Small GTPases of the Ras-like (Ral) family are crucial for signalling

Small GTPases of the Ras-like (Ral) family are crucial for signalling functions in both normal and cancer cells; however their role in a developing organism is poorly understood. The loss of RalA also causes a cell non-autonomous phenotype owing to reduced Jak/Stat signalling in neighbouring follicle cells. As a result border-cell assembly and migration as well as the polarization of the oocyte are defective. Thus is required in organizing centres to control proper patterning and migration egg chambers using a set of 311 independent P-element-mediated lethal mutations targeting individual genes on the underscreened X chromosome (Bourbon homologue of human genes (Fig 1D). Another insertion PG89 in the first intron of (Fig 1D) was analysed and showed phenotypes similar to those of PG69 (data not shown). As PG69 and PG89 are Gal4-expressing enhancer-trap lines (Bourbon mutant background. Expression of TH-302 UAS-(a wild-type form of RalA) or UAS-(a constitutively activated form; Sawamoto (a dominant-negative form; Sawamoto gene. Furthermore a crossreacting polyclonal antibody directed against the human RALB protein allowed detection of RalA in wild type which was strongly reduced in mutant ovaries taken from a viable combination (Fig 1E). Figure 2 Expression of RalA and sequence determinants for RalA subcellular localization. (A) Expression of RalA in border cells (BCs) and follicle cells was detected using a hybridization and specific enhancer-trap lines (Fig 2A-D″). The RalA protein is localized to the plasma membrane in ovaries and we showed that a carboxy-terminal sequence containing a putative CAAX box (198-CTLL-STOP) important for prenylation of H-Ras and other small GTPases is essential for RalA subcellular localization and function. Indeed the deletion or hiding of this motif was sufficient to abolish both the rescue activity and plasma membrane localization of RalA (Fig 2E H-J). RalA has been shown to be downstream from Rap1 for bristle development (Mirey function we analysed mosaic clusters containing zero one or two mutant PCs with wild-type or mutant oBCs. We found that the BC cluster formed and migrated normally when all oBCs were mutant for (Fig 3A-B′) indicating that function is not required in oBCs. Consistently mosaic analysis showed an TH-302 essential function of in PCs with a varied phenotype depending on the number of mutant PCs present Rabbit polyclonal to Catenin T alpha. in the BC cluster. When only one of TH-302 the two PCs was mutant the BC formed but did not TH-302 migrate (Figs 1C 3 In this case the number of oBCs was reduced with an average of 3.5±0.7 oBCs (and enhancer-trap expression (Fig 4G-H′; supplementary Fig 1 online). Note that the expression of these oBC markers remained unaffected when only oBCs were mutant for (data not shown) indicating a cell non-autonomous function of in PCs to control recruitment of oBCs and migration of the cluster. Figure 3 RalA is essential in polar cells for border-cell recruitment and migration. (A) Mosaic border-cell (BC) cluster in which the two polar cells (PCs; arrows in B B′) are wild type whereas outer BCs (oBCs; enclosed by dotted lines) are … Figure 4 RalA controls polar-cell and terminal follicle cell differentiation. Specific markers for polar cells (PCs; Fas3 A101-lacZ PZ80-lacZ) and terminal follicle cells (Slbo-lacZ) were used to analyse cell fate in homozygous … To analyse further the role of in PC function we stained egg chambers using a set of PC-specific markers. These markers are expressed both at the anterior and posterior poles so clones made at either side of the egg chamber have been analysed indifferently. When anterior or posterior PCs were mutant for is essential for proper PC differentiation (Fig 4A-F). The effect of mutations on Fas3 protein was stage specific: before stage 6-7 Fas3 staining was normal whereas later on staining was strongly reduced or absent suggesting that RalA controls the maintenance of PC differentiation. It has been shown that PCs originate from a larger group of pre-PCs in which cell number is reduced from 3-5 to 2 through apoptosis at early stages (Besse & Pret 2003 To assess whether de-differentiated PCs could initiate apoptosis we stained egg chambers with antibodies raised against activated caspase 3 (Casp3). Interestingly some of the mutant PCs (20% was recently shown to protect cells from apoptosis in the sensory lineage (Balakireva development. Altogether our results identify as a new regulator to maintain PC fate and survival. RalA shows a cell non-autonomous phenotype originating from the PC. PCs are essential anteriorly for recruiting a ring of around six oBCs that make a mature BC cluster which depends.