To understand the function of SJs in the Drosophila wing imaginal disc epithelium, we first looked at the subcellular localization of the SJ component Nrx-IV using a GFP-tagged protein trap line previously shown to reflect Nrx-IV endogenous localization. SJs are typically localized at the apical region of the cell, below Adherens Junctions (AJs) and its associated cortical actin belt. Therefore, we compared Nrx-IV localization to F-actin in XZ sections. As expected, Nrx-IV is apically localized, just below F-actin (Fig. S1A-C).
To investigate the function of Nrx-IV, we knocked down the expression in the wing imaginal disc. For that we used the Gal4/UAS system to express double-stranded RNA (dsRNA) against Nrx-IV specifically in the posterior compartment of the wing disc using the hedgehog-Gal4 (hh-Gal4) driver. Using this approach, the anterior compartment of the same wing disc serves as an internal control.
The SJs form a stable protein complex in which the knockdown of a single component leads to mislocalization of the other components, thus compromising SJ function. Thus, to validate the Nrx-IV RNAi we checked whether its expression affects the localization of another SJ core component, Coracle, known to bind Nrx-IV (Fig. S1D-L’). As expected, in control wing discs Coracle is localized mostly at the apical region of cells (Fig. S1D’). We knocked-down Nrx-IV using two different RNAi lines. In both cases we observed a reduction of Coracle levels in the posterior compartment, where Nrx-IV RNAi was expressed (Fig. S1G, J). Additionally, Coracle localization was impaired: it was localized evenly along the lateral membrane and not mostly apically as in the control (more evident with the Nrx-IV RNAi dsRNA-HMS00419 line, Fig. S1G’, white arrowhead).
To assess the impact of Nrx-IV knockdown on epithelial integrity and polarity, we examined the localization of F-actin and the AJ protein E-cadherin on fixed wing discs. E-cadherin localization does not seem to be affected in Nrx-IV knockdown: both in control and Nrx-IV knockdown wing discs, E-cadherin was mostly localized at the apical-most region of cells (Fig. S2). F-actin staining revealed that, in contrast to control wing discs (Fig. 1A, A’), the posterior region of Nrx-IV knockdown wing discs displayed an abnormal shape (Fig. 1F, F’, K, K’, see wing disc pouch region, yellow dashed lines). The Nrx-IV RNAi posterior compartment typically presented a depression close to the anterior-posterior boundary (Fig. 1F’, K’, yellow arrowheads). Moreover, in some RNAi expressing cells, F-actin accumulated in more basolateral regions, whereas in control cells F-actin localized at the apical region (Fig 1F’, K’, white arrows). These data suggest that Nrx-IV is required for epithelial sheet organization but not for AJ localization.
Looking at the localization of the nuclear marker we noticed that, in contrast to controls, several Nrx-IV RNAi expressing cells presented fragmented nuclei and had delaminated from the epithelial layer (Fig. 1H’, M’, Fig. S2F’, white arrowheads), but did not seem to express E-cadherin (Fig. S2D’-F’, white arrowheads). This suggests that the absence of Nrx-IV might induce cell death. To explore this, we stained controls and Nrx-IV RNAi wing discs for the apoptosis marker activated Caspase-3. We found that, indeed, the delaminating cells expressing Nrx-IV RNAi are positively stained for activated Caspase-3 (Fig. 1I-I’, J-J’, N-N’, O-O’, white arrows). This indicates that Nrx-IV knockdown induces cell death. Consistent with this, the posterior compartment in Nrx-IV RNAi wing discs appears to have less cells than controls (compare Fig. 1A’-E’ to F’-J’).
Altogether, our results suggest that SJs, and in particular Nrx-IV, are essential to maintain epithelial organization and survival in the wing imaginal disc. Recent studies have already proposed that SJ components might have an important role in epithelial morphogenesis independently of their barrier function. Namely, it has been shown that Nrx-IV, together with other SJ molecules (Yurt, Coracle, and the Na+/K+-ATPase), acts to maintain apical-basal polarity during early embryonic stages, before the SJ barrier function is established, but not during late embryogenesis, when the SJ barrier function is fully established. Other SJ proteins have also been shown to regulate cell shape and rearrangements during salivary gland formation and wound closure. Finally, SJs regulate the mechanical properties of the embryonic epidermis. On the other hand, we show that Nrx-IV loss of function does not seem to affect AJ localization, which is also supported by previous studies. Therefore, future experiments should further address how apical-basal polarity is influenced by Nrx-IV or other SJs components in the wing imaginal disc.
In what concerns post-embryonic epithelia, our study is the first showing that a core SJ component might influence the actin cytoskeleton. Whereas in vertebrates a link between OJs and F-actin has been clearly demonstrated, in invertebrates this connection is still poorly understood. More studies are thus needed to understand the molecular mechanisms that regulate the interactions between OJs and the cytoskeleton.
The cell death phenotype we observed upon Nrx-IV knockdown also points to a role of SJs in cell survival and tissue integrity. Previous studies have reported that clones of mutant cells for several SJ components do not survive to adult stages. Although cell death has not been assessed in these studies, their results support the hypothesis that SJs are required for cell survival. Interestingly, one study has also reported that the SJ transmembrane protein Neuroglian stabilizes epithelial integrity in the ovarian follicular epithelium and that its loss of function leads to abnormal cell polarity and delamination, although the molecular mechanisms are still not completely understood.