortly after their nuclei enter mitosis, the small fraction of buds that form within the germ plasm are reorganized into PGCs. To capture the transformation of buds into cells, we developed a 4D-imaging assay (Fig. 1a) that revealed and quantified the localization of green fluorescent protein (GFP), fused to either of two known cleavage furrow components, Myosin-II regulatory light chain-GFP (MRLC, Drosophila Sqh; now called ‘Myosin-GFP’) and Anillin-GFP (Drosophila Scraps), along with a kusabira orange fused germ plasm marker, Vasa-KO21-23 (Fig. 1b and c). We found that both Anillin-GFP and Myosin-GFP were enriched at the neck of posterior buds (hereafter termed the ‘bud furrow’, BF)(Fig. 1d and e, Supplementary Fig. 1e). When nuclei within these buds entered mitosis, the BF constricted beneath the chromosomes, in a plane parallel to the mitotic spindle. During anaphase, a second cleavage furrow (hereafter termed the ‘anaphase furrow’, AF) assembled orthogonally to both the mitotic spindle and BF (Fig. 1b and c, Supplementary Fig. S1b and f, Supplementary Video S1 and S2). Although the AF ingressed asymmetrically, it divided the bud into two daughter cells in a manner similar to a cytokinetic furrow (Fig. 1c, Supplementary Fig. S1f). In contrast, BF cleavage separated the bud from the embryo, asymmetrically partitioning the germ plasm, marked by Vasa-KO, into the PGCs (Fig. 1c and Supplementary Video S2). Following their constriction, these paired furrows (AF-BF) resolved into a tripartite midbody-like structure that attached the newly formed cells to the embryonic cortex (Supplementary Fig. S1c and d). We conclude that the constriction of two orthogonally paired furrows remodels one bud into two PGCs (Fig. 1f).

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