The loss of Dlg5 alters cell autonomously follicle cell growth
We performed a reverse genetics screen to identify new genes involved in Drosophila follicular epithelium development, a tissue used as a generic model for various aspects of epithelium biology [18, 19]. Follicle cells form a monolayer epithelium surrounding germline cyst with the apical domain facing the germline. Follicle undergoes a rapid growth through 14 developmental stages, with a 1000-fold volume increase. Follicle cell growth is associated with proliferation until stage 6, then follicle cells become endoreplicative and larger. During the screen, we noticed that clones expressing RNAi against Dlg5 were small and the cells appeared also smaller than wild-type cells, especially after stage 6 (Fig 1A). This defect was quantified at stages 9-10A, showing an average reduction of 33% of the cell surface (Fig 1B). A similar defect was observed with a different RNAi line (Fig 1C). A P-element insertion in the 5’UTR of Dlg5 was available. This insertion was lethal and homozygous mitotic clones for this mutant also give small follicular cells (Fig 1D). However, the defect obtained with this mutant appeared more variable than with the RNAi lines, suggesting that it may correspond to a hypomorphic mutant. We generated P-element excisions and most of them restored the viability of the stock indicating that its lethality was associated with this insertion in Dlg5 gene. We also obtained several lethal imprecise excisions, Dlg5Ex5,Ex8,Ex13,Ex14 all, except Ex8, deleting the start codon and part of the coding sequence (Fig 1H). However, they also deleted part of the neighboring annotated gene (CG4970). This gene is only expressed in testis and is very poorly conserved, with no known domains and no ortholog in other insect species. Trans-heterozygous between a Minos element inserted in the coding sequence of this gene (MimicMI02472) and the deletions that we generated complement perfectly in terms of viability and fertility, indicating no essential function of CG4970. We also obtained rescue of Dlg5ex13 mutation lethality using a transgene with Dlg5 coding sequence under ubiquitin promoter and fused to GFP added in N-terminal (Ubi:GFP-Dlg5). Thus, we assumed that Dlg5ex13 could be considered as a bona fide null mutant. Mitotic clones for this allele contain cells with a reduced size (Fig 1E,G), a defect also rescued by Ubi:GFP-Dlg5 (Fig 1F,G), confirming the cell autonomous role of Dlg5 in follicle cell growth.
Dlg5 has a general function in growth
We next wondered whether Dlg5 function in cell growth could apply to other tissues. We induced Dlg5 knock-down specifically in the wing disc pouch, which corresponds to the future cells of the fly wing, using Nubbin:Gal4 . It led to a dramatic reduction of the wing size (Fig 2A-B, D). We also induced mitotic clones for Dlg5Ex13 in the wing disc and quantified several parameters. Of notice, the mutant cells tend to form a row of cells rather to extend the clone in all directions, a defect reminiscent of what has been recently described for other mutants generating small cells  (Fig 2C). Mutant clones were systematically smaller than their twin and contained fewer cells (Fig2 E,G). Moreover, the mutant cells had, in average, a size reduced by 40% (Fig 2F). DCP-1 staining did not reveal apoptotic cells in Dlg5 mutant clones suggesting that the lower cell number per clone correspond to a growth and proliferation decrease (Fig 2H).
Finally, we looked at a transheterozygous combination of Dlg5 null alleles. First instar larvae hatch, are able to crawl around and are still alive 48 hours after egg deposition. However, their growth is strongly impaired, indicating a systemic requirement for Dlg5 (Fig 2J-K). Thus, altogether these results show that Dlg5 owns a general growth function, as its mammal counterpart, and that this function is performed in a cell-autonomous manner.
We aimed to define how Dgl5 modulates growth. It has been reported that Dlg5 modulated hippo pathway in the wing. However, this pathway is usually described as controlling of cell proliferation rather than cell growth. The Hippo signaling pathway regulates cell proliferation by inactivating Yorkie (Yki), the Drosophila Homolog of YAP. We therefore induced yki RNAi, and, as expected, its loss of function markedly reduces wing size and the estimated number of cells in the whole wing (Fig 3A-D). Importantly, we also noticed a reduction of cell size in the same range than what has been observed with the loss of Dlg5 (Fig 3E). Thus the hippo pathway also modulates cell size and could therefore explain Dlg5 contribution in this tissue. Yki is known to be required for normal follicle cell proliferation . We induced null mutant clones and checked for cell size defects at stages 9-10. Comparing the cell surface indicates a moderate but significant effect of yki (Fig 3F-G). Together, these data clearly establish a role for yki in the control of cell size. However, this defect appears not stronger than the one induced by the loss of Dlg5 by RNAi (Fig 1B). Moreover, inhibition of hippo pathway has not been described as affecting systemic growth, leading to the hypothesis that Dlg5 may modulate growth by at least another means. Looking for other potential growth actors affected by the loss of Dlg5, we noticed that this gene was picked-up in a RNAi screen for TORC1 pathway regulators in S2 cells as affecting the level of S6K protein . However, S6K level was unchanged in follicular cells or wing disc mutant cells for Dlg5ex13 (Fig 3H,L). Moreover, phosphorylation level of S6 were not affected in Dlg5 mutant follicle cells, indicating that Dlg5 does not modulate S6K activity and more generally the Tor pathway in this tissue (Fig 3I,L). We also, checked Insulin/PI3K pathway activity, which when affected, give similar defect both at the cellular and the systemic levels, but we did not observe any alteration of Phospho-Akt in Dlg5 mutant cells (Fig 3J,L). Thus, how Dlg5 influences growth in these cells remains to determine. Nonetheless, we noticed a reduction of Myc expression in Dlg5 mutant follicle cells, suggesting that it is required for the efficient signaling of one of the multiple pathways controlling Myc levels (Fig 3K,L) . Importantly, Myc levels were never affected in mutant follicle cells for yki, demonstrating that Dlg5 effect is independent of Hippo pathway (Fig 3M). However, similar reduction was not detected in wing disc Dlg5 mutant cells (Fig 3N), indicating that Myc regulation cannot account for Dlg5 effect on growth in all tissues.
Dlg5 is required for the localization of apical polarity determinants
The fact that Dlg5 regulates growth both in mammals and in fly prompted us to check for an epithelial polarity phenotype, since such a defect has also been observed in Dlg5 mutant mouse. These defects have been detected for instance in the kidney or the lung, where a partial mislocalization of the apical determinant aPKC, a key component of the apical PAR complex, has been observed [10, 11]. Moreover, knock-down of Dlg5 in follicle cells also give similar phenotypes . In follicular cells mutant for null mutant Dlg5Ex13 we saw a semi-penetrant reduction of the apical level of aPKC, the apical domain of these cells being inwards, at the contact with the germline (12/19 clones) (Fig 4A). The level of the apical determinant Crumbs (Crb) is also reduced (fig 4B). However, this apical reduction of apical determinants was never associated with an extension of lateral markers, such as Coracle (Cora), to the apical domain or to a mispositioning of the adherens junctions. Nonetheless, we pointed out that Cora was often upregulated in the mutant cells (21/33 clones) (Fig 1C,D), a defect never observed with another septate junction marker such as Dlg (n=9) (Fig 4D). Moreover, we never spotted multi-layers or round mutant cells. Also, mutant cells tend to flatten, a defect more often observed in young follicles (Fig 4C). Thus, although it is not sufficient to induce a complete loss of cell polarity in this tissue, Dlg5 null mutation can affect apical polarity determinants and cell morphology.
To characterize endogenous Dlg5 localization we generated an antibody against the third and fourth PDZ domains. In follicle cells, Dlg5 antibody reveals a dotty pattern, similarly to what has been described in mammals (Fig 5) . This signal is specific because the antibody gives no signal in Dlg5 mutant follicle cells (Fig 6G). These dots were observed both inside the cell and at the cell cortex. Moreover, Dlg5 pattern was dynamic depending on the stages. During early stages (2-8) Dlg5 localizes at apical and lateral membranes at stage 1, and appears therefore in apical sooner than Crb (Fig 5A). Then the apical localization progressively decreases, and is barely detectable at stage 9 (Fig 2B). Consequently, at later stages this cortical localization is restricted to the lateral domain. Because Dlg5 is present apically as key apical determinants such as aPKC and Crb and affect their localization, we compared their localization in the apical plain of the follicle cells with higher resolution using Airyscan. We observed that aPKC and Crb are usually colocalized, especially at the marginal zone, an area of cell–cell contact apical to the adherens junctions (Fig 5C,F). This observation comes as confirmation that these proteins cooperate to define the apical domain . In contrast, no evident colocalization is observed between Dlg5 and those two proteins and their localization even tend to be exclusive in the marginal zone, indicating that Dlg5 is not stably associated with these apical determinants (Fig 5D,E,G,H).
Dlg5 is required for N-cadherin localization independently of its effect on cell polarity
Looking at the Adherens junction, we found that E-Cad level was not affected (n=18) (Fig 6A). Follicle cells also expressed N-Cad, which is integrated in adherens junction, and Dlg5 has been functionally and molecularly linked to this cadherin in mammals [10, 12, 25]. We therefore looked at N-Cad and spotted an extremely strong and fully penetrant reduction in Dlg5 mutant cells (n>20) (Fig 6B). This effect correlates with the strength of Dlg5 loss of function because the reduction is weaker in hypomorphic conditions (Fig 6C). However, Dlg5 overexpression does not increase N-Cad levels at cell contacts and has no visible impact on cell size (Fig 6D). Thus, Dlg5 loss of function has a dramatic effect on N-Cad, but not on E-Cad, in the same cell type and at the same developmental stages. It indicates therefore a very specific effect on N-Cad membrane delivery or stability.
We therefore looked at a potential colocalization between N-Cad and Dlg5. Both are present mainly at the cell periphery. However, at the adherens junction plain of the cells Dlg5 is mainly found as medioapical dots whereas N-Cad surrounds the cells (Fig 6F). Just above, less N-cad is observed whereas Dlg5 becomes more enriched at the cortex. As a result, only a weak colocalization between the two proteins is observed with N-Cad being globally more apical than Dlg5 (Fig 6F). Thus, although Dlg5 has a very strong and specific impact on N-Cad, their potential association is likely transitory. We also noticed that when we induced Dlg5 mutant cell clones, Dlg5 disappears from the cell cortex even at the boundary with wild-type cells (Fig 6G). Classical interpretation for such observation is that Dlg5 is associated with proteins performing homophilic interactions between cells and that it is required for their localization, explaining its absence on the wild-type cell side. However, Dlg5 localization is not affected in N-Cad mutant clones (Fig 6H). Thus, Dlg5 is probably associated with another protein performing homophilic interactions and that is mainly localized at the lateral domain of the follicle cells.
The loss of Dlg5 affects both apical and adherens junction proteins. Since there is important cross-talks between the protein complexes acting at these two sites, we wondered whether those two defects were linked [26-28]. First, the loss of N-Cad had no effect on aPKC apical level, in agreement with the previous proposition that N-Cad and E-Cad are redundant in the follicle cells to maintain adherens junction and epithelial polarity (Fig 6I) . Thus, Dlg5 impact on N-Cad does not explain the loss of apical proteins. Second, the reduction of apical determinant Crb and aPKC is much less expressive and penetrant than the loss of N-Cad and it is therefore unlikely the cause of such a defect. Patj mutation, a component of the Crb complex, leads to a very similar mild effect to Dlg5 mutation on aPKC and Crb apical levels and on cell morphology . However, patj mutant cells show a normal level of N-Cad (Fig 6J). Thus, the reduction of apical determinants and of N-Cad observed in Dlg5 mutant cells correspond to two independent functions of the protein.