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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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Germ cell-less is a rate-limiting component of bud furrow constriction(a) Time-lapse micrographs (single optical sections) showing the BF diameter revealed with Anillin-GFP in wt, gcl mutant and Gcl-overexpressing embryos (EP-gcl) between t=-322s and t=-196s prior to AF formation (see Supplementary Video 6, 7 and 8). (b) Quantification of the BF diameter between t=-322s and t=-196s in wt, gcl mutant and EP-gcl embryos. c, BF rate of constriction between t=-322s and t=-196s in wt, gcl mutant and EP-gcl buds calculated from the slopes of the lines shown in (b). Total number of embryos analyzed for c, d, e: wt = 7, gcl = 6, EP-gcl = 8, where 1 or 2 buds were measured in each embryo. One-way Anova: p < .05 for wt vs gcl and p < .001 for wt vs EP-gcl. Scale bars = 5 μm
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Figure 4: Germ cell-less is a rate-limiting component of bud furrow constriction(a) Time-lapse micrographs (single optical sections) showing the BF diameter revealed with Anillin-GFP in wt, gcl mutant and Gcl-overexpressing embryos (EP-gcl) between t=-322s and t=-196s prior to AF formation (see Supplementary Video 6, 7 and 8). (b) Quantification of the BF diameter between t=-322s and t=-196s in wt, gcl mutant and EP-gcl embryos. c, BF rate of constriction between t=-322s and t=-196s in wt, gcl mutant and EP-gcl buds calculated from the slopes of the lines shown in (b). Total number of embryos analyzed for c, d, e: wt = 7, gcl = 6, EP-gcl = 8, where 1 or 2 buds were measured in each embryo. One-way Anova: p < .05 for wt vs gcl and p < .001 for wt vs EP-gcl. Scale bars = 5 μm

Mentions: Since Gcl overexpression within the germ plasm directs additional posterior buds to undergo PGC formation38, we considered two final models, in which Gcl acts as either a permissive or instructive signal for BF cleavage. To distinguish between these two models, we filmed BF cleavage in control, gcl mutant and Gcl overexpressing (EP-gcl) embryos using our 4D-imaging assay (Supplementary Video 9, 10 and 11). We measured the BF diameter at several time points and calculated the rate of furrow constriction (Fig. 4a and b). If Gcl acts as a permissive cue, we would expect equal rates of constriction in control and Gcl overexpressing embryos. However, we found that the rate of constriction was proportional to the amount of Gcl present within the germ plasm (Fig. 4c). Indeed, even in control embryos, where the highest concentration of Gcl protein is found in the most posterior buds, we found a direct correlation between the position of the bud and the degree of BF constriction (Supplementary Fig. S5). Thus, we conclude that Gcl activity instructs BF cleavage by regulating the rate of furrow constriction.


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

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

Germ cell-less is a rate-limiting component of bud furrow constriction(a) Time-lapse micrographs (single optical sections) showing the BF diameter revealed with Anillin-GFP in wt, gcl mutant and Gcl-overexpressing embryos (EP-gcl) between t=-322s and t=-196s prior to AF formation (see Supplementary Video 6, 7 and 8). (b) Quantification of the BF diameter between t=-322s and t=-196s in wt, gcl mutant and EP-gcl embryos. c, BF rate of constriction between t=-322s and t=-196s in wt, gcl mutant and EP-gcl buds calculated from the slopes of the lines shown in (b). Total number of embryos analyzed for c, d, e: wt = 7, gcl = 6, EP-gcl = 8, where 1 or 2 buds were measured in each embryo. One-way Anova: p < .05 for wt vs gcl and p < .001 for wt vs EP-gcl. Scale bars = 5 μm
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
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Figure 4: Germ cell-less is a rate-limiting component of bud furrow constriction(a) Time-lapse micrographs (single optical sections) showing the BF diameter revealed with Anillin-GFP in wt, gcl mutant and Gcl-overexpressing embryos (EP-gcl) between t=-322s and t=-196s prior to AF formation (see Supplementary Video 6, 7 and 8). (b) Quantification of the BF diameter between t=-322s and t=-196s in wt, gcl mutant and EP-gcl embryos. c, BF rate of constriction between t=-322s and t=-196s in wt, gcl mutant and EP-gcl buds calculated from the slopes of the lines shown in (b). Total number of embryos analyzed for c, d, e: wt = 7, gcl = 6, EP-gcl = 8, where 1 or 2 buds were measured in each embryo. One-way Anova: p < .05 for wt vs gcl and p < .001 for wt vs EP-gcl. Scale bars = 5 μm
Mentions: Since Gcl overexpression within the germ plasm directs additional posterior buds to undergo PGC formation38, we considered two final models, in which Gcl acts as either a permissive or instructive signal for BF cleavage. To distinguish between these two models, we filmed BF cleavage in control, gcl mutant and Gcl overexpressing (EP-gcl) embryos using our 4D-imaging assay (Supplementary Video 9, 10 and 11). We measured the BF diameter at several time points and calculated the rate of furrow constriction (Fig. 4a and b). If Gcl acts as a permissive cue, we would expect equal rates of constriction in control and Gcl overexpressing embryos. However, we found that the rate of constriction was proportional to the amount of Gcl present within the germ plasm (Fig. 4c). Indeed, even in control embryos, where the highest concentration of Gcl protein is found in the most posterior buds, we found a direct correlation between the position of the bud and the degree of BF constriction (Supplementary Fig. S5). Thus, we conclude that Gcl activity instructs BF cleavage by regulating the rate of furrow constriction.

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