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Damage to the Drosophila follicle cell epithelium produces "false clones" with apparent polarity phenotypes.

ABSTRACT: The Drosophila follicular epithelium, which surrounds developing egg chambers, is a well-established model for studying epithelial polarity because it is continuously generated from adult stem cells, making it easy to generate homozygous mutant clones in a heterozygous background. Mutant clones are usually marked by the loss of Green Fluorescent Protein (GFP) expression, which distinguishes them from their green, wild-type neighbours. Here we report that damage to the epithelium during dissection can produce groups of GFP-negative cells that resemble mutant clones. Furthermore, several polarity factors, such as aPKC and Discs large, are not localised in these damage-induced false clones. This phenotype is identical to that reported for several mutants, including ampk and Dystroglycan mutant clones under conditions of energetic stress. Using more reliable systems to mark ampk and Dystroglycan null clones such as the MARCM system, we found that neither protein is required for epithelial polarity under low energy conditions. Thus, our previous report of a specific low energy polarity pathway is an artefact of the increased damage caused by dissecting the small ovaries of starved flies. However, ampk mutant cells are larger than normal under both starvation and well-fed conditions, indicating that AMPK restricts follicle cell growth even when dietary sugar is not limiting. We suspect that several other reports of mutants that disrupt follicle cell polarity may also be based on the phenotype of damage-induced false clones, and recommend the use of positively marked clones to avoid this potential artefact.

SUBMITTER: Haack T

PROVIDER: S-EPMC3863415 | biostudies-literature | 2013 Dec

REPOSITORIES: biostudies-literature

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Damage to the Drosophila follicle cell epithelium produces "false clones" with apparent polarity phenotypes.

Haack Timm T Bergstralh Dan T DT St Johnston Daniel D

Biology open 20131215 12

The Drosophila follicular epithelium, which surrounds developing egg chambers, is a well-established model for studying epithelial polarity because it is continuously generated from adult stem cells, making it easy to generate homozygous mutant clones in a heterozygous background. Mutant clones are usually marked by the loss of Green Fluorescent Protein (GFP) expression, which distinguishes them from their green, wild-type neighbours. Here we report that damage to the epithelium during dissectio ...[more]

PMID: 24337115

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Project description:BACKGROUND:Bioelectrical signals are known to be involved in the generation of cell and tissue polarity as well as in cytoskeletal dynamics. The epithelium of Drosophila ovarian follicles is a suitable model system for studying connections between electrochemical gradients, patterns of cytoskeletal elements and axial polarity. By interactions between soma and germline cells, the transforming growth factor-? homolog Gurken (Grk) establishes both the anteroposterior and the dorsoventral axis during oogenesis. RESULTS:In the follicular epithelium of the wild-type (wt) and the polarity mutant grk, we analysed stage-specific gradients of membrane potentials (Vmem) and intracellular pH (pHi) using the potentiometric dye DiBAC4(3) and the fluorescent pH-indicator 5-CFDA,AM, respectively. In addition, we compared the cytoskeletal organisation in the follicular epithelium of wt and grk using fluorescent phalloidin and an antibody against acetylated ?-tubulin. Corresponding to impaired polarity in grk, the slope of the anteroposterior Vmem-gradient in stage S9 is significantly reduced compared to wt. Even more striking differences in Vmem- and pHi-patterns become obvious during stage S10B, when the respective dorsoventral gradients are established in wt but not in grk. Concurrent with bioelectrical differences, wt and grk exhibit differences concerning cytoskeletal patterns in the follicular epithelium. During all vitellogenic stages, basal microfilaments in grk are characterised by transversal alignment, while wt-typical condensations in centripetal follicle cells (S9) and in dorsal centripetal follicle cells (S10B) are absent. Moreover, in grk, longitudinal alignment of microtubules occurs throughout vitellogenesis in all follicle cells, whereas in wt, microtubules in mainbody and posterior follicle cells exhibit a more cell-autonomous organisation. Therefore, in contrast to wt, the follicular epithelium in grk is characterised by missing or shallower electrochemical gradients and by more coordinated transcellular cytoskeletal patterns. CONCLUSIONS:Our results show that bioelectrical polarity and cytoskeletal polarity are closely linked to axial polarity in both wt and grk. When primary polarity signals are altered, both bioelectrical and cytoskeletal patterns in the follicular epithelium change. We propose that not only cell-specific levels of Vmem and pHi, or the polarities of transcellular electrochemical gradients, but also the slopes of these gradients are crucial for cytoskeletal modifications and, thus, for proper development of epithelial polarity.

Project description:In this study, we hypothesized that if the same deficit can be caused by damage to one or another part of a distributed neural system, then voxel-based analyses might miss critical lesion sites because preservation of each site will not be consistently associated with preserved function. The first part of our investigation used voxel-based multiple regression analyses of data from 359 right-handed stroke survivors to identify brain regions where lesion load is associated with picture naming abilities after factoring out variance related to object recognition, semantics and speech articulation so as to focus on deficits arising at the word retrieval level. A highly significant lesion-deficit relationship was identified in left temporal and frontal/premotor regions. Post-hoc analyses showed that damage to either of these sites caused the deficit of interest in less than half the affected patients (76/162 = 47%). After excluding all patients with damage to one or both of the identified regions, our second analysis revealed a new region, in the anterior part of the left putamen, which had not been previously detected because many patients had the deficit of interest after temporal or frontal damage that preserved the left putamen. The results illustrate how (i) false negative results arise when the same deficit can be caused by different lesion sites; (ii) some of the missed effects can be unveiled by adopting an iterative approach that systematically excludes patients with lesions to the areas identified in previous analyses, (iii) statistically significant voxel-based lesion-deficit mappings can be driven by a subset of patients; (iv) focal lesions to the identified regions are needed to determine whether the deficit of interest is the consequence of focal damage or much more extensive damage that includes the identified region; and, finally, (v) univariate voxel-based lesion-deficit mappings cannot, in isolation, be used to predict outcome in other patients.

Project description:BACKGROUND:Alterations of bioelectrical properties of cells and tissues are known to function as wide-ranging signals during development, regeneration and wound-healing in several species. The Drosophila follicle-cell epithelium provides an appropriate model system for studying the potential role of electrochemical signals, like intracellular pH (pHi) and membrane potential (Vmem), during development. Therefore, we analysed stage-specific gradients of pHi and Vmem as well as their dependence on specific ion-transport mechanisms. RESULTS:Using fluorescent indicators, we found distinct alterations of pHi- and Vmem-patterns during stages 8 to 12 of oogenesis. To determine the roles of relevant ion-transport mechanisms in regulating pHi and Vmem and in establishing stage-specific antero-posterior and dorso-ventral gradients, we used inhibitors of Na+/H+-exchangers and Na+-channels (amiloride), V-ATPases (bafilomycin), ATP-sensitive K+-channels (glibenclamide), voltage-dependent L-type Ca2+-channels (verapamil), Cl--channels (9-anthroic acid) and Na+/K+/2Cl--cotransporters (furosemide). Either pHi or Vmem or both parameters were affected by each tested inhibitor. While the inhibition of Na+/H+-exchangers (NHE) and amiloride-sensitive Na+-channels or of V-ATPases resulted in relative acidification, inhibiting the other ion-transport mechanisms led to relative alkalisation. The most prominent effects on pHi were obtained by inhibiting Na+/K+/2Cl--cotransporters or ATP-sensitive K+-channels. Vmem was most efficiently hyperpolarised by inhibiting voltage-dependent L-type Ca2+-channels or ATP-sensitive K+-channels, whereas the impact of the other ion-transport mechanisms was smaller. In case of very prominent effects of inhibitors on pHi and/or Vmem, we also found strong influences on the antero-posterior and dorso-ventral pHi- and/or Vmem-gradients. For example, inhibiting ATP-sensitive K+-channels strongly enhanced both pHi-gradients (increasing alkalisation) and reduced both Vmem-gradients (increasing hyperpolarisation). Similarly, inhibiting Na+/K+/2Cl--cotransporters strongly enhanced both pHi-gradients and reduced the antero-posterior Vmem-gradient. To minor extents, both pHi-gradients were enhanced and both Vmem-gradients were reduced by inhibiting voltage-dependent L-type Ca2+-channels, whereas only both pHi-gradients were reduced (increasing acidification) by inhibiting V-ATPases or NHE and Na+-channels. CONCLUSIONS:Our data show that in the Drosophila follicle-cell epithelium stage-specific pHi- and Vmem-gradients develop which result from the activity of several ion-transport mechanisms. These gradients are supposed to represent important bioelectrical cues during oogenesis, e.g., by serving as electrochemical prepatterns in modifying cell polarity and cytoskeletal organisation.

Dataset Information

Damage to the Drosophila follicle cell epithelium produces "false clones" with apparent polarity phenotypes.

Publications

Damage to the Drosophila follicle cell epithelium produces "false clones" with apparent polarity phenotypes.

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