eyes shut: Biological Overview | References
Gene name - eyes shut
Synonyms - Spacemaker (Spam)
Cytological map position - 22E1-22E1
Function - secreted ECM protein
Symbol - eys
FlyBase ID: FBgn0031414
Genetic map position - 2L:2,311,693..2,358,181 [+]
Classification - Laminin G domain; Calcium-binding EGF-like domain
Cellular location - secreted
|Recent literature||Mahato, S., Nie, J., Plachetzki, D. C. and Zelhof, A. C. (2018). A mosaic of independent innovations involving eyes shut are critical for the evolutionary transition from fused to open rhabdoms. Dev Biol. PubMed ID: 30243673
A fundamental question in evolutionary biology is how developmental processes are modified to produce morphological innovations while abiding by functional constraints. This question was addressed by investigating the cellular mechanism responsible for the transition between fused and open rhabdoms in ommatidia of apposition compound eyes; a critical step required for the development of visual systems based on neural superposition. Utilizing Drosophila and Tribolium as representatives of fused and open rhabdom morphology in holometabolous insects respectively, three changes were identified that are required for this innovation to occur. First, the expression pattern of the extracellular matrix protein Eyes Shut (EYS) was co-opted and expanded from mechanosensory neurons to photoreceptor cells in taxa with open rhabdoms. Second, EYS homologs obtained a novel extension of the amino terminus leading to the internalization of a cleaved signal sequence. This amino terminus extension does not interfere with cleavage or function in mechanosensory neurons, but it does permit specific targeting of the EYS protein to the apical photoreceptor membrane. Finally, a specific interaction evolved between EYS and a subset of Prominin homologs that is required for the development of open, but not fused, rhabdoms. Together, these findings portray a case study wherein the evolution of a set of molecular novelties has precipitated the origin of an adaptive photoreceptor cell arrangement.
|Laffafian, A. and Tepass, U. (2019). Identification of genes required for apical protein trafficking in Drosophila photoreceptor cells. G3 (Bethesda). PubMed ID: 31649044
Drosophila melanogaster photoreceptor cells are highly polarized epithelial cells. Their apical membrane is further subdivided into the stalk membrane and the light-sensing rhabdomere. The photo-pigment Rhodopsin1 (Rh1) localizes to the rhabdomere, whereas the apical determinant Crumbs (Crb) is enriched at the stalk membrane. The proteoglycan Eyes shut (Eys) is secreted through the apical membrane into an inter-rhabdomeral space. Rh1, Crb, and Eys are essential for the development of photoreceptor cells, normal vision, and photoreceptor cell survival. Human orthologs of all three proteins have been linked to retinal degenerative diseases. This study describes an RNAi-based screen examining the importance of 237 trafficking-related genes in apical trafficking of Eys, Rh1, and Crb. Twenty-eight genes were found that have an effect on the localization and/or levels of these apical proteins, and several factors were analyzed in more detail. The Arf GEF protein Sec71 was shown to be required for biosynthetic traffic of both apical and basolateral proteins, that the exocyst complex and the microtubule-based motor proteins dynein and kinesin promote the secretion of Eys and Rh1. Syntaxin 7/Avalanche was shown to controls the endocytosis of Rh1, Eys, and Crb.
The formation of epithelial luminal cavity is a fundamental process in animal development. Each ommatidium of the Drosophila retina forms an epithelial lumen, the interrhabdomeral space, which has a critical function in vision as it optically isolates individual photoreceptor cells. Ommatidia containing an interrhabdomeral space have evolved from ancestral insect eyes that lack this lumen, as seen, for example, in bees. In a genetic screen, eyes shut (eys) was identified as a gene that is essential for the formation of matrix-filled interrhabdomeral space. Eys is closely related to the proteoglycans agrin and perlecan and secreted by photoreceptor cells into the interrhabdomeral space. The honeybee ortholog of eys is not expressed in photoreceptors, raising the possibility that recruitment of eys expression has made an important contribution to insect eye evolution. These findings show that the secretion of a proteoglycan into the apical matrix is critical for the formation of epithelial lumina in the fly retina (Husain, 2006).
Many internal organs such as lungs, liver, pancreas, and kidneys contain epithelial tubes in which polarized epithelial cells surround a luminal space. Lumen formation and the maintenance of a specific lumen size, shape, and composition are important determinants of organ function. Work on models, such as the branching network of epithelial tubes that constitutes the respiratory (tracheal) system of flies or mammalian epithelial Madin-Darby canine kidney (MDCK) cells grown in a three-dimensional matrix, has begun to illuminate the cellular and molecular mechanisms of tube formation. The different strategies that are employed to create tubes hold in common that polarized epithelial architecture of the surrounding cells is either maintained or generated during tube biogenesis so that the lumen is bound by the apical surfaces of those epithelial cells. This suggests that the formation as well as the size, shape, and secretory activity of the apical membrane have pivotal functions in lumen biogenesis (Husain, 2006).
Formation of epithelial tubes is also an important aspect of nervous system morphogenesis. Neurulation in vertebrates involves the invagination of the neuroepithelium, which gives rise to the neural tube. The lumen of the neural tube can be the direct result of the invagination process or may develop secondarily through cavitation or cord hollowing. For example, neurulation in zebrafish embryos leads to the formation of a neural keel in which the apical surfaces of opposing neuroepithelial cells are in direct contact so that no lumen is apparent initially. The lumen opens later in development when the apical surfaces retract from each other. Thus, while biogenesis of the apical membrane as a result of epithelial polarization can go hand in hand with lumen formation, both processes may also be separated temporally, indicating that in addition to apical membrane formation other mechanisms are needed to open a luminal cavity. The lumen of the neural tube gives rise to the ventricular space of the adult central nervous system and the subretinal space of the retina, which is bound by the apical surfaces of photoreceptor cells (PRCs) and Müller glia cells on one side and the retinal pigment epithelium on the other. The subretinal space has an important function in vision, as some of its components contribute to the recycling of the photopigment (Husain, 2006).
Each ommatidium of a fly eye contains a luminal space, the interrhabdomeral space (IRS), that has a critical function in vision. The visual system of flies is built following the principle of neural superposition, an architecture that is believed to have evolved from an apposition compound eye type found in most insects; an apposition compound eye creates a mosaic in the retina of visual information. Fly eyes, by multiply sampling the same visual field, are more sensitive to light while retaining the resolving power of an apposition eye with the same number of ommatidia. In apposition eyes, such as those of bees, each ommatidium samples a different area in the visual field. All PRCs within one ommatidium collect light from the same area and their photosensitive membranes, the rhabdomeres, are not required to be optically isolated and tightly adhere to each other at the center of the ommatidium, forming a 'fused rhabdome'. The main PRCs of an ommatidium in apposition eyes (PRCs R2-4 and R6-8 of bees) project axons to the same interneuronal cartridge in the first optic ganglion, the lamina. This eye architecture implies that the sensitivity of apposition eyes is determined by the diameter (the aperture) of a single ommatidium. In contrast, in neural superposition eyes of flies, the main PRCs within each ommatidium (PRCs R1-6 in flies) detect light from different areas in the visual field. This requires two critical morphological changes. First, fly ommatidia display an 'open rhabdom,' where PRCs are optically isolated from each other, a function provided by the IRS. Second, each PRC projects an axon to a different interneuronal cartridge. Each cartridge still receives input from six PRCs that sample the same area in the visual field, but these PRCs are located in six different neighboring ommatidia. The result of these changes in the structure of the visual system is that the fly eye retains its resolving powerthe number of areas sampled in the visual field equals the number of ommatidiabut has a larger aperture, and thus greater sensitivity as each area in the visual field is sampled by six ommatidia rather than one (Husain, 2006).
A number of factors have been identified in recent years that are involved in shaping epithelial tubes and generating lumina of correct dimensions. However, a coherent view of the mechanisms that control lumen morphogenesis is still missing and, in particular, information about how a luminal cavity is opened up after opposing apical membranes have been established is unknown. This study used the Drosophila retina as a model to address this question. The study characterized the expression and function of eyes shut (eys), a gene required for the formation of the IRS. Eys protein is secreted into the matrix-filled IRS and is essential for the opening of a luminal cavity (Husain, 2006).
The formation of the IRS is a critical step in the development of a functional fly retina. The data suggest that the secretion of the proteoglycan Eys is essential for opening up a luminal cavity between the apical membranes of PRCs. In the absence of Eys function, PRC apical membranes remain attached to each other at both the rhabdomere and the stalk membrane. Except for the lack of an IRS, PRCs appear to undergo normal differentiation including axon pathfinding. The often seen fragmentation of individual rhabdomeres into two or three blocks of microvilli may be a secondary consequence of rhabdomeres forming while they are in direct contact with rhabdomeres of other PRCs. A molecular defect was also detected in the lumen that surrounds the sensory dendrite of mechanosensory organs, although no defect in lumen integrity or mechanosensation was identified. No Eys expression was detected in any other epithelia or lumina, suggesting that Eys plays a specific role in the formation of apical lumina of sensory epithelia (Husain, 2006).
Eys is not a component of basement membranes, like other proteoglycans, and extracellular matrix proteins such as Laminin, Collagen IV, or Perlecan could not be detected in the IRS, suggesting that the Eys-containing apical matrix of the IRS has a composition clearly distinct from basal extracellular matrix. At present, the possibility cannot be rule out that Eys is simply a secreted ligand that does not interact with the IRS matrix, although this seems unlikely, since Eys is distributed throughout the IRS and is not restricted to the plasma membrane. Recently, several proteins of the apical matrix, apical cell membrane, or membrane-associated cytoskeleton were shown to contribute to epithelial lumen morphogenesis of Caenorhabditis elegans tissues and the Drosophila tracheal system. Lumen morphogenesis of Drosophila trachea also depends on several components of the septate junction, which forms a paracellular diffusion barrier at the basolateral membrane. However, in all of these mutants at least some luminal space forms, suggesting that lumen formation may depend on multiple independent pathways once epithelial cells have polarized and an apical, nonadhesive membrane is established. Eys is distinct from other lumen morphogenesis mutants reported; in its absence a luminal cavity fails to form completely although epithelial polarity of PRCs is normal and the apical membranes appear intact and of normal dimensions (Husain, 2006).
The observations suggest that Eys is secreted through the stalk membrane, a region of the apical membrane between the rhabdomere and the zonula adherens, that topologically corresponds to the subapical region of epithelial cells; Eys remains associated with the stalk membrane in protein-positive eys mutants. Moreover, the loss of exocyst function, which is required for excretory vesicle delivery to the rhabdomere (Beronja, 2005), does not compromise Eys secretion. Modulating the length of the stalk membrane by changing the activity of Crumbs or βH-Spectrin causes abnormalities in the dimensions of the IRS but a matrix-filled lumen forms (Pellikka, 2002). Interestingly, a portion of the enlarged IRS that forms as a result of Crumbs overexpression is often not filled with matrix as detected by transmission electron microscopy (TEM), and the matrix remains attached to the distended apical membrane, leaving an empty central space. This suggests that the IRS does not have to be filled with matrix throughout to maintain an open lumen (Husain, 2006).
It is hypothesized that the recruitment of eys expression by dipteran PRCs was an important element in the transition from the ancestral apposition compound eye to the neural superposition eye of flies. The eys ortholog of A. gambia is well conserved. Many mosquito species display an IRS similar to flies, whereas A. gambia has a highly modified ommatidial structure that is adapted to nocturnal vision. In contrast, A. mellifera ommatidia do not show an IRS, and honeybees have an Eys ortholog that apparently lacks potential GAG attachment sites and some protein domains compared to the fly and mosquito proteins. Dipterans have orthologs of perlecan but not of agrin, raising the possibility that Eys is a modified version of agrin. This seems unlikely, however, as the bee genome encodes well-conserved perlecan and agrin orthologs in addition to Am-Eys. Since no clear eys ortholog was detected in genomes of animals outside the insects, it is speculated that eys (as well as the closely related gene SP2353) may have arisen through gene duplication from either agrin or perlecan genes. Interestingly, bee PRCs lack stalk membranes, raising the possibility that stalk membranes, through which Eys is secreted, and the IRS matrix may have coevolved (Husain, 2006).
The creation of a luminal space often goes hand in hand with cell polarization and apical membrane formation. In Drosophila PRC differentiation, these processes are temporally separated, allowing individual aspects of lumen formation to be analyzed independently. In addition to de-adhesion of apical membranes, secretion of Eys through the stalk membrane into the IRS is required to open a luminal space. Water import may play an important role in generating a luminal cavity in tissues such as the lung epithelia, which seems to be caused by an ionic gradient that generates osmotic pressure. As a highly glycosylated proteoglycan, Eys could promote lumen expansion by attracting water. However, such a simple model for Eys function is not supported by the observation that an Eys protein that lacks only the fourth LamG domain and remains glycosylated, as suggested by immunoblot analysis, is secreted normally but remains at the stalk and is incapable of opening a lumen. Some of the C-terminal LamG and EGF domains in agrin and perlecan are known to interact with cellular receptors such as dystroglycan and integrin. Interaction of Eys with a receptor could promote its spreading from the stalk to the rhabdomere to fill the IRS. Alternatively, this interaction could elicit a cellular response that is essential for IRS formation, such as the secretion of additional lumen components (Husain, 2006).
Eyes differ markedly in the animal kingdom, and are an extreme example of the evolution of multiple anatomical solutions to light detection and image formation. A salient feature of all photoreceptor cells is the presence of a specialized compartment (disc outer segments in vertebrates, and microvillar rhabdomeres in insects), whose primary role is to accommodate the millions of light receptor molecules required for efficient photon collection. In insects, compound eyes can have very different inner architectures. Fruitflies and houseflies have an open rhabdom system, in which the seven rhabdomeres of each ommatidium are separated from each other and function as independent light guides. In contrast, bees and various mosquitoes and beetle species have a closed system, in which rhabdomeres within each ommatidium are fused to each other, thus sharing the same visual axis. To understand the transition between open and closed rhabdom systems, the role of Drosophila genes involved in rhabdomere assembly was isolated and characterized. This study shows that Spacemaker, a secreted protein expressed only in the eyes of insects with open rhabdom systems, acts together with Prominin and the cell adhesion molecule Chaoptin to choreograph the partitioning of rhabdomeres into an open system. Furthermore, the complete loss of spacemaker (spam termed eyes shut by Flybase) converts an open rhabdom system to a closed one, whereas its targeted expression to photoreceptors of a closed system markedly reorganizes the architecture of the compound eyes to resemble an open system. These results provide a molecular atlas for the construction of microvillar assemblies and illustrate the critical effect of differences in a single structural protein in morphogenesis (Zelhof, 2006).
During ommatidium biogenesis the apical membranes of different photoreceptor cells separate from one another and concomitantly produce the rhadomeric structure needed for housing the phototransduction machinery. This process involves the precise coordination of membrane-membrane adhesion events both within and between rhabdomeres, and culminates in the production of about 60,000 microvilli per cell, each 1-2 microm in length and 50 nm in diameter. The generation of the inter-rhabdomeral space (IRS), by which rhabdomeres of the photoreceptor neurons partition from each other, is an essential event in the transition of compound eyes from a closed to an open system. This evolutionary change resulted in a considerable improvement in angular sensitivity, thus allowing the detection of smaller moving objects. Adult viable ethylmethane sulphonate-mutagenized Drosophila lines were screened for defects in rhabdomere structure and topology by examining deep-pseudopupil phenotypes and performing an electron-microscopic ultrastructural analysis of pupal and adult eyes in candidate lines. Only 2 of 40 different complementation groups, spacemaker and prominin, had eyes with apparently normal microvilli but showing a striking failure of the rhabdomeres to separate from each other, including a marked loss of IRS, irregular rhabdomere morphology and improper rhabdomeral contacts. Interestingly, loss-of-function mutations in spam completely eliminated the IRS, thus generating ommatidia resembling the compound eyes of animals with closed rhabdom systems (Zelhof, 2006).
To understand how spam and prom function, both mutants were mapped and the candidate genes were cloned and tested for rescue of the mutant phenotypes by P-element germline transformation. spam encodes a 2,165-amino-acid polypeptide containing several protein motifs commonly found in extracellular molecules: an amino terminus consisting of seven epidermal growth factor (EGF)-like repeats, a linker region containing multiple sites for glycosaminoglycan addition, and a carboxy terminus including four alternating repeats of EGF-like and Laminin G domains. prom encodes a 910-amino-acid Prominin (prom)-like molecule, a family of evolutionarily conserved transmembrane proteins often associated with microvilli but of unknown function (Zelhof, 2006).
What is the role of Spam? Sequence analysis of spam indicates that it might function as an extracellular protein. If Spacemaker is a secreted molecule, it should localize to the IRS. Indeed, immunocytochemical studies showed that Spam is selectively localized to the IRS. However, mosaic analysis reveals that even though Spam is capable of diffusing throughout the ommatidium, near wild-type levels of the protein are still required to ensure complete rhabdomere separation. To determine when spam function is required, its transcriptional unit was placed under the control of an inducible heat-shock promoter (UAS-spacemaker and heat shock (HS)-GAL4) and mutant animals were subjected to temperature shifts at various times during development. The results demonstrated that spam expression at 36-64 h after puparium formation (APF), a window of time coincident with the initiation of rhabdomere biogenesis, is sufficient and necessary for complete rescue of the phenotype. Late expression (about 72 h APF), using either HS-GAL4 or a late-expressing rhodopsin promoter (Rh1-GAL4), leads only to a partial rescue: Spam is still secreted into the IRS and the apical stalk membranes (the base of the rhabdomeres) are still capable of separating, but the rhabdomeres themselves remain fused in the centre of the ommatidium. Notably, this phenotype closely resembles that of prom mutants, suggesting that they might both participate in a common morphogenesis programme (Zelhof, 2006).
If prom and spam function in the same pathway, genetic interactions between these two loci would be expected. Indeed, although both spam and prom are recessive genes, the spam prom double heterozygote shows a failure of the rhabdomeres to separate and the loss of a continuous IRS; these results indicate that the two gene products probably act together in the biogenesis of the IRS. To explore the function of prom, its spatial and temporal expression pattern was examined. Like Spam, Prom is present at the beginning of rhabdomere biogenesis (48 h APF), when it decorates the entire photoreceptor apical surface. By the time of eclosion, however, Prom is selectively localized to the stalk membrane and the tips of the microvilli; this is best illustrated by simultaneous labelling with Chaoptin, a photoreceptor adhesion protein expressed throughout the perimeter of microvilli. Given that Spam is an IRS-secreted molecule and Prom is a rhabdomere integral membrane protein, whether these two proteins interact was examined. When Drosophila tissue-culture cells are transfected with spam, the protein is secreted into the medium. However, if the same cells are instead transfected together with prom, Spam then localizes selectively to the exterior surface of the plasma membrane. These results indicate that Prom might function as a binding partner for Spam. To demonstrate that Prom in fact recruits extracellular Spam to the plasma membrane, a mixing experiment was performed: RFP-labelled cells expressing spam were incubated with green fluorescent protein (GFP)-labelled cells transfected with prom. As proposed, secreted Spam now specifically localizes to the surface of prom-expressing cells. Taken together, these findings substantiate prom as a candidate spam receptor (Zelhof, 2006).
What is the function of prom and spam in orchestrating open rhabdomere development? Microvilli are interconnected by a network of homophilic interactions that ensures their tight and regular packing within rhabdomeres. However, such an arrangement would also render microvilli vulnerable to inter-rhabdomeric adhesion. It is suggested that secretion of spam into the IRS forces the separation of the stalk membranes, pushing the rhabdomeres apart, and that the recruitment of spam to the microvillar surface by the binding of prom prevents inter-rhabdomere adhesion. This model is consistent with the phenotypes of spam and prom mutants, and makes three significant predictions: first, overexpression of spam should increase the volume of the IRS by pushing rhabdomeres further away from each other; second, in mosaic ommatidia containing prominin mutant cells there should be a significant loss of spam binding and accumulation surrounding the mutant rhabdomeres; and third, the fused rhabdomeres of prom mutants should be rescued by independently reducing or abolishing inter-rhabdomere interactions. As predicted, overexpression of spam markedly expands the IRS of wild-type photoreceptors, and the presence of prom in mosaic ommatidia promotes the recruitment of spam selectively around wild-type rhabdomeres. To alter inter-rhabdomere adhesion, what molecule was responsible for the inappropriate fusion of rhabdomeres in prom and spam mutants needed to be determined. It was reasoned that Chaoptin, a photoreceptor-specific glycosylphosphatidylinositol-linked membrane protein expressed very early during photoreceptor morphogenesis and required for crosslinking microvilli by means of homophilic interactions, might be a strong candidate (Krantz, 1990; Van Vactor, 1988; Reinke, 1988). It was recognized that the early expression of Chaoptin would ensure the proper packing of microvilli within rhabdomeres and that the subsequent expression of spam and prom would guarantee that individual rhabdomeres remain separated in open rhabdom systems. Indeed, removing just one copy of the gene encoding Chaoptin (chp) in a prom mutant background (prom/prom;chp/ +) is sufficient for a drastic suppression of the prom mutant phenotype. These data demonstrate that the capacity of spam to push rhabdomeres apart is very sensitive to the amount of adhesive force available (for example Chaoptin). Given these results, it was anticipated that further reducing the levels of spam in this prom/prom;chp/+ genetic background (that is, bringing the ratio of spam to chp back to normal levels) should revert the 'rescued phenotype' to the mutant state. As proposed, the rhabdomeres are now stuck together. Thus, in this genetic background, there is sufficient Spam to partition the stalk membranes but not enough to overcome the Chaoptin-dependent adhesiveness between adjacent rhabdomeres. A final prediction of these findings is that early expression of prom and spam should result in the development of broken-up rhabdomeres, probably as the Spam/Prom complex outcompetes the adhesive force linking microvilli. The data validate this postulate. Together, these results demonstrate that Spam, Prom and Chaoptin orchestrate the assembly of microvilli, ensure the structural integrity and the partitioning of rhabdomeres, and guarantee the construction of an open rhabdom system (Zelhof, 2006).
Are these molecules responsible for the transformation between closed and open rhabdom systems? To examine this possibility, expression of Spam, Prom and Chaoptin homologues was examined in other insect species containing open or closed rhabdoms. First, analysis of Anopheles gambiae eyes, a closed system, showed that both Prom and Chaoptin are present but Spam is absent from pupal eyes, suggesting that Spam might be the critical component for partitioning rhabdomeres. To lend further credibility to this hypothesis, spam expression was isolated and examined in several other species, two with open rhabdomere systems (the housefly Musca domestica and the mosquito Toxorhynchites amboinensis) and two with closed ones (the honeybee Apis mellifera and the flour beetle Tribolium castaneum). As predicted, spam is conspicuously absent from the eyes of insects with closed rhabdomere systems, even though spam expression is still detected in the body of all of these species, probably reflecting an evolutionarily conserved role (Avidor-Reiss, 2004; Zelhof, 2006 and references therein).
Taken together, these results substantiate spam expression as a powerful indicator of visual system architecture and suggest that targeting spam/prom function to a closed rhabdom system may well transform it into an open system. To test this final postulate, focus was placed on the ocellar photoreceptors of Drosophila. Ocelli are simple eyes located at the vertex of the fly head and involved in navigation. They are composed of 70-90 photoreceptor neurons organized in a near-close rhabdomeric arrangement. Flies were engineered in which spam expression was targeted to the ocelli under the control of either a weak (HS-GAL4/UAS-spam at 29°C) or a strong (glass multimer reporter (GMR)-GAL4, UAS-spam) promoter, and eye morphology was examined by electron microscopy. Overexpression of spam produced a complete reorganization of ocellar eye architecture ranging from the creation of a novel IRS surrounding otherwise structurally intact rhabdomeres to the generation of an extensive IRS accompanied by the disintegration of the rhabdomeres into their individual microvilli (GMR-GAL4, UAS-spam) (Zelhof, 2006).
Taken together, these studies delineate a vital evolutionary role for spam in the construction of insect eyes, assign candidate functions to three important proteins and provide a mechanistic path for the assembly of rhabdomeres. Interestingly, human prominin-1 localizes to the base of the outer segments, a subcellular compartment responsible for producing new discs, and frameshift mutations in hProm-1 leads to severe retinal degeneration. It is suggested that, much as in Drosophila, prom has a key function in the morphogenesis of discs by ensuring their unimpeded folding and stacking into the outer segment (Zelhof, 2006).
Haltom, A. R., Lee, T. V., Harvey, B. M., Leonardi, J., Chen, Y. J., Hong, Y., Haltiwanger, R. S. and Jafar-Nejad, H. (2014). The protein O-glucosyltransferase Rumi modifies Eyes shut to promote rhabdomere separation in Drosophila. PLoS Genet 10: e1004795. PubMed ID: 25412384
The protein O-glucosyltransferase Rumi/POGLUT1 regulates Drosophila Notch signaling by adding O-glucose residues to the Notch extracellular domain. Rumi has other predicted targets including Crumbs (Crb) and Eyes shut (Eys), both of which are involved in photoreceptor development. However, whether Rumi is required for the function of Crb and Eys remains unknown. This study reports that in the absence of Rumi or its enzymatic activity, several rhabdomeres in each ommatidium fail to separate from one another in a Notch-independent manner. Mass spectral analysis indicates the presence of O-glucose on Crb and Eys. However, mutating all O-glucosylation sites in a crb knock-in allele does not cause rhabdomere attachment, ruling out Crb as a biologically-relevant Rumi target in this process. In contrast, eys and rumi exhibit a dosage-sensitive genetic interaction. In addition, although in wild-type ommatidia most of the Eys protein is found in the inter-rhabdomeral space (IRS), in rumi mutants a significant fraction of Eys remains in the photoreceptor cells. The intracellular accumulation of Eys and the IRS defect worsen in rumi mutants raised at a higher temperature, and are accompanied by a approximately 50% decrease in the total level of Eys. Moreover, removing one copy of an endoplasmic reticulum chaperone enhances the rhabdomere attachment in rumi mutant animals. Altogether, these data suggest that O-glucosylation of Eys by Rumi ensures rhabdomere separation by promoting proper Eys folding and stability in a critical time window during the mid-pupal stage. Human EYS, which is mutated in patients with autosomal recessive retinitis pigmentosa, also harbors multiple Rumi target sites. Therefore, the role of O-glucose in regulating Eys may be conserved (Haltom, 2014 PubMed).
Maintaining cell shape and tone is crucial for the function and survival of cells and tissues. Mechanotransduction relies on the transformation of minuscule mechanical forces into high-fidelity electrical responses. When mechanoreceptors are stimulated, mechanically sensitive cation channels open and produce an inward transduction current that depolarizes the cell. For this process to operate effectively, the transduction machinery has to retain integrity and remain unfailingly independent of environmental changes. This is particularly challenging for poikilothermic organisms, where changes in temperature in the environment may impact the function of mechanoreceptor neurons. Thus, it was of interest to discover how insects whose habitat might quickly vary over several tens of degrees of temperature manage to maintain highly effective mechanical senses. Drosophila mutants were screened for defective mechanical responses at elevated ambient temperatures, and a gene, spam, was identified whose role is to protect the mechanosensory organ from massive cellular deformation caused by heat-induced osmotic imbalance. This study shows that Spam protein forms an extracellular shield that guards mechanosensory neurons from environmental insult. Remarkably, heterologously expressed Spam protein also endowed other cells with superb defense against physically- and chemically-induced deformation. The mechanical impact of Spam-coating was studied and it was shown that Spam-coated cells are up to ten times stiffer than uncoated-controls. Together, these results help explain how poikilothermic organisms preserve the architecture of critical cells during environmental stress, and illustrate an elegant and simple solution to such challenge (Cook, 2008).
Fly mechanoreceptor neurons (MRNs) are essential for a number of critical functions such as hearing, proprioception, flight control and touch sensing, and their mis-function leads to uncoordination and loss of mechanoreceptor responses. To identify components of the machinery that preserve the functional integrity of the mechanosensory apparatus at high environmental temperatures, a genetic screen was carried out for temperature-sensitive uncoordinated flies; it was anticipated that loss-of-function mutations in such components may render MRN function highly susceptible to the elevated temperature. Approximately 12,000 ethylmethane sulphonate-mutagenized homozygous lines6 were examined for intact locomotor responses at RT, but defective behavior after 1 hr at 37°C . One mutant line, 2649, had no apparent defects at RT, including walking, feeding and flying, but upon shifting to the restrictive temperature the flies gradually lost the ability to fly, to stand upside-down, and to climb the walls, until eventually they could only lie and sporadically move their legs, wings and mouth-parts in an uncoordinated manner. Genetic mapping and transformation rescue experiments proved that the mechanosensory defects of line 2649 are due to a non-sense mutation in the spacemaker gene (Cook, 2008)
Recently, it was shown that spam encodes an extracellular protein required for creating the intra-rhabdomeral space (IRS) in the compound eyes of insects with open rhabdom systems7. There, Spam provides the extracellular substrate to sustain the precise arrangement of rhabdomeres within each ommatidium. Notably, the other sites of Spam expression are on mechanosensory and chemosensory neurons. To directly examine the impact of loss-of-function mutations in spam on mechanosensory transduction, electrophysiological recordings were performed from bristle mechanoreceptors (touch) and antennal chordotonal organs (hearing) from control and mutant flies. Sensory bristles were given calibrated mechanical stimuli while recording transduction currents with a voltage-clamp apparatus. At 21°C, control flies and spam mutants displayed robust inward currents in response to bristle deflections. In contrast, 30 min of exposure to 37°C reduced mechanoreceptor response amplitudes in spam mutant animals by over 80%. The same heat exposure also nearly abolished all mechanoreceptor antennal responses, while having no significant effect on control flies (Cook, 2008).
Next, the ultrastructure was examined of MRN in control flies and spam mutants at both permissive and non-permissive temperatures. Drosophila mechano- and chemosensory neurons house their entire sensory apparatus in a ciliated outer-segment that forms the neuronal sensory endings. In the case of MRNs, this outer segment is bathed in an extracellular fluid (lymph) which provides the proper ionic environment for the generation of mechanoreceptor currents. Remarkably, spam mutants, but not control flies, experience a dramatic deformation of their MRN in response to heat treatment: the entire neuronal cytoplasm invades the lymph space, such that the region that normally contained only the cilium and extracellular fluid now becomes filled with cellular material from the MRN cell body (Cook, 2008).
How does exposure to elevated temperatures have such a dramatic effect on the morphology of spam MRNs? Changes in molecular thermal motion between 21°C and 37°C are too small, and unlikely to account for the phenotype. Therefore a prominent secondary effect of heat was considered: water loss by evaporation. To investigate how much water is lost during the heat exposure, the weight of control and mutant flies was measured at 15 min intervals. All flies lose ~20% of their total weight after 60 min at 37°C (~25% of their water content), yet only the mutants display the mechanosensory defect. To determine whether the heat-induced deformation of MRN in spam mutants is indeed a consequence of water loss, spam flies were placed either in a control petri dish or in a dish at over 90% humidity, and subjected to the 60 minutes treatment at 37°C. Notably, only the flies in the dry chamber were affected by heat; exposure to high humidity during the high-temperature treatment completely prevented the manifestation of the mutant phenotype, both morphologically and behaviorally. These data demonstrate that the mutant's mechanosensory deficit does not arise from an effect of temperature per se, but is instead triggered by excessive water evaporation at high temperature. Why does water loss lead to deformation of the MRN only in spam mutants? It was hypothesized that the rapid loss of water from the animal's circulatory system (hemolymph) would increase its osmolarity, leading to an outflow of water from the sensory lymph. The new imbalance between the MRN cytoplasm and the lymph would cause the deformation of the MRN cytosol, which if not contained (as in the absence of Spam protein), would then invade the lymph space. This proposed mechanism anticipates that hypertonic shock to the hemolymph of spam mutants, but not wild type animals, should mimic the effect of high temperature on the morphology and function of MRNs (i.e. hypertonic shock should induce a similar osmotic imbalance between the endolymph and the MRN cytoplasm). A high-osmolarity solution was injected to the abdomen of spam and control flies and these flies were prepared for EM examination. As hypothesized, only spam flies showed deformation of the MRN and loss of mechanosensory responses, substantiating the mechanism of deformation and the role of Spam in maintaining cell-shape (Cook, 2008).
In photoreceptor neurons, Spam is secreted into the inter-rhabdomeral space where it forms the extracellular medium that organizes and preserves the separation of rhabdomeres. It was reasoned that in mechanoreceptor neurons the role of Spam might be a variation on this theme, perhaps this time serving as a cellular exoskeleton that provides structural rigidity to the MRN, thus ensuring the preservation of cell shape under environmental stress. This postulate makes two significant predictions: First, Spam protein should be specifically localized within the fly's mechanoreceptor organ, at locations that might be particularly vulnerable to osmotic pressure changes. Second, if Spam functions as a mechanical barrier that protects MRN from deformation, it should be possible to engineer cells that are coated with Spam and make them resistant to osmotic insult and deformation pressures. Indeed, Spam protein concentrates at two specific sites in MRN: one, right at the interface between the MRN cell body and the lymph space, the very domain that collapses at high temperature in mutant animals, and at a second site close to the ciliary dilation, possibly helping sustain the two ciliary processes at the proper position. To generate cells that are decorated by a layer of Spam, advantage was taken of Spam's ability to directly bind the membrane receptor Prominin. Therefore, Drosophila tissue culture cells expressing and secreting Spam were incubated with GFP-labeled cells transfected with Prominin. As expected, secreted Spam specifically decorated the surface of Prominin-expressing cells; to identify those cells that are entirely (or nearly completely) coated, immunofluorescent staining was performed with anti-Spam antibodies. Cellular deformation was induced by subjecting control and coated-cells to hyper- and hypo-osmotic solutions. As predicted, control cells undergo significant swelling following hypo-osmotic shock, and severe shrinking in the presence of hyper-osmotic solutions. In contrast, coated cells were largely resistant to these treatments and showed only minor changes in shape and size (not surprisingly, poorly coated cells were indistinguishable from controls). Next, the impact of Spam on chemically-induced cell shape changes was examined. Control cells were subjected to latrunculin A and dramatic changes were elicited in cell morphology. However, Spam coated cells retained their normal spherical shape, even after extensive actin remodeling resulting from the latrunculin A treatment. Collectively, these studies demonstrate that Spam coating of the plasma membrane endows cells with exquisite protection against osmotically- and chemically-induced transformations in cell shape (Cook, 2008).
How robust are Spam-treated cells? The stiffness of Spam-coated and control cells was directly examined by measuring their mechanical properties. In these experiments, a glass filament of known bending constant is continuously pressed against the cell using a linear piezoelectric drive. The force applied to the tip of the probe by the resistance of the cell to indentation is then calculated by optically measuring the bending of the glass probe. The major source of stiffness in cells is the actin cytoskeleton. Therefore, to eliminate the contribution of the cystoskeleton and explore the specific effect of Spam, experimental and control samples were first treated with cytochalasin D for 120 min. The results demonstrate that Spam-coated cells exhibit stiffness that is approximately 10 times that of control cells (Cook, 2008).
Together, these studies have revealed a remarkable solution to the problem of maintaining cellular integrity and structure under duress. They also provide a salient example of evolution employing the same protein to satisfy two very different needs: the building of compound eyes in open rhabdom systems, and the preservation of cell shape in mechano- and chemoreceptor organs. Interestingly, both entail the production and assemblage of a rigid substrate, thus highlighting the fundamental role of Spam in tissue morphogenesis (in one scenario to ensure the partitioning and maintenance of the rhabdomere complex, and in the other to guarantee the mechanical integrity of sensory neurons). Finally, it is worth noting that the ability to assemble a 'cell-wall' surrounding an animal cell may provide the foundation for important applications in cell engineering, where resistance to osmotic pressures may be warranted, or where preservation of cell and tissue structure (or tone) may be needed (Cook, 2008).
In patients with autosomal-recessive retinitis pigmentosa (arRP), homozygosity mapping was performed for detection of regions harboring genes that might be causative for RP. In one affected sib pair, a shared homozygous region of 5.0 Mb was identified on chromosome 6, within the RP25 locus. One of the genes residing in this interval was the retina-expressed gene EGFL11. Several genes resembling EGFL11 were predicted just centromeric of EGFL11. Extensive long-range RT-PCR, combined with 5'- and 3'- RACE analysis, resulted in the identification of a 10-kb transcript, starting with the annotated exons of EGFL11 and spanning 44 exons and 2 Mb of genomic DNA. The transcript is predicted to encode a 3165-aa extracellular protein containing 28 EGF-like and five laminin A G-like domains. Interestingly, the second part of the protein was found to be the human ortholog of Drosophila eyes shut (eys), also known as spacemaker, a protein essential for photoreceptor morphology. Mutation analysis in the sib pair homozygous at RP25 revealed a nonsense mutation (p.Tyr3156X) segregating with RP. The same mutation was identified homozygously in three arRP siblings of an unrelated family. A frame-shift mutation (pPro2238ProfsX16) was found in an isolated RP patient. In conclusion, a gene, coined eyes shut homolog (EYS), has been identified consisting of EGFL11 and the human ortholog of Drosophila eys, which is mutated in patients with arRP. With a size of 2 Mb, it is one of the largest human genes, and it is by far the largest retinal dystrophy gene. The discovery of EYS might shed light on a critical component of photoreceptor morphogenesis (Collin, 2008).
Using a positional cloning approach supported by comparative genomics, a previously unreported gene, EYS, was reported at the RP25 locus on chromosome 6q12 commonly mutated in autosomal recessive retinitis pigmentosa. Spanning over 2 Mb and containing 43 exons, this is the largest eye-specific gene identified so far. EYS is independently disrupted in four other mammalian lineages, including that of rodents, but is well conserved from Drosophila to man and is likely to have a role in the modeling of retinal architecture (Abd El-Aziz, 2008).
The unfolded protein response (UPR) is composed by homeostatic signaling pathways that are activated by excessive protein misfolding in the endoplasmic reticulum. Inositol-requiring enzyme-1 (Ire1) signaling is an important mediator of the UPR, leading to the activation of the transcription factor Xbp1. This study shows that Drosophila Ire1 mutant photoreceptors have defects in the delivery of rhodopsin-1 to the rhabdomere and in the secretion of Spacemaker/Eyes Shut into the interrhabdomeral space. However, these defects are not observed in Xbp1 mutant photoreceptors. Ire1 mutant retinas have higher mRNA levels for targets of regulated Ire1-dependent decay (RIDD), including for the Fatty acid transport protein (Fatp). Importantly, the downregulation of fatp by RNAi rescues the rhodopsin-1 delivery defects observed in Ire1 mutant photoreceptors. These results show that the role of Ire1 during photoreceptor differentiation is independent of Xbp1 function and demonstrate the physiological relevance of the RIDD mechanism in this specific paradigm (Coelho, 2013).
The endoplasmic reticulum (ER) is the cell organelle where secretory and membrane proteins are synthesized and folded. When the folding capacity of the ER is impaired, the presence of incorrectly folded (misfolded) proteins in the ER causes ER stress and activates the unfolded protein response (UPR), which helps to restore homeostasis in the ER. In higher eukaryotes, the activation of the UPR is accomplished via three signaling pathways induced by ER-resident molecular ER stress sensors: protein kinase (PKR)-like ER kinase (PERK),activating transcription factor 6 (ATF6), and inositol-requiring enzyme 1 (Ire1). Being conserved in all eukaryotes, Ire1 contains an ER luminal domain, which is involved in the recognition of misfolded proteins, and cytoplasmic endoribonuclease and kinase domains, which are involved in the activation of downstream pathways. Activated Ire1 mediates the nonconventional splicing of an intron from X box binding protein 1 (Xbp1) mRNA (or HAC1 mRNA, the yeast Xbp1 ortholog), causing a frameshift during translation, thereby introducing a different carboxyl domain in the Xbp1 protein. Xbp1spliced is an effective transcription factor that regulates the expression of ER chaperones and other target genes (Coelho, 2013).
In addition to mediating Xbp1 mRNA splicing, cell culture studies demonstrated that Ire1 promotes the degradation of mRNAs encoding ER-targeted proteins, a process called RIDD (regulated Ire1-dependent decay), to reduce the load of ER client proteins during ER stress. The cytosolic domain of mammalian IRE1 binds Traf2 (tumor necrosis factor receptor-associated factor 2), an upstream activator of the c-Jun N-terminal kinase (JNK) signaling pathway. This IRE1/ Traf2 interaction is also independent of Xbp1 splicing and may lead to the activation of apoptosis after prolonged ER stress (Coelho, 2013 and references therein).
In the Drosophila photoreceptor cells, the rhabdomere is the light-sensing organelle, a stack of photosensitive apical microvilli that is formed during the second half of pupal development. The rhabdomere is formed in the apical domain of each photoreceptor cell, which after a 90 rotation extends its apical domain along the proximal-distal axis of the retina. The growth of the rhabdomere requires the delivery of large amounts of membrane and proteins into this structure, imposing a considerable demand to the cellular mechanisms controlling protein folding and membrane production in the ER (Coelho, 2013).
Among the proteins targeted to the developing rhabdomeres are the rhodopsins, the light-sensitive proteins, and other proteins involved in the transduction of the light stimuli. Rhodopsin-1 (Rh1) is a seven transmembrane domain protein that starts to be expressed by 78% of pupal life and is delivered to the rhabdomeres of the outer photoreceptors (R1–R6), in a trafficking process that requires the activity of Rab11, MyosinV, and dRip11. The delivery of Rh1 to the rhabdomere is required for rhabdomere morphogenesis because in Rh1-null mutants, the rhabdomere does not form, causing degeneration of the photoreceptors (Coelho, 2013).
In mammalians, the microRNA mir-708 is upregulated by CCAAT enhancer-binding protein homologous protein (CHOP) to control rhodopsin expression levels and prevent an excessive rhodopsin load into the ER . In Drosophila, Ire1 signaling is activated in the photoreceptors upon expression of Rh1 folding mutants or in ninaA mutations that cause the accumulation of misfolded Rh1 in the ER. However, the role of Ire1 signaling during normal photoreceptor differentiation remains unknown. This study shows that Ire1 signaling is activated in the photoreceptors during pupal stages of Drosophila development. Ire1 mutant photoreceptors have defects in the delivery of Rh1 to the rhabdomere and the secretion of Spacemaker/Eyes Shut (Spam/Eys) into the interrhabdomeral space (IRS). Surprisingly, Xbp1-null mutant photoreceptors have a milder phenotype with no defects in Rh1 delivery into the rhabdomere or Spam/Eys secretion. Targets of RIDD are upregulated in Ire1 mutant retinas, including the fatty acid transport protein (fatp), a known regulator of Rh1 protein levels. Finally, it was shown that the regulation of fatp levels by RIDD is critical for normal Rh1 delivery into the rhabdomere (Coelho, 2013).
Studies in mammalian systems revealed that the Ire1/Xbp1 signaling pathway is important during development for the differentiation of secretory cells. For example, Xbp1 'knockout' mice have defects in the differentiation of antibody-secreting plasma cells and secretory cells of the exocrine glands of the pancreas. Presumably, in these cases, activation of Ire1/Xbp1 signaling is required to increase the capacity of the ER to fold and process the high load of secreted proteins (Coelho, 2013).
The present results demonstrate that Ire1 signaling is required for photoreceptor differentiation and rhabdomere morphogenesis, a process that also imposes a high demand to the capacity of the ER to fold proteins such as Spam/Eys and Rh1. As shown, Ire1 mutant photoreceptors have defects in the secretion of Spam/Eys to the IRS and in the delivery of Rh1 to the rhabdomere. However, activation is seen of the Xbp1-EGFP reporter starting at 48 hr of pupal development, well before when the Spam/Eys secretion and Rh1 delivery defects are observed. Presumably, the folding of other unidentified proteins during these earlier stages might also require Ire1 signaling. It is noteworthy though, that in mutant B lymphocytes modified to lack antibody production, Ire1 is still activated (and Xbp1 spliced) upon lymphocyte differentiation to plasma cells. Activation of Ire1/Xbp1 signaling in this context seems to be part of the process of plasma cell differentiation, independently of the accumulation of misfolded proteins in the ER lumen (Coelho, 2013).
Ire1 function is also required for the regulation of the membrane lipids. In mammalians, Ire1/Xbp1 signaling regulates the biosynthesis of phospholipids and other lipids. A study in yeast demonstrated that Ire1 is activated by 'membrane aberrancy,' a condition of stress caused by the experimental depletion of inositol. Activation of Ire1 in this case occurs by a mechanism that is distinct from the one involving the recognition of misfolded proteins by the luminal domain of Ire1. Furthermore, Ire1 can be activated by direct binding of flavonoids, such as quercetin, to a pocket present in the cytoplasmic domain of Ire1, in a mechanism that is also independent of the binding of misfolded proteins to Ire1. The present results do not clarify if Ire1 activation in the photoreceptors during pupal stages results from the accumulation of misfolded proteins in the ER lumen or an imbalance in the membrane lipids (Coelho, 2013).
The results demonstrate that Ire1 signaling is required for photoreceptor differentiation and rhabdomere morphogenesis in an Xbp1-independent manner. Studies using cell culture paradigms demonstrated that, in addition to mediating Xbp1 mRNA splicing, Ire1 also promotes RIDD, the degradation of mRNAs encoding ER-targeted proteins, but the physiological significance of the RIDD mechanism is unknown. Quantitative RT-PCR results show that RIDD targets are upregulated in Ire1 mutant eyes, including fatp, a regulator of Rh1 protein levels. The results show that regulation of fatp mRNA by RIDD is critical for rhabdomere morphogenesis because the experimental downregulation of fatp mRNA by RNAi rescues the Rh1 rhabdomere delivery defect observed in Ire1 mutants (Coelho, 2013).
Rh1 protein levels and Rh1 delivery to the rhabdomere are very sensitive to the levels of sphingolipids and phosphatidic acid. Increased fatp levels may lead to an increase in the levels of fatty acids and, subsequently, phosphatidic acid, which is known to downregulate Rh1 protein levels and cause rhabdomere morphogenesis defects. High levels of phosphatidic acid disrupt the Arf1-dependent transport of membrane to the developing rhabdomere. The results show that phosphatidic acid levels are elevated in Ire1 mutant retinas, and lowering phosphatidic acid levels by expression of LPP rescues the defects observed in Ire1 mutants, demonstrating that Ire1/fatp-dependent regulation of fatty and phosphatidic acids levels is important for rhabdomere morphogenesis in Drosophila. In addition, it is possible that the increase in phosphatidic acid levels in Ire1 mutant photoreceptors is also caused by the activation of PERK because upregulation was observed of the PERK pathway mediator ATF4 in Ire1 mutant photoreceptors, and in cell culture models, it was shown that PERK is able to phosphorylate diacylglycerol and generate phosphatidic acid. In conclusion, the results, using well-characterized genetic tools (Ire1 and Xbp1-null mutations) and a developmental paradigm (photoreceptor differentiation in the Drosophila pupa), demonstrate the physiological relevance of Xbp1-independent mechanisms downstream of Ire1 signaling (Coelho, 2013).
Search PubMed for articles about Drosophila Eyes shut
Abd El-Aziz, M. M., et al. (2008). EYS, encoding an ortholog of Drosophila spacemaker, is mutated in autosomal recessive retinitis pigmentosa. Nat. Genet. 40(11): 1285-1287. PubMed ID: 18836446
Avidor-Reiss, T. et al. (2004). Decoding cilia function: defining specialized genes required for compartmentalized cilia biogenesis. Cell 117: 527-539. PubMed ID: 15137945
Beronja, S., Laprise, P., Papoulas, O., Pellikka, M., Sisson, J. and Tepass, U. (2005). Essential function of Drosophila Sec6 in apical exocytosis of epithelial photoreceptor cells. J. Cell Biol. 169(4): 635-46. PubMed ID: 15897260
Collin, R. W. J., et al. (2008). Identification of a 2 Mb human ortholog of Drosophila eyes shut/spacemaker that is mutated in patients with retinitis pigmentosa. Am. J. Hum. Genet. 83(5): 594-603. PubMed ID: 18976725
Cook B, Hardy RW, McConnaughey WB, Zuker CS. (2008). Preserving cell shape under environmental stress. Nature 452(7185): 361-4. PubMed ID: 18297055
Husain, N., et al. (2006). The Agrin/Perlecan-related protein Eyes Shut is essential for epithelial lumen formation in the Drosophila retina. Dev. Cell 11: 483-493. PubMed ID: 17011488
Krantz, D. E. and Zipursky, S. L. (1990). Drosophila chaoptin, a member of the leucine-rich repeat family, is a photoreceptor cell-specific adhesion molecule. EMBO J. 9: 1969-1977. PubMed ID: 2189727
Pellikka, M., et al. (2002). Crumbs, the Drosophila homologue of human CRB1/RP12, is essential for photoreceptor morphogenesis. Nature 416: 143-149. PubMed ID: 11850625
Reinke, R., Krantz, D. E., Yen, D. and Zipursky, S. L. (1988). Chaoptin, a cell surface glycoprotein required for Drosophila photoreceptor cell morphogenesis, contains a repeat motif found in yeast and human. Cell 52: 291-301. PubMed ID: 3124963
Van Vactor, D., Krantz, D. E., Reinke, R. and Zipursky, S. L. (1988). Analysis of mutants in chaoptin, a photoreceptor cell-specific glycoprotein in Drosophila, reveals its role in cellular morphogenesis. Cell 52: 281-290. PubMed ID: 2449286
Zelhof, A. C., Hardy, R. W., Becker, A. and Zuker, C. S. (2006). Transforming the architecture of compound eyes. Nature 443(7112): 696-9. PubMed ID: 17036004
date revised: 25 May 2010
Home page: The Interactive Fly © 2009 Thomas Brody, Ph.D.
The Interactive Fly resides on the
Society for Developmental Biology's Web server.