Academic journal article Genetics

Germ Cell Segregation from the Drosophila Soma Is Controlled by an Inhibitory Threshold Set by the Arf-GEF Steppke

Academic journal article Genetics

Germ Cell Segregation from the Drosophila Soma Is Controlled by an Inhibitory Threshold Set by the Arf-GEF Steppke

Article excerpt

ABSTRACT Germline cells segregate from the soma to maintain their totipotency, but the cellular mechanisms of this segregation are unclear. The Drosophila melanogaster embryo forms a posterior group of primordial germline cells (PGCs) by their division from the syncytial soma. Extended plasma membrane furrows enclose the PGCs in response to the germ plasm protein Germ cell-less (Gcl) and Rho1-actomyosin activity. Recently, we found that loss of the Arf-GEF Steppke (Step) leads to similar Rho1-dependent plasma membrane extensions but from pseudocleavage furrows of the soma. Here, we report that the loss of step also leads to premature formation of a large cell group at the anterior pole of the embryo . These anterior cells lacked germ plasm, but budded and formed at the same time as posterior PGCs, and then divided asynchronously as PGCs also do. With genetic analyses we found that Step normally activates Arf small G proteins and antagonizes Rho1-actomyosin pathways to inhibit anterior cell formation. A uniform distribution of step mRNA around the one-cell embryo cortex suggested that Step restricts cell formation through a global control mechanism. Thus, we examined the effect of Step on PGC formation at the posterior pole. Reducing Gcl or Rho1 levels decreased PGC numbers, but additional step RNAi restored their numbers. Reciprocally, GFP-Step overexpression induced dosage- and Arf-GEF-dependent loss of PGCs, an effect worsened by reducing Gcl or actomyosin pathway activity. We propose that a global distribution of Step normally sets an inhibitory threshold for Rho1 activity to restrict early cell formation to the posterior.

KEYWORDS germline segregation; cell division; Arf-GEF activity; Rho1 inhibition; Drosophila

EMBRYONIC speci fi cation of primordial germline cells (PGCs) distinguishes them from somatic cells to maintain the totipotency of the germline (Seydoux and Braun 2006; Hayashi et al. 2007; Strome and Lehmann 2007; Johnson et al. 2011). Although this segregation occurs across animals, its cellular bases remain unclear.

The early Drosophila embryo segregates the germline from the soma through an extreme form of asymmetric cell division. The very early embryo is a syncytium of dividing nuclei. At nuclear cycle 9, a group of nuclei are recruited from the subcortex to the posterior pole of the syncytium, and each induces transient, shallow, dome-like buds at the embryo surface. During nuclear cycle 10, these posterior cells bud again and then divide fully from the remaining syncytium. This asymmetric division forms the PGCs at the posterior pole of the embryo. The remaining somatic nuclei continue dividing as a syncytium until 13 rounds of nuclear division are complete, at which point they too divide into separate cells through the process of cellularization that forms the blastoderm (Foe and Alberts 1983).

The asymmetric division of PGCs from the soma is dictated by germ plasm deposited maternally at the posterior pole (Wilson and Macdonald 1993; Lehmann and Ephrussi 1994; Mahowald 2001). Specifically, the germ plasm protein Germ cell-less (Gcl) promotes activity of Rho1 and downstream actomyosin pathways to form extended plasma membrane furrows that encase single PGCs laterally and then basally (Cinalli and Lehmann 2013). Once the lateral membranes form, their basal tips expand perpendicularly to form basal membranes beneath each nucleus. These basal membranes have been termed "bud furrows" and are coated with cytoskeletal networks composed of actin, nonmuscle myosin II, and Anillin. These furrows form independently of spindles, and other than positive roles for Gcl and Rho1 (Cinalli and Lehmann 2013), it is unclear how bud furrows form and what prevents their formation elsewhere around the embryo.

We recently reported that early embryo depletion of the plasma membrane Arf-guanine nucleotide exchange factor (Arf-GEF) Steppke (Step) leads to premature basal membrane formation, but for pseudocleavage furrows that transiently separate somatic, syncytial nuclei (Lee and Harris 2013). …

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