Ontogenesis of complex multicellular organisms involves the generation of different cell types. One mechanism by which cell fate diversity can be achieved is asymmetric cell division. During asymmetric cell division, progenitor cell polarity directs the orientation of the mitotic spindle and the asymmetric localization of cell fate regulators, thereby ensuring unequal segregation of these regulators [1], [2].

Each mechanosensory bristle located on the notum of Drosophila is composed of four different cells. These cells originate from a single sensory organ precursor cell (SOP) via a fixed lineage comprising four stereotyped asymmetric cell divisions [3]. The SOP first divides within the plane of the epithelium to generate a posterior pIIa cell and an anterior pIIb cell [4]. Two regulators of Notch receptor signaling, Numb and Neuralized (Neur), localize at the anterior cortex of dividing SOPs and segregate into the anterior daughter cell [5], [6]. Numb inhibits Notch whereas Neur positively regulates the signaling activity of the Notch ligand Delta. This therefore leads to Notch inhibition in the anterior cell that adopts the pIIb fate and Notch activation in the posterior cell that becomes pIIa.

By analogy with the role of atypical Protein Kinase C (aPKC) in neuroblasts [7], asymmetric localization of Numb and Neur at the anterior cortex of dividing SOPs is thought to depend on the kinase activity of aPKC, which localizes at the posterior pole. In brief, aPKC phosphorylates and inhibits Lethal (2) giant larvae (Lgl) at the posterior cortex, such that active nonphosphorylated Lgl protein is restricted to the anterior cortex where it promotes the cortical localization of Numb and Neur [7], [8]. Direct phosphorylation of Numb by aPKC may further contribute to restricting the localization of Numb to the anterior cortex [9]. Additionally, drug studies have shown that depolymerization of microfilaments prevents cortical localization of Numb and Neur in dividing SOPs, indicating that actin plays an essential role in their polar distribution [6], [10]. Lastly, anterior localization of Numb probably also involves its interaction with Partner of Numb (Pon). The Pon protein contains an N-terminal Numb-interacting domain, a central coiled-coil domain, and a C-terminal localization domain (LD) that is sufficient for its asymmetric localization in neuroblasts and SOPs. Pon colocalizes with Numb and is required, at least in neuroblasts, for its asymmetric localization [11], [12].

Phosphatidylinositol 4,5-bisphosphate (PIP2) is a phospholipid present at the inner leaflet of the plasma membrane that has a wide range of proposed functions [13]. PIP2 directly interacts with several actin regulators [14] as well as with proteins known to be involved in the process of asymmetric division, including Par3 [15], Neuralized [16] and Numb [17]. PIP2 is mostly produced by type I (PIP5KIs) phosphatidylinositol phosphate 5-kinases that use phosphatidylinositol 4-phosphate (PI(4)P) as a substrate [13]. The Drosophila genome encodes three predicted PIP5KIs: PIP5K59B, CG17471 and skittles (sktl) [18], [19]. Recently, the sktl gene has been shown to play a critical role in PIP2 synthesis in the oocyte (Gervais et al., unpublished). Interestingly, the sktl gene appears to be expressed in SOPs [18]. To begin studying the potential role of PIP2 in asymmetric cell division, we have examined here the localization of Sktl and PIP2 reporters in dividing SOPs. Our analysis indicates that PIP2 and Sktl are distributed at the cortex of dividing SOPs with no clear sign of planar asymmetry. However, in the course of these experiments, we have observed that an increased accumulation of Pon, which is known to accumulate at the anterior cortex in mitotic SOPs, resulted in the posterior localization of Sktl. We discuss here the practical implications and possible biological significance of this overexpression phenotype.

We have shown that PIP2 and the PIP2-producing kinase Sktl are distributed at the cortex of dividing SOPs with no asymmetry within the plane of the epithelium. Whether PIP2 and Sktl regulate SOP polarization and/or polar localization of cell fate determinants in dividing SOPs remains to be further studied. Of note, we have not been able to analyze the phenotypes associated with a loss of sktl activity in the notum since sktl mutant clones fail to grow in wing imaginal discs (C.P., unpublished results).

We have also shown that overexpression of Pon can alter the spatial distribution of PIP2 binding proteins and of endogenous Sktl in dividing SOPs. This unexpected observation raises at least two questions and should also serve as a note of caution for studies using Pon as a marker of polarity.

Multiple mechanisms may explain how Pon overexpression alters the distribution of Sktl. One hypothesis is that both proteins compete for transport machinery components and/or cortical anchoring sites. Endogenous Pon would not affect the transport and/or anchoring of Sktl but overexpression of Pon could saturate the system. Also, the posterior pole accumulation of the PIP2-binding proteins PH::GFP and ENTH::GFP in Pon-overexpressing cells can be interpreted to suggest that overexpression of Pon results either in lower PIP2 concentration at the anterior pole or in higher concentration of competing PIP2-interacting proteins. It would also be interesting to examine whether the localization of the PIP2-producing kinase is itself regulated by PIP2 levels.

How may endogenous Pon regulate protein distribution at the cortex of asymmetrically dividing progenitors? A known function of Pon is to recruit Numb, most likely via direct protein-protein interaction [11], [12]. In this case, Pon plays a positive role by recruiting specific proteins at one pole of the dividing cell. However, our observation that Sktl accumulates at the posterior cortex when Pon is overexpressed raises the hypothesis that endogenous Pon may also normally exclude proteins from the membrane domain where it localizes.

Finally, our observation that Pon and RFP::Pon^LD alter the distribution of both PIP2 reporters and Sktl is relevant since both RFP::Pon^LD and GFP::Pon^LD have often been used to study asymmetric cell division of neural precursor cells in Drosophila [see: [8], [11], [26]] including detailed quantitative aspects of asymmetric protein localization [27]. As accumulation of free PIP2-binding sites at the posterior pole of RFP::Pon^LD-expressing cells is likely to cause subtle deviations from the wild-type, our observation should serve as a note of caution when interpreting studies that use RFP::Pon^LD as a marker.

The following transgenes were expressed using neur^P72Gal4 [26] combined with a pTub-GAL80^ts transgene: ENTH::GFP [22], [28], PH::GFP [21], RFP::Pon^LD [24], Sktl::GFP (Gervais, in press), mycPon [11], mycMira [29], mycNumb [30].

Live GFP imaging was carried out as described in [26]. All images were acquired on SP2 and SP2AOBS confocal microscopes. Quantification of GFP signal intensity was performed using the plot profiling function of ImageJ 1.32.

Pupal nota were dissected from staged pupae and processed as previously described [3]. Primary antibodies were: rabbit anti-Sktl (Gervais et al. unpublished; 1∶500), guinea-pig anti-Senseless (Sens; gift from H. Bellen; 1∶3000), rabbit anti-GFP (Molecular Probes; 1∶1000) and mouse anti-Myc (9E10; DSHB; 1∶500). Cy3- and Cy5-coupled secondary antibodies were from Jackson's Laboratories and Alexa-488 coupled secondary antibodies were from Molecular Probes.