In the present study we show that scrib/dlg function in both anterior and posterior patterning of the follicular epithelium. While removal of scrib or dlg function from the FCs at the posterior terminal completely blocked specification and differentiation of the PFCs, loss-of-function mutations in scrib/dlg at the anterior domain resulted in a partially penetrant phenotype of defective AFC cell fate induction as indicative of absence of the stretched and centripetal cell types at stage 10 of egg chambers. The differential regulation of the PFC and AFC cell differentiation by scrib/dlg could be attributable to the distinct signaling basis underlying the follicular patterning at the two terminals. In the case of posterior patterning, the combinatorial and sequential activities of JAK/STAT, EGFR and Notch signaling pathways play key roles in this process. The fully penetrant phenotype of aberrant PFC fate specification in posterior scrib/dlg mutant clones can be explained by the fact that inactivation of scrib or dlg completely perturb the EGFR signaling. Further investigation demonstrated that scrib/dlg mutation also causes defects in coordinated activation of JAK/STAT and Notch pathways in each multilayered clone cell, as evident in localized activity of JAK signaling and Notch signaling in the outer layer and inner layer respectively (Fig 4B, C, E, G, H, and 4J). Thus, the mutant cells at the posterior terminals generally do not adopt a terminal cell fate either. At the anterior, AFC fate induction requires JAK and Notch signaling activities. Current data, however, do not exclude a possibility that other unknown signals are involved in specifying the distinct AFC cell types, e.g. the stretched and centripetal cells[1, 6, 7]. We observed that while JAK signaling activity is present in the FCs surrounding the polar cells, preferentially in outer cells of the multilayered clones at the anterior terminals, activation of Notch pathway occurs in almost all mutant cells. The combined pattern of JAK and Notch signaling activity in the anterior mutant cells provides a good basis that the border cell, at least Slbo-expressing cell fate, can be induced in the most anterior region of the multilayered clones. However, our clonal analysis revealed specification of the stretched or centripetal cell types in corresponding mutant clones at the anterior is disrupted, albeit to a lesser extent. Considering the presence of JAK signaling activity in the mutant clone cells, we assume that loss-of-function mutation of scrib/dlg may perturb other unidentified signals implicated in patterning of the stretched and centripetal cell subpopulations.
We have identified in this report aberrant EGFR signaling pathway as the mechanism underlying defective PFC fate induction in scrib/dlg mutant FCs. In addition to EGFR pathway, loss of scrib or dlg function at the terminal domains can differentially affect JAK and Notch signaling activities in the multilayered clone cells. JAK signaling was absent in inner cells of the multilayered clones at the two terminals. Conversely, loss of Notch activity was localized to outer cells of the posterior multilayered clones. This discrepancy is likely to be linked to the spatial and temporal control of each signaling activity with respect to growth and patterning of the FC layer during oogenesis. Starting from stage 6 of oogenesis, Notch signaling is activated in all epithelial FCs for inducing the mitotic-to-endocycle transition [12, 13]. Given that at this time the multilayered cells have been formed in scrib/dlg mutant clones at the terminals, it can be imagined that activation of Notch pathway occurs only in the mutant cells directly contacting the Delta-producing germ cell, as shown in the inner cells of the posterior mutant clones (Fig 4E, G, H and 4J). This scenario was further justified by the recent report that loss of lgl causes the same aberrant Notch signaling pattern in the posterior multilayered FCs as scrib/dlg mutation does . Surprisingly, the multilayered scrib/dlg mutant clones at the anterior display a distinct Notch signaling pattern in which almost all FCs regardless of their spatial relation with the oocyte are positive for Notch activation (Fig 6E, F and 6G). Although at this point we do not understand the basis for this controversial pattern of Notch activity in the mutant clones at different terminals, this mechanism may partly underlie the observation in the present study that loss-of-function mutations in scrib/dlg differentially affect anterior and posterior patterning of the epithelium.
Likewise, the distinct pattern for JAK/STAT pathway activity between the inner and outer cells in scrib/dlg mutant clones is probably due to the spatial location of the ligand sources relative to the multilayered cells. Indeed, analysis of the polar cell positioning in the mutant clones revealed that the polar cells are in close proximity to the single layer of outer cells that retain JAK signaling activity (scrib2, 95.3%, n = 43; dlgm52, 88.9%, n = 27) (see Additional file 1) (Fig 4B, C). The positioning of the polar cells led us to argue that inner cells in the multilayered clones do not respond to the signaling ligand Unpaired secreted from polar cells, presumably due to their spatial relation with the ligand source. However, unlike the Notch pathway, JAK signaling is activated in FCs at the terminal domains of the egg chambers at early oogenesis after the polar/stalk cells are specified [6, 7, 22]. This temporal regulation might be alternatively responsible for the distinct JAK/STAT activity pattern in the multilayered clone cells. In this model, we assume that JAK/STAT pathway is activated in the mutant FCs at the terminal domains prior to occurrence of the cell overaccumulation. Thus, the presence of JAK signaling activity in single outer cell layer of the multilayered scrib/dlg mutant clones may indicate the initial activation of JAK/STAT pathway induced by the polar cells for specifying the terminal fate at early oogenesis. On the contrary, the inner cells deriving from the overaccumulation fail to respond to Unpaired ligand. Further studies in this direction will better define the underlying mechanisms for defective follicular patterning elicited by loss of scrib or dlg.
The phenotypic effects of scrib/dlg mutation in posterior patterning of the epithelium are similar to those of the Hpo pathway deficiency [24–26]. Further characterization of the patterning defects, however, reveals distinct underlying mechanisms for these two instances. In the case of the Hpo pathway, loss of the pathway component Hippo, Salvador, or Warts disrupts Notch signaling in all mutant FCs at the posterior via interfering with endocytosis of the Notch receptor, thereby resulting in aberrant PFC cell specification and differentiation at mid-oogenesis [24–26]. By contrast, activation of the Notch signaling is evident in inner cells of the multilayered scrib/dlg mutant clones at the posterior (Fig 4E, G, H and 4J) and almost all the multilayered clone cells at the anterior (Fig 6E, F and 6G). Furthermore, the fully disrupted EGFR pathway associated with the posterior patterning defects conferred by loss of scrib or dlg clearly distinguishes the Hpo pathway from scrib/dlg in the signaling basis for the mutant phenotype of defective PFC fate induction ([24–26] and this paper). In Drosophila, scrib/dlg are known to encode scaffolding proteins that are localized at the septate (basolateral) junctions of epithelial cells, and regulate the apico-basal cell polarity[31, 35, 50–52]. Previous studies have demonstrated that mutations in scrib/dlg disrupt the epithelial polarity in the FCs at the terminal domains of egg chambers, exhibiting mislocalized cell polarity proteins [31, 48, 53]. Based on this fact, one would surmise that the polarity defects observed in the posterior scrib/dlg mutant FCs perturb the apical accumulation of EGFR receptors, rendering these cells incompetent to respond to EGF signals due to failure of EGFR activation. However, further studies in this paper disapproved this simple scenario. Instead, we found that ERK is di-phosphorylated in the posterior FCs lacking scrib or dlg, suggesting that the EGFR in the mutant cells can still be activated in response to Grk signal. Thus, this finding points out that blocking in signal transduction from the activated ERK to the downstream targets elicited by loss of scrib or dlg may result in failure of the mutant FCs at the posterior to respond to EGFR signals.
Once the specified FC cell types are induced, each cell population will undergo a unique morphogenetic change and execute respective functions . Remarkably, we observed a concurrent defect in morphogenesis of those anterior scrib/dlg mutant clone cells with aberrant patterning of the stretched or centripetal cell subpopulation (Fig 5E, G and Fig 6I, J). It would be interesting and important to determine whether scrib/dlg is implicated in morphogenesis of the patterned follicular epithelium as well. For this purpose, we need to identify a scrib or dlg mutant allele for certain genetic background in which the morphogenesis can be uncoupled from the patterning process. Under such circumstance could we generate scrib/dlg mutant clones with proper patterning of the AFC cell types, and then test how the subsequent morphogenesis occurs in the specified cell subpopulations, e.g. the stretched and centripetal cells. Likewise, a hypomorphic scrib or dlg allele with certain reduced activity could be of great value to understanding better how the specified PFCs function in polarization of the oocyte at mid-oogenesis. Interestingly, we found that RNAi-mediated knockdown of the endogenous dlg expression alone in the follicular epithelium can disrupt the oocyte polarity as indicative of mislocalization of Stau, but properly induce the PFC fate (our unpublished data). This unexpected observation led us to propose that expression of this dlg
transgene may specifically perturb the process in which the specified PFCs control formation of the oocyte polarity. Thus, screening a Drosophila mutant library such as the transgenic RNAi library for gene(s) modifying the dlg
phenotype would unveil the mechanisms responsible for involvements of dlg in regulation of the PFC function.