The Maintenance Of A Monolayered Epithelium

Published Date: 02 Nov 2017

To establish the AP axis in drosophila, the follicle cells are required to respond to a signal from the oocyte to repolarise the microtubule cytoskeleton of the oocyte, allowing the localisation of maternal determinants. For follicle cells to be capable of repolarising the oocyte they must differentiate, mature and maintain their polarity within a monolayered epithelium, and we are interested in exactly what factors contribute to these series of events. Notch and the salvador-hippo-warts pathway are both necessary for the maturation of follicle cells and the control of proliferation however in hippo, spindle misalignment and multilayering also occur possibly suggesting that proper spindle alignment is crucial for maintaining a monolayer. This has since been disputed due to the observation of a varying array of mutants that cause multilayering that all have a different set of phenotypes. I have investigated the phenotypes associated with α-spectrin mutants showing that multilayering does not always result in failure to polarise the oocyte and that dlg may play a key role in maintaining a monolayer. I also have looked at Notch mutants, where multilayers were occaisionally observed, suggesting that notch may also be required to maintain a monolayer.

Introduction

Drosophila melanogaster has been an intensively studied model organism for over one hundred years resulting in a wealth of knowledge for this organism and a huge range of well-developed genetic techniques. To investigate the maintenance of a monolayered epithelium I will be using the Drosophila melanogaster follicular epithelium cells as a model due to its suitable characteristics and ease of preparation. The ovaries are the largest organs in Drosophila, both of which contain approximately 18 ovarioles, a chain of developing eggs at a number of stages (Bastock and Johnston 2008). Oocyte development can be described in 14 distinct stages (fig. 1A) determined by the appearance of the chambers providing a good opportunity for studying effects that are under different temporal control.

In the germarium, a cyst cell undergoes an asymmetric division, with one cell remaining as a stem cell and the other becoming a cystoblast. This cell is then surrounded by somatic cells (which develop into the follicle cells), as it undergoes 4 rounds of mitosis with incomplete cytokinesis to form 16 cells, two of these cells have four connections to their neighbours, and one of these becomes the oocyte which locates in the posterior of the egg chamber (Bastock and Johnston 2008). This is due to selective adhesion, caused by high levels of DE-cadherin in the posterior follicle cells, as a result of talin down regulation which usually acts to suppress translation of cadherins (Bécam et al. 2005). As the oocyte leaves the germarium, the follicle cells surrounding the germline cells become specialised into three types: polar, interfollicular and epithelial follicle cells (fig. 1A). The interfollicular and polar cells are specialised by Notch and JAKSTAT signalling, and undergo no further mitoses after leaving the germarium, however are important for further signalling (polar cells) and separating the egg chambers (interfollicular cells)( Ogienko et al 2007). The epithelial follicle cells go through a number of mitoses until stage 6 where delta up-regulation in the oocyte activates notch signalling for the second time in the follicle cells. This inactivates string (a Cdc25 phosphtase) resulting in down regulation of cdc25 causing the cells to enter an endocycle (Deng et al 2001).

The next stage of signalling requires gurken secretion from the posterior of the egg chamber due to its association with the nucleus and this signals to torpedo (EGFR) in the follicle cells which specifies the fate of these cells as posterior follicle cells (PFC)( Riechmann and Ephrussi 2001). Previous studies have shown that cells must be capable to respond to this signal to become posterior follicle cells and this involves signalling pathways such as JAK STAT, Notch and Salvador-Warts-Hippo (Lopez-Schier et al 2001) (Meignin et al 2007). The anterior cells become specified through secretion of unpaired from the polar cells however I shall not be focusing on these throughout my study (Poulton and Deng 2007). Once these cells have been specified, they signal back to the oocyte, resulting in the establishment of the anterior-posterior axis (AP axis). This exact signal from the PFCs has not been identified and is currently an active area of research.

Between stages 6 and 9 the follicle cells mature and change shape from cuboidal to columnar and many changes occur within the oocyte (Horne-Badovinac and Bilder 2005). On receiving the signal from the PFCs the microtubule organising centre (MTOC) is disassembled and Par1N1 localises at the posterior. This acts to recruit a few microtubule plus ends to the posterior which are enough to transport oskar to the posterior, which then acts in a positive feedback loop by recruiting more Par1N1 (Zimyanin et al 2007). When the microtubules have become polarised, a number of maternal determinants and the nucleus are localised to specific sites of the egg via transport by motor proteins (fig. 1B) and this polarisation is completed by stage 9 in type egg chambers.

In normal development, the follicle cells (FCs) form a monolayer of highly polarised epithelium both to protect and signal to the oocyte. Direct contact between FCs and the oocyte is critical for the signalling and therefore disruption of this, results in severe defects (Poulton and Deng 2007). A monolayered epithelium is integral to many other organisms and found within many organs and their maintenance is critical to their function. An example can be found in the kidneys where a monolayer of epithelium are key is forming a barrier between the body and the external environment and to allow exchange of key ions. If the integrity of this monolayer is lost, it results in pathologies. By understanding what factors contribute to maintaining this layer in drosophila follicle cells, may provide clues to how regulation is achieves in other organisms and could assist with treating diseases. Many essential genes involved are conserved from flies to humans and although in humans there is often a greater number of homologues making the system more complicated, the fly provides a simpler base of knowledge to start with (Reiter et al 2001).

As previously mentioned the signalling between the follicle cells (particularly the posterior) and the oocyte it critical to the polarisation of the egg, despite that we do not know the true signal. It is clear that the posterior follicle cells must have matured through a series of previous signalling so that they are competent to receive the gurken signal from the oocyte. Three models were presented by Poulton and Deng that were 1) a secreted signal, despite this being a conceptually attractive model, no evidence for this has been found, 2) A complex that forms at the basal side of follicle cells causes a signal transduction, it has been suggested the possibly down regulation of dystroglycan (DG) in response to EGFR signalling allows correct localisation of Laminin A (lan) at the basal side however further studies would be required to confirm and 3) a change in adhesion between the PFCs and the Oocyte may occur. In many of the pathways I am addressing, not only do they form large multilayers, but oocyte polarisation is lost suggesting that there may be a link between these two processes.

The key functions of epithelium are to provide an interface between different environments, therefore maintaining polarity is critical. The cells of epithelial sheets have distinctly defined apical, lateral and basal edges and these are characterised by a number of complexes and are established and maintained through a network of processes. In Drosophila there are three main polarity complexes which are Par6/aPKC/Crumbs/PatJ/Stardust localised apically, Bazooka localises at the adherens junctions and Scribble/Discs large/ lethal giant larvae that localise at the septate junctions along the lateral edge (Fig 1C). For delivery of these proteins to the correct domain, there is selective sorting by the intracellular vesicular trafficking, which is further supported by a polarised cytoskeleton and specific membrane identities (PIP2 localises apically, which PIP3 localises basally). The maintenance of polarity is achieved in a number of ways, for example many of the polarity complexes antagonise each other and therefore they are mutually exclusive and Baz recruits PTEN which converts PIP3 to PIP2, maintaining the membrane identity. The septate junctions are also important in maintaining polarity as they prevent lateral diffusion of proteins whereas the adherens junctions (AJ) are critical in maintaining cell-cell adhesion. If polarity is disrupted, it has a huge effect on the functionality of the cells and therefore I shall address where disruption occurs by staining for atypical (novel) Protein Kinase C (nPKC) and Discs large (Dlg).

My lab have been looking at a number of genetic pathways that cause loss of polarity or a multilayering phenotype and determining what other phenotypes may be involved, as shown in table 1 and I will address a number of these mutants.

Notch signalling is required at two stages, however I shall focus of the latter where it is required for cells to exit mitosis. Notch is a transmembrane receptor that transduces short range signals by regulating transcriptional complexes such as CSL to control many processes such as cell fate, differentiation and proliferation. It has a number of ligands such as delta and serrate and its ability to bind to these is determined by the extent of action by fringe- a glycosyltransferase. On binding, a conformational change allows the protease ADAM10 to cleave the extracellular domain and then γ-secretase cleaves, resulting in the active NICD to bring about the effect (Kopan 2012). In notch mutant clones cells fail to undergo the switch from mitosis to endocycling, resulting in overproliferation and lack of differentiation, resulting in cells being unable to respond to the gurken signalling and therefore cannot signal back to the nucleus.

The hippo-salvador-warts pathway is a tumour suppressor pathway that is important in organ size regulation. The core pathway consists of a kinase cascade where hippo(hpo)activity is enhanced by Salvador (sav) allowing it to phosphorylate warts (wts). This results in the phosphorylation of yorkie(yki), a transcriptonal coactivator, which exposes a binding site for 14-3-3 phosphopeptic binding protein which acts to retain yki within the cytoplasm, preventing transcription of genes for cell cycle and growth. There are a number of upstream regulators of this pathway such as fat (ft) which is a transmembrane protein that transduces information regarding the level of cell-cell contact- contact inhibition. Expanded (ex) and merlin (mer) are two other upstream regulators both of which associate with crumbs and may also integrate information about cell-cell contacts. Loss of proteins required in this pathway result in huge over growth of the tissues due increased rate of proliferation and reduction in apoptosis (due to lower levels of drosophila inhibitor of apoptosis 1 (diap1) (Hamaratoglu et al 2006). This pathway has been implicated as a tumour suppressor pathway and it is thought to have important implications in cancer- the observation that a human homologue of hpo can rescue loss of hippo in flies illustrate that there is significant conservation in this pathway and much information can be gained from this model system (Wu et al 2003). Mutations of this pathway have been shown to cause a failure of the oocyte to polarise, maturation defects, and overproliferation leading to multilayers forming at the anterior and posterior of the egg chambers (MacDougall et al 2001)(Meignin et al 2007) . Studies by Meignin et al found that spindles were misaligned. As spindle orientation can determine whether a cell remains within the same plane, and as spindle mis-orientation has not been observed in notch (unpublished data), this was thought to be a causal factor in the formation of multilayers. Spindle misalignment was also observed in integrin mutants, however no overproliferation occurred (Fernandez-Minan et al 2007) suggesting that spindle misorientation may be the key factor that caused multilayers, and suggests these pathways may act in regulating spindle orientation. Recent studies on inscuteable (unpublished data), a protein required to link spindle orientation to the polarity of a cell, have shown no multilayers within the PFCs however 100% spindle misorietation, strongly suggesting that other factors must also be involved in generating multilayers.

Another gene that causes multilayering is α-spectrin, and this will be one of the main focuses of my study. Previous work by Lee et al (1997) has investigated the multilayering phenotype in α-spectrin, however the allele they were using had a large deficiency and so effects may not have been exclusive to α-spectrin and I shall also use a number of different methods to clarify results further. α-spectrin is a member of the spectrin-based membrane skeleton (SBMS) . In drosophila, the SBMS consists of a single 278kDa form of α-spectrin which associates to form a heterodimer, either with β-spectrin to form (αβ)2 which localises basolaterally or with βH-spectrin to form (αβH)2 which localises at the apical membrane. These dimers act as molecular scaffolds for nucleating protein complexes at the membrane (Zarnesku et al 1999). To achieve this, the proteins have a number of conserved domains. Both contain spectrin repeats which are triple helix bundles in a tandem array that provide rigidity and structure to the cell (Djinovic-Carugo. et al 2002.) β-spectrin also contains an N terminal actin binding domain and a PH domain for association with the cytoskeleton and membrane respectively (Byers. 1992). βH-spectrin comprises of more repeats and also has an SH3 and similarly a PH domain to allow interactions with other proteins (Dubreuil 1990). The functions of these dimers have been demonstrated to be quite distinct, specifically βH-spectrin is exclusively affected in α-spectrin mutants which leads to multi-layering.

To investigate the role of these proteins I will use FLT/FRT recombinase. This is an elegant approach that generates small mutant follicle cell clones (where "clone refers to a small subset of cells that have lost the gene of interested), in a heterozygous background. This provides internal clones for comparison whilst also allowing careful observation of the cell autonomous effect the mutation has. One of the cell lines contains the flipase protein (FLP) under a heat shock promoter, whereas the other cell line contains the flipase recognition site (FRT) associated with target genes and a reporter gene so that you can tell in which cells the recombination event has occurred. On heatshock FLP is transcribed causing a recombination between chromosomes during mitosis and removing the target gene and reporter. Heat shock of varying severity induces clones of different sizes (Golic 1989) .

My results show that α-spectrin is not implicated in the polarising signal from the PFCs to the oocyte however it does cause mutilayering and suggests that loss of dlg localisation may be a factor in maintaining a monolayer. I also show that despite rare observations of multilayers in notch, they may be formed however soon undergo cell death and this highlights a difference in multilayering and may lead to the identification of a signal that must be lost to allow multilayers to survive. Avoiding apoptosis is a key characteristic of cancer cells and further study may shed light on new targets to prevent the apoptotic route being disabled.

Materials and Methods

Fly stocks

The following lines were used: yw hs-Flp; +/CyO; P{w[+mC]=Ubi-GFP.nls}3L1 P{Ubi-GFP.nls}3L2 P{w[+mW.hs]=FRT(w[hs])}2A/TM6B and FRT101GFP/FMC7; Flp/Cyo; MKRS/+. The mutant lines were: w;; a-spectrinRG41 e FRT2A/TM3, Sb, e and yw N55e11 FRT101/ FM7

Generating clones

Flies were crossed and left to lay eggs at 25oC for 2-3 days. At the 3rd instar larvae stage clones were induced by subjecting samples to 2 hours of heat shock at 37oC for three consecutive days. To induce larger clones this was sometimes repeated at the adult stage. 3 or 4 days later flies were then selected for dissection. α-spectrin and Notch mutant follicle cells were generated by the FRT/FLP recombinase system (Xu and Rubin 1993) and in all cases identified by the loss of GFP expression.

Antibody staining

Selected flies were fattened on yeast overnight at 25oC. Dissection was carried out in PBT (PBS + 0.2% Tween) in less than 15 minutes, ovarioles were separated and then fixed for 20 minutes in 4% Paraformaldehyde/PBT, rehydrated in PBT and then blocked in PBT-10 (10%BSA in PBT) for at least 45 min. Antibodies were added in PBT-1 (1%BSA in PBT) and left at 4oC overnight. After three washes in PBT samples were incubated with secondary antibody (1:200) for 2 hours. Samples were finally washed with PBS three times and mounted on slides in Vectashield+DAPI for visualisation of DNA. The following antibodies were used: rabbit anti-nPKC (1:1000) (Insight Biotechnology), mouse anti-Dlg (1:500) (DSHB), chicken anti-GFP (1:2000) (Abcam), rabbit anti-PH3 (1:500) (Upstate Biotechnology), rabbit anti-Staufen (1:3000), mouse anti-Hnt (1:15) (DSHB). Secondary antibodies were AlexaFluor 488, AlexaFluor 568, and AlexaFlour 647 coupled (molecular probes).

Microscopy

Samples were visualised using the Sp5 upright Leica microscope with 40x and 63x objective. Images were processed in image J 1.46r to adjust channel level (NIH, USA)

Statistics

Students t-test was performed for comparison of No.PH3 +ve cells for control and α-spectrin mutants using Microsoft excel. Normal distribution and equal variance was assumed with a two-tailed test.

Results

To assist with the understanding of whether there is a link between these pathways required for the maintenance of a functional epithelia and to establish what factors are required in preventing a multilayer, I have sequentially addressed the areas that have yet to be studied , or where results have been conflicting.

Characterisation of Notch55e11 epithelial morphology

In hippo and a-spectrin mutants multilayering in the posterior and anterior of the somatic follicle cells surrounding the oocyte is observed (fig. 2A,B), whereas in notch mutants this is rarely the case (14.3% n=21) (fig. 2C,D). It is clear however that in these notch mutants, the follicle cells are affected and distinct changes in the cell morphology can be observed in all follicle cells, not just the anterior and posterior as is the case in a-spectrin follicle cell clones (FCCs and where "clone" refers to a group of mutant cells marked by the lack of GFP in a heterozygous background). Figure 2D shows a lateral mutant clone where cells are smaller in size than their neighbouring wildtype cells, and they also appear to have become flattened, losing their normal columnar appearance. These cells have also lost the expression of hindsight (hnt –a transcription factor that is upregulated by notch at stage 6 (Sun and Deng 2007) and has previously been used to demonstrate maturation (Meignin et al 2007) showing that maturation in these cells has not occurred due to loss of Notch. In 14.3% of cases (n=21), multilayers were observed (fig. 2E,F). On closer inspection, the cells in these ectopic layers do not have a normal morphology with a condensed and fragmented nuclear appearance suggesting that cell death has occurred. Further, when staining for polarity markers atypical (novel) Protein Kinase C (nPKC) and Discs large (Dlg), staining is completely lost from the lateral and apical domain suggesting a loss of cell polarity and possibly break down of the plasma membrane(fig. E’’’). Comparison between the single and multi-layered epithelium within the same egg chamber shows similar appearance for cells adjacent to the nucleus(fig. 2E’ and E’’’). In figure 2E’’’ there are two distinct nuclear morphologies. The oocyte adjacent nuclei, although possibly smaller than controls, look normal. In comparison, nuclei in the ectopic layers appear condensed and fragmented. This was not always the case, as can be seen in figure 2F where all cells have lost normal morphology and have condensed nuclei despite being adjacent to the oocyte. Similar to the case of hippo and a-spectrin these multilayers are only observed at the posterior end (fig. 2F,F’’) suggesting a positional factor that causes multilayers, possibly an increase in tension. The low number of chambers with multilayers observed suggests that multilayering in Notch may trigger apoptosis and so the egg chambers become non-viable and do not survive.

α-Spectrin is required for proliferation control before stage 6

Studies into the Salvador-Warts-Hippo pathway and Notch have shown that FCCs do not correctly exit mitosis and switch to an endocycle, and so proliferation is still detected past stage 6 of oogenesis. This is not the case for a-spectrin, as no phosphohistone 3 (PH3) a mitotic marker is detected past stage 6 (fig. A,A’). Despite this, we hypothesised that some degree of overproliferation would be expected as there is a greater number of total cells (Lee et al. 1997) in mutant egg chambers indicating that a greater rate of proliferation may occur before stage 6. To investigate this we counted the number of PH3 positive cells in both control egg chambers (heterozygous background where no heat shock had induced clones) and in large mutant clones (>90% mutants cells within the egg chamber) as presented in fig. 3B. At both of the chosen stages (3/4 and 5/6) it is clear that the average number of PH3 positive cells is greater in the large clones, and in both cases approximately equal variance is displayed. To investigate the statistical significance I applied an unpaired t-test assuming equal variances. At both stages a significant result was achieved, 0.0002 at stage 3/4 and 0.0009 at stage 5/6. At stage 3/4 it was also noted that in egg chambers where only approx. 50% of cells were mutant, a higher mean than wildtype was recorded (data not shown), however this was far lower than the large mutants suggesting that the overproliferation phenotype is cell autonomous. A better way of investigating this may have been to compare the ratio of wild type cells undergoing apoptosis in comparison to FCCs as was done by Zhao and colleagues (2008b).

nPKC localisation in α-spectrin mutants appears disrupted when contact with both the oocyte and the basement membrane is lost

Maintaining polarity is crucial to the function of epithelia particularly in this system. To achieve axis formation a number of signals are directed between the oocyte and the follicle cells and direct cells contact must be achieved as in many cases, such a notch/delta signalling, the ligand and receptor are transmembrane proteins. To ensure signalling is achieved, these proteins must be localised in the appropriate location and this requires cells to have established polarity. To determine whether polarity was disrupted in α-spectrin and Notch FCCs we looked at the localisation of nPKC and Dlg. In controls (fig. 4B) it was clear that nPKC localisation had been established by stage 6 and remained the same until stage 10 characteristically being brighter at the anterior and posterior ends, and so results compile these different stages. All effects observed were cell autonomous and so clone sizes have not been distinguished, however distinct results were found in bilayers and multilayers as shown in the graph. A previous study for α-spectrin suggested that epithelial polarity was affected (Lee et al. 1997) however they were using a construct with a large deficiency and so there may have been an affect that was not specific to the loss of α-spectrin. In hippo FCCs normal nPKC localisation (fig. B’) was maintained in oocyte adjacent cells however in multilayers this localisation was lost (Meignin et al. 2007) as appears to be the case here. In oocyte adjacent cells, loss of nPKC very rarely occurred, mis-localisation in monolayers (fig. 4C) only occurred in 3.8% (n=26) of cases indicating that loss of α-spectrin does not directly affect the epithelial polarity, and in bilayers and multilayers despite mislocalisation being recorded, this was never obsered in the oocyte adjacent layers (Fig. 4D’’ and E’’) . In bilayers the ectopic layer often maintained a level of polarity (fig. 4D’’), however occaisionally nPKC was not visible or became laterally localised in 44.1% (n=34). It must be taken into consideration that in some cases a number of focal planes must be studied to assess localisation of nPKC in all the ectopic cells, despite great care, this may introduce false readings. Multilayering correlated with a far more severe phonotype with 100% mislocalisation of nPKC in non-oocyte adjacent layers. When looking closely at these samples it is clear that cells that directly adjoining the oocyte or the basement membrane establish a level of polarity (fig. 4E’’ arrows), however central cells often have ectopic nPKC localisation either laterally, more diffuse or ectopic accumulation. (fig. 4E’’ arrow heads). Further it often appeared that the nPKC concentration was greater at the apical edge of the posterior most cells in the multilayer. At this resolution it is impossible to tell whether the nPKC detected belongs to the apical side of one cell, or the basal side of the adjacent cell and so this increased intensity may be two apical domains meeting, such as in rosette formation however I shall address this possibility later.

Dlg localisation is disrupted in α-spectrin mutant cell

Unlike nPKC localisation, Dlg (fig. 5B,B’) is affected in oocyte adjacent layers cell autonomously (fig. 5C’,C’’). By stage 9, Dlg localisation becomes more concentrated at the apical side of the lateral edge. Whether this change is a loss of lateral accumulation becoming more diffuse along the membrane or a complete loss of membrane association is unclear however due to phenotypes observed in Dlg mutants (Li et al 2009), I would suggest that Dlg has just become more diffuse. Figure A shows that this phenotype is observed in most cases, (mono 82.6% [n=23] bi 90% [n=10] multi 91.0% [n=11]). This is an interesting result as it does not show the same result as nPKC, the other epithelial polarity marker, which possibly suggests the loss of the tight accumulation at septate junctions does not affect the epithelial polarity, however may affect a different pathway Dlg is involved in, which I will discuss later. http://www.ncbi.nlm.nih.gov/pubmed/9144922

Notch shows similar polarity phenotypes to a-spectrin

In Notch, Dlg shows a very similar result to that observed in a-spectrin, becoming more diffuse along the membrane cell autonomously in FCCs in 81.3% (n=16) of egg chamber(fig. 6A’’, B’). This result possibly suggests that a-spectrin is affected in notch mutants and staining for a-spectrin in notch mutants could reveal if there is a link. nPKC localisation displays a slightly different phenotype where it becomes concentrated along the apical edge of FCCs in 68.8% (n=16) of cases. In small mutant clones this concentration is more obvious and in these cases the membrane also appears to be contracted with a greater curvature than neighbouring wildtype follicles (fig. 6B’’).

Oocyte polarity is rarely affected in α-spectrin mutants

Considering the defects observed in the follicle cells of α-spectrin FCCs, we were interested to discover whether this disrupted the signalling between the oocyte and the posterior follicle cells leading to a failure in oocyte polarisation and so we moved on to address whether oocyte polarity was affected. Initially we looked at whether nuclear migration was successfully achieved by stage 9. In anterior or lateral clones nuclear migration was successful in 100% of cases, consistent with the knowledge that signalling to polarise the oocyte comes from the posterior follicle cells (MacDougal et al. 2001). In posterior clones, the phenotype wasn’t that much more penetrant, no defects were observed in partial clones and in full posterior clones only 5.7% (n=35) showed a defect. Notably, both of these cases displayed severe multilayering, however in some other samples with multilayering, nuclear migration was successful (fig 7D). We then also stained for staufen localisation, a protein that always colocalises with oskar mRNA and is required for oskar localisation and function (Micklem et al. 2000) and found this to be more sensitive to disruption of oocyte polarity as in many cases where staufen localisation was lost, nuclear migration had successfully occurred (fig. 7G). Wildtype localisation (fig. 7C) was always observed in A or L clones and often in full (fig. 7E) or partial posterior clones (fig. 7F). In partial posterior clones 4.6% (n=24) showed mislocalisation and in full posterior clones 33.3% (n=24) were affected, again, all those affected displayed a multi-layered epithelium. When analysing the images it was clear that when there was a partial clone, staufen localisation was biased towards the wildtype cells (fig. 7F).

Discussion

Does the loss of notch result in cell death or a change in cell fate?

Notch is known to be required for the maturation and the halting of mitosis at stage six in follicle cells and so the loss of it results in a failure to send the polarising signal to the oocyte and overproliferation. From my images it is clear that Notch FCCs also display disruption in epithelial morphology with the underlying remaining unknown. In small posterior clones cells fail to become columnar and remain cuboidal, in lateral clones cells appear to become squamous, and in large posterior clones multilayering is occasionally observed, however in comparison to multilayers in hippo and α-spectrin the cells look like they are undergoing cell death.

As the follicle cells mature they change from a cuboidal morphology to a columnar shape and so I feel the small posterior clones can be explained by the FFCs failing to mature, clearly shown by the lack of Hindsight expression (Hnt) (fig. 2D’). In comparison, the change in the lateral cell morphology is reminiscent of flattening of anterior stretched cells that surround the nurse cells. Previous experiments have demonstrated that stretched cells are specified by JAK/STAT signalling by unpaired secretion from the polar cells and not as a result of being stretched by the migrating border cells. This signal is sufficient to change the fate of posterior follicle cells to stretched cells in the absence of EGFR signalling, also indicating that the nurse cells are not necessary for this change in fate (Gonza´lez-Reyes 1995). To assess whether the cells have differentiated into stretched cells the enhancer trap line MA33 with the β-galactosidase reporter could be used (Gonzalez-Reyes and Johnston 1998). It has also been shown that BMP signalling is required in the anterior terminal FCs with decapentaplegic (dpp) being expressed before and during flattening (Dobens and Raftery 2000). This indicates that receiving this signal is required for the cells to differentiate into stretched cells and so antibody staining for dpp in clones could shed light on to whether the cells are capable to respond to these signals and therefore whether the observed change in morphology is due to responding to the BMP signal and differentiating, or another effect.

In previous studies it has not been reported that notch forms multilayers, however I propose that this may be due to apoptosis occurring if a multilayer forms resulting in them being rare. It has been shown that the hippo pathway activates the apoptotic pathway through lowering levels of drosophila apoptotic deficient 1 (diap1) relieving the inhibition on caspase3 and therefore in hippo mutants apoptosis may not be induced in multilayers due to maintained high levels of diap1 whereas in Notch FCCs this pathway is still intact and therefore apoptosis is induced. To identify if this is the case, we could stain egg chambers for diap1 to see if there is downregulation, and caspase3 to see if there is upregulation in FCCs. To further identify whether it is apoptosis that is inhibiting the formation of multilayers in Notch FCCs we could attempt to ‘rescue’ the presence of multilayers by inhibiting apoptosis. A previous study by Avery et al demonstrated that upf1 and upf2 loss of function mutants (members of the nonsense-mediated RNA decay mechanism) caused both inhibition of cell growth and induction of apoptosis. The latter had been difficult to study and was achieved by over-expressing p35, a baculovirus protein that acts to inhibit apoptosis (Hay et al. 1994) resulting in rescue of the clone size (Avery et al 1994). By over-expressing p35 in Notch FCC you would inhibit apoptosis and be able to determine whether the lack of multilayers is due to apoptosis, potentially unmasking the fact that Notch is also an important player when considering the maintenance of a monolayer.

Polarity results are fairly inconsistent

Previously in Notch mutants it has been found that Armadillo (Arm) has correct apical localisation (Deng et al 2001). I looked at the localisation of nPKC which appeared to be concentrated in the Notch FCCs. This increased intensity could be explained by a local increase of nPKC due to the increased proliferation observed resulting in cells being more compact and therefore I believe this should not be considered as an epithelial polarity defect.

In hippo and integrin mutants it has been found that in follicle cells adjacent to the oocyte, polarity is maintained however in ectopic layers there is loss of this apical localisation (Meignin et al 2007 and Fernandez-Minan 2007), which is consistent with the results for nPKC localisation in α-spectrin FCCs. This suggests that contact is required with the oocyte to establish and/or maintin polarity. As integrins are required for adhesion between different cells types and play a role in signal transduction, they were considered as having a role in generating polarity in the epithelial cells. Studies into myospheroid (mys –gene that encodes integrins in drosophila) loss of function in the follicular epithelial cells found that loss of integrins in the follicle cells resulted in no polarity defect, hypothesising that they may just be required in the oocyte, however when chambers were mutant in both the germline and somatic cells, FCs still maintained polarity (Fernandez-Minan 2007).

In development of the drosophila midgut it was found that there was no requirement for integrins, however basal adhesion was required to maintain polarity (Devenport and Brown 2004). When looking at nPKC localisation in multilayers in both hippo and α-spectrin is does appear that in both the oocyte adjacent layer and the layer in direct contact with the basement membrane localisation is maintained, whereas in central layers apicobasal polarity is disrupted with the loss of precise apical localisation of nPKC. At the resolution of my images and without a cell membrane marker, whether the nPKC is at the apical of one cell or the basal edge of another is hard to distinguish. I found that the intensity of nPKC was often greater at these border (fig. 4E’’ arrows), possibly indicating the presence of two apical faces adjacent to each other. This structure is reminiscent of rosette formation that is observed during development of eplithelial sheets (Blankenship et al 2006). This cell rearrangement results in cells intercalating and changing from a short wide sheet, to a long thin sheet of cells, and if this was occurring in the follicular epithelium when multilayers occur, it could explain the lack of multilayers found at later stages. To investigate whether this is a possible explanation, we would need to first establish in which cell nPKC is localised using greater image resolution and membrane markers. To then identify whether the structures observed behave like rosettes it would be necessary to visualise one resolving in live imaging, however as courrently egg chambers cannot survive long after having been removed from the fly, this may not be possible.

In comparison to the apical markers, Dlg, a lateral marker, showed defects in its localisation in both monolayers and multilayers and this phenotype also extended to the lateral domain. Although not a total disruption of epithelial morphology, the distinct apical lateral concentration established by stage 9 is lost and becomes more diffuse along the membrane. I have interpreted this as becoming more diffuse rather than a loss of dlg as appears to be the case in a number of images, due to previous studies into the result of complete loss of Dlg. In dlg mutants there is a loss of follicle cell patterning and loss of oocyte polarity, which does not occur in α-spec mutants (Li et al 2009). Loss of tight Dlg localisation has not been described in previous studies, however I found it to be the case for both α-spec and Notch FCCs. For notch mutants, the explanation may again be that the cells do not mature and as this concentration only occurs by stage 9, lack of maturation may prevent the downstream signalling required for the Dlg positive domain to shrink as is observed in control cells. In α-spectrin one could speculate that α-spectrin may indirectly recruit dlg to the membrane due to its role in the SBMS and its association with septate junctions, however no current research shows that these proteins interact. Dlg loss of function results in overproliferation of the epithelia, both before and after stage 6 (Zhao et al 2008).This was assayed by the ratio of PH3+ve cells and bromodeoxyuridine (BrdU – incorporated in cells going through S phase) incorporation in control and mutant cells. In notch and hippo proliferation is observed past stage 6 however these studies have not investigated whether greater proliferation occurs before stage 6 compared to controls. In α-spectrin no proliferation post stage 6 is observed however accelerated proliferation does occur earlier and this over proliferation may be linked to the localisation of dlg being more diffuse. I have noted that the phenotype I have observed occurs at stage 9, whereas the effect I am suggesting it has occurs before stage 6. I propose that dlg localisation may be affected at earlier stages however this may be a more subtle phenotype that was missed as we were focusing on stage 9 egg chambers. To investigate whether the less concentrated accumulation of dlg at septate junctions is the cause for overproliferation in α-spec FCCs we could try over expressing dlg and see whether this increased concentration is sufficient to prevent overproliferation. This may not be successful if the cell is unable to localise this additional dlg.

In dlg mutants overproliferation is seen both before and after stage 6, indicating that the loss of dlg also disrupts notch signalling as the switch to the endocyle does not occur. As no proliferation is seen after stage 6 in α-spec mutants it suggests that either dlg is not what is causing this over proliferation or that there are multiple pathways involved in the localisation of dlg and when one is lost, other pathways may compensate producing a less severe phenotype. It will be interesting to readdress overproliferation in the other mutants that cause multilayering as there may be a divide between those that show proliferation either before or after stage 6 or both, indicating the presence of multiple levels of regulation. This could be addressed by counting the proportion of cells in a clone compared to control cells that are PH3+ before stage 6.

What factors determine if oocyte polarity is affected?

To establish oocyte polarity a signal is required from the posterior follicle cells to break down the mitotic organising centre (MTOC) and reorganise the microtubule cytoskeleton so that it is polarised with the positive ends in the posterior to facilitate the transport of the nucleus and key maternal determinants to the correct locations (MacDougall et al 2001). It has been established that for polarisation to occur the posterior follicle cells must have matured and be capable to respond to gurk, and signal back to the nucleus although the exact nature of the signal is unknown. In hippo and notch mutants neither nuclear migration nor localisation of determinants occurs. Both of these mutants FCs fail to mature, and we were interested to see if multilayering alone had an effect on oocyte polarisation. In α-spectrin the oocyte polarity is rarely affected and therefore one can assume the α-spectrin is not directly involved in the polarising signal. An interesting feature is that occasionally in multi-layered egg chambers oocyte migration is disrupted. Two cells that have an important role at many stages of signalling in oogenesis are the posterior polar cells (PPC) and may play a dominant role in signalling back to the oocyte. To further investigate whether these cells are key for the polarisation signal would be to count whether the PPCs are still in contact with the oocyte and whether these cells are wild type or mutant. I would therefore propose that the occasional loss of oocyte polarity observed in α-spec mutants is not a direct effect of a loss of α-spectrin however it is more likely to be a secondary effect to multilayering that causes the detachment of the PFCs from the oocyte adjacent layer. It is clear that it is not just the PPCs that are important for the oocyte polarising signal as in hippo mutants with wts FCCs but wildtype PPCs oocyte polarisation is still not achieved (personal communication), and therefore suggests that both cell types must be present for the polarising signal.

What are the key factors in maintaining a monolayer

Taking into consideration all of the results to date, it is clear that this is a very complex system, with many regulatory levels. I have compiled the results to date into table 2 and currently there is no clear answer to what the causative factor is for multilayering. This may be due to us not yet identifying the key pathway required for maintaining a monolayer, or that in each mutant numerous factors act together and within the regulatory network as a whole there may be many levels of redundancy.

Tension could potentially play a role in multilayer formation- multilayers only occur in the anterior and posterior of egg chambers where there is greatest curvature, and therefore possibly greatest tension, suggesting it may play a role in multilayering. Our lab have recently begun investigation by staining for p-sqh to reveal if ectopic tension correlates with the formation of multilayers.

Like many biological processes, it is becoming clear that there is a complex network, rather than a pathway involved in regulating the maintenance of monolayers. I propose that modelling this may assist with further understanding. This will allow the integration of multiple results, also considering temporal and spatial regulation, painting a clearer picture of what is occurring. Further, it must be considered that in different cell types, different pathways may contribute to different degrees and therefore extending the model to different systems will be informative for establishing how conserved the mechanism for maintaining a monolayer is between different cells and different species. The expansion of this mechanism will prove important when trying to understand loss of epithelial monolayers in human pathologies. Many of the pathways we are studying are tumour suppressor pathways and understanding how these pathways interlink with other pathways within the cell will be critical in identifying further targets for cancer therapy.

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