InteractiveFly: GeneBrief

pickled eggs: Biological Overview | References

Gas2-like proteins harbour putative binding sites for both the actin and the microtubule cytoskeleton and could thus mediate crosstalk between these cytoskeletal systems. Family members are highly conserved in all metazoans but their in vivo role is not clear. The sole Drosophila Gas2-like gene, CG3973 (pigs), was recently identified as a transcriptional target of Notch signalling and might therefore link cell fate decisions through Notch activation directly to morphogenetic changes. A null mutant was generated in CG3973 (pigs): pigs¹ mutants are semi-viable but adult flies are flightless, showing indirect flight muscle degeneration, and females are sterile, showing disrupted oogenesis and severe defects in follicle cell differentiation, similar to phenotypes seen when levels of Notch/Delta signalling are perturbed in these tissues. Loss of Pigs leads to an increase in Notch signalling activity in several tissues. These results indicate that Gas2-like proteins are essential for development and suggest that Pigs acts downstream of Notch as a morphogenetic read-out, and also as part of a regulatory feedback loop to relay back information about the morphogenetic state of cells to restrict Notch activation to appropriate levels in certain target tissues (Pines, 2010).

During development, signalling pathways are repeatedly used to assign different cell fates among identical precursors. Differentiating cells then undergo regulated changes in gene expression and cell morphology appropriate for their function in a tissue. Cell shape changes are largely mediated by the cytoskeleton. Downstream effectors of developmental signalling pathways thus have to impinge on the cytoskeleton to exert the desired changes (Pines, 2010).

Spatial and temporal coordination of cytoskeletal elements can be performed by 'cytolinker' proteins that have the ability to interact with more than one cytoskeletal system at a time. Only two proteins in Drosophila contain a combination of an actin-binding Calponin homology (CH) domain and a microtubule-binding domain of the Gas2 family. One is the fly spectraplakin Short stop, and spectraplakins serve many important functions during tissue morphogenesis in both flies and mammals. The other protein is encoded by the gene CG3973, which was named pigs (‘pickled eggs’, referring to the mutant phenotype). There are four close paralogues of Pigs in mammals, and these are the only proteins in mammals to have Gas2 domains apart from the two spectraplakins MACF1 and BPAG1 (DST -- Human Gene Nomenclature Database). The first relative of Pigs identified in mammals was called Growth arrest specific 2 (Gas2), which also gave the domain its name. Although subsequent studies did not confirm a role for Gas2 in growth arrest induction, three further relatives were found and named Gas2-like 1 (Gas2L1), Gas2L2 and Gas2L3. All have CH and Gas2 domains and can associate with both the actin and the microtubule cytoskeleton in tissue culture (Goryunov, 2007). However, the in vivo role of Gas2 or the Gas2-like proteins is unclear, and to date, no loss-of-function analysis has been reported in any species. As these proteins share with the spectraplakins the presence of both a Gas2 domain and an actin-binding CH domain, it is suspected that they serve a similarly important, albeit non-overlapping, function in the regulation of the cytoskeleton. Thus, Drosophila, with only one Gas2-like homologue, seems an excellent system to analyse the function of this class of proteins, avoiding the possibility of redundant functions between paralogues masking phenotypes (Pines, 2010).

The pigs (CG3973) gene has recently been identified as containing sites occupied by the transcription factor Supressor of Hairless [Su(H)] upon activation of Notch signalling (Krejci, 2009), suggesting that pigs could act as a direct downstream target of Notch. The Notch receptor is activated through one of its membrane-bound ligands, such as Delta, on neighbouring cells. The intracellular domain of Notch (NICD) is then cleaved and released, free to act as a transcriptional co-activator together with Su(H). Notch signalling is required for many cell fate decisions during development, but relatively few genes directly activated by Notch signalling have been identified over the years. A recent study has identified what could be a comprehensive set of direct targets of Notch activation in Drosophila myogenic cells (Krejci, 2009). This study used genome-wide chromatin immunoprecipitation (ChIP) analysis with Su(H) antibodies to identify genomic regions occupied by Su(H). The first intron of pigs contained a peak of Su(H)-bound sites and thus might be a new target (Pines, 2010).

As most previously described targets of Notch are themselves transcription factors, it was intriging to find a potential cytolinker protein as a possible direct target, and thus effector, of Notch signalling. This study set out to analyse the phenotypes observed in the absence of Pigs function and to elucidate its potential role downstream of Notch. This study shows that, in addition to modifying the cytoskeleton and cell shape, Pigs appears to act as part of a feedback loop that negatively regulates the activity of Notch during morphogenesis in certain tissues (Pines, 2010).

The data demonstrate that not only is the Gas2-like protein Pigs essential for development in the fly and is a direct target of Notch signalling, but also that Pigs is probably part of a negative regulatory feedback loop that restricts Notch signalling to appropriate levels in certain tissues. These findings are intriguing, since the domain composition of Pigs would suggest it to function as a cytoskeletal crosslinker protein, helping to coordinate actin and microtubules and aiding tissue morphogenesis. Although neither a strong disruption of either actin nor microtubule cytoskeleton was observed in early stages of oogenesis in pigs¹ mutant ovaries, at late stages during the dumping phase, actin cages appeared highly disrupted. Also, already at early stages of oogenesis in the germarium, cell shapes of FCs were very irregular. As cell shape is largely determined by the cytoskeleton, this suggests that, although not visible at the level of light microscopic analysis, cytoskeletal function is impaired in pigs¹ mutant cells (Pines, 2010).

The ChIP data and reporter assays indicate that Pigs is directly regulated by Notch in at least some tissues, and in both the muscles and the ovaries, the phenotypes are compatible with Pigs being an effector of Notch. However, assays of Notch pathway activity in pigs mutants indicate that Pigs is a negative regulator of Notch activity. How can this paradox be resolved? Three alternate models for Pigs function at a molecular level can be envisaged. Pigs might facilitate cytoskeletal rearrangements induced by Notch signalling by stabilising the cytoskeleton at certain subcellular sites, and the execution of necessary changes could induce further signalling factors to terminate Notch signalling. Alternatively, Pigs could directly link Notch signalling to the cytoskeleton through sequestering Notch at a particular subcellular localisation (i.e., through linkage to the cytoskeleton), and this could bring Notch in proximity to factors that switch off the signalling appropriately. In a third scenario, the morphological changes downstream of Notch could be independent of Pigs, but Pigs could act as a molecular 'sensor' to determine if the actin and the microtubule cytoskeleton have rearranged in an appropriate fashion. In support of the second scenario, the localisation of Notch (based on detection of the intracellular domain), is changed in pigs¹ mutant FCs that are attempting to interdigitate and encapsulate a germline cyst (Pines, 2010).

Although Pigs is a cytoskeleton-associated protein, the phenotypes observed in pigs¹ mutant ovaries are not those generally seen in mutants for structural cytoskeletal proteins. FCs mutant for actin regulators such as CAP, Cofilin (Twinstar -- FlyBase), Profillin (Chickadee -- FlyBase), Ena or Abl show alterations in the actin cytoskeleton and, in some cases, multi-layering of the follicular epithelium, but do not lead to phenotypes resembling Notch-misregulation. This supports the notion that the function of Pigs confers more than just cytoskeletal changes in the FCs (Pines, 2010).

Pigs is likely to be only one of several downstream effectors of Notch in the tissues where it is directly regulated, and it remains to be proven that it is a target in the ovary. emc is a previously characterised effector of Notch in the ovary and has overlapping phenotypes with pigs¹ (Adam, 2004), supporting the model that pigs is one of several Notch targets in the ovary. This also explains the observation that the defects in pigs¹ mutant ovaries are milder than those of Notch alleles; the overall pigs loss-of-function phenotype is expected to represent just a subset of the defects caused by Notch loss-of-function. Furthermore, pigs function is needed only in a subset of tissues that depend on Notch signalling for their differentiation. For example, no sensory organ defects were observed in the pigs¹ deletion, and therefore it would be concluded that Pigs does not function downstream of Notch in the differentiation of this tissue. Consistent with this, a recent genome-wide analysis of genes involved in Notch signalling that focused on phenotypes in the external sensory organs of the notum did not identify pigs as a candidate (Pines, 2010).

The data presented in this study suggest that Pigs could serve a dual function in specific Notch-dependent processes: aiding morphological changes downstream of a differentiation signal combined with a regulatory role that allows that signal to be terminated when appropriate. Thus, the Notch-Pigs interaction provides an opportunity to further dissect the link between Notch-induced differentiation, cell shape and control of the cytoskeleton (Pines, 2010).

Search PubMed for articles about Drosophila Pickled eggs

Adam, J. C. and Montell, D. J. (2004). A role for extra macrochaetae downstream of Notch in follicle cell differentiation. Development 131: 5971-5980. PubMed ID: 15539491

Goryunov, D., et al. (2007). Molecular characterization of the genetic lesion in Dystonia musculorum (dt-Alb) mice. Brain Res. 1140: 179-187. PubMed ID: 16725123

Krejci A., Bernard F., Housden B. E., Collins S. and Bray S. J. (2009). Direct response to Notch activation: signaling crosstalk and incoherent logic. Sci. Signal. 2: ra1. PubMed ID: 19176515

Pines, M. K., Housden, B. E., Bernard, F., Bray, S. J. and Röper, K. (2010). The cytolinker Pigs is a direct target and a negative regulator of Notch signalling. Development 137(6): 913-22. PubMed ID: 20150280

date revised: 4 December 2010

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