InteractiveFly: GeneBrief

roughest: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | Evolutionary Homologs | References

Gene name - roughest

Synonyms - irregular optic chiasma C, irreC-roughest

Cytological map position - 3C5

Function - surface receptor

Keywords - axon guidance, eye

Symbol - rst

FlyBase ID: FBgn0003285

Genetic map position - 1-2.2

Classification - Ig-C2-type-domain protein, transmembrane domain

Cellular location - surface transmembrane

NCBI link: Entrez Gene

rst orthologs: Biolitmine
Recent literature
Linneweber, G. A., Winking, M. and Fischbach, K. F. (2015). The cell adhesion molecules Roughest, Hibris, Kin of Irre and Sticks and Stones are required for long range spacing of the Drosophila wing disc sensory sensilla. PLoS One 10: e0128490. PubMed ID: 26053791
The development of external sensory organs requires complex cell-cell communication in order to give each cell a specific identity and to ensure a regular distributed pattern of the sensory bristles. In a variety of processes the heterophilic Irre Cell Recognition Module, consisting of the Neph-like proteins: Roughest, Kin of irre and of the Nephrin-like proteins: Sticks and Stones, Hibris, plays key roles in the recognition events of different cell types throughout development. In the present study these proteins are apically expressed in the adhesive belt of epithelial cells participating in sense organ development in a partially exclusive and asymmetric manner. Using mutant analysis the GAL4/UAS system, RNAi and gain of function an involvement was found of all four Irre Cell Recognition Module-proteins in the development of a highly structured array of sensory organs in the wing disc. The proteins secure the regular spacing of sensory organs showing partial redundancy and may function in early lateral inhibition events as well as in cell sorting processes. Comparisons with other systems suggest that the Irre Cell Recognition module is a key organizer of highly repetitive structures.
Machado, M. C. R., Valer, F. B., Lima, C. A. C. and Ramos, R. G. P. (2018). Transcriptional cross-regulation of Irre Cell Recognition Module (IRM) members in the Drosophila pupal retina. Mech Dev. PubMed ID: 30030087
How cell adhesion molecules coordinate dynamic expression in space and time - an essential requirement for their function - is regulated at the genomic and transcriptional levels constitutes an important, albeit still little understood question. The Irre Cell Recognition Module (IRM) is a highly conserved phylogenetically group of structurally related single pass transmembrane glycoproteins belonging to the immunoglobulin superfamily that in Drosophila melanogaster are encoded by the genes roughest (rst), kin-of-irre (kirre), sticks-and-stones (sns) and hibris (hbs). Their cooperative and often partly redundant action are crucial to major developmental processes such axonal pathfinding, myoblast fusion and patterning of the pupal retina. In this latter system rst and kirre display a tightly regulated complementary transcriptional pattern so that lowering rst mRNA levels leads to a concomitant increase in kirre mRNA concentration. This study investigated whether other IRM components are similarly co-regulated and the extent changes in their mRNA levels affect each other as well as their collective function in retinal patterning. The results demonstrate that silencing any of the four IRM genes in 24% APF retinae changes the levels all other group members although only kirre and hbs mRNA levels are increased. Furthermore, expression, in a rst null background, of truncated versions of rst cDNA in which the portion encoding the intracellular domain has been partially or completely removed not only can still induce changes in mRNA levels of other IRM members but also result in Kirre mislocalization. Taken together, these data point to the presence of a highly precise and fine-tuned control mechanism coordinating IRM expression that may be crucial to the functional redundancy shown by its components during the patterning of the pupal retina.
Valer, F. B., Machado, M. C. R., Silva-Junior, R. M. P. and Ramos, R. G. P. (2018). Expression of Hbs, Kirre and Rst during Drosophila ovarian development. Genesis. PubMed ID: 30114331
The Irre Cell-Recognition Module (IRM) is a group of evolutionarily conserved and structurally related transmembrane glycoproteins of the immunoglobulin superfamily. In Drosophila melanogaster it comprises the products of the genes roughest (rst; also known as irreC-rst), kin-of-irre (kirre; also known as duf), stick-and-stones (sns) and hibris (hbs). In this model organism, the behavior of this group of proteins as a partly redundant functional unit mediating selective cell recognition was demonstrated in a variety of developmental contexts, but their possible involvement in ovarian development and oogenesis has not been investigated, notwithstanding the fact that some rst mutant alleles are also female sterile. This study shows that IRM genes are dynamically and, to some extent, coordinately transcribed in both pupal and adult ovaries. Additionally, the spatial distribution of Hbs, Kirre and Rst proteins indicates that they perform cooperative, although largely non-redundant, functions. Finally, phenotypical characterization of three different female sterile rst alleles uncovered two temporally separated and functionally distinct requirements for this locus in ovarian development: one in pupa, essential for the organization of peritoneal and epithelial sheaths that maintain the structural integrity of the adult organ and another, in mature ovarioles, needed for the progression of oogenesis beyond stage 10.
Ben-Zvi, D. S. and Volk, T. (2019). Escort cell encapsulation of Drosophila germline cells is maintained by Irre cell recognition module proteins. Biol Open 8(3). PubMed ID: 30837217
Differentiation of germline stem cells (GSCs) in the Drosophila ovary is induced by somatic escort cells (ECs), which extend membrane protrusions encapsulating the germline cells (GCs). Germline encapsulation requires activated epidermal growth factor receptor (Egfr) signaling within the ECs, following secretion of its ligands from the GCs. This study shows that the conserved family of irre cell recognition module (IRM) proteins is essential for GC encapsulation by ECs, with a requirement for roughest (rst) and kin of irre (kirre) in the germline and for sticks and stones (sns) and hibris (hbs) in ECs. In the absence of IRM components in their respective cell types, EC extensions are reduced concomitantly with a decrease in Egfr signaling in these cells. Reintroducing either activated Egfr in the ECs, or overexpressing its ligand Spitz (Spi) from the germline, rescued the requirement for IRM proteins in both cell types. These experiments introduce novel essential components, the IRM proteins, into the process of inductive interactions between GCs and ECs, and imply that IRM-mediated activity is required upstream of the Egfr signaling.
Valer, F. B., Spegiorim, G. C., Espreafico, E. M. and Ramos, R. G. P. (2022). The IRM cell adhesion molecules Hibris, Kin of irre and Roughest control egg morphology by modulating ovarian muscle contraction in Drosophila. J Insect Physiol 136: 104344. PubMed ID: 34896373
The Irre Cell Recognition Module (IRM) is an evolutionarily conserved group of transmembrane glycoproteins required for cell-cell recognition and adhesion in metazoan development. In Drosophila melanogaster ovaries, four members of this group - Roughest (Rst), Kin of irre (Kirre), Hibris (Hbs) and Sticks and stones (Sns) - play important roles in germ cell encapsulation and muscle sheath organization during early pupal stages, as well as in the progression to late oogenesis in the adult. Females carrying some of the mutant rst alleles are viable but sterile, and previous work has identified defects in the organization of the peritoneal and epithelial muscle sheaths of these mutants that could underlie their sterile phenotype. In this study, besides further characterizing the sterility phenotype associated with rst mutants, the role of the IRM molecules Rst, Kirre and Hbs was investigated in maintaining the functionality of the ovarian muscle sheaths. Knocking down any of the three genes in these structures, either individually or in double heterozygous combinations, not only decreases contraction frequency but also irregularly increases contraction amplitude. Furthermore, these alterations can significantly impact the morphology of eggs laid by IRM-depleted females demonstrating a hitherto unknown role of IRM molecules in egg morphogenesis.

The roughest mutation was isolate in 1932 as an X-ray induced mutation that produces reduced viability, rough and bulging eyes and irregularly shaped facets in terms of both their size and arrangement (FlyBase). The irregular chiasm C (irreC) mutants, (irreCUB883 and In(1)irreC1R34) are, respectively, a P-element induced mutation and an x-ray-generated inversion, both isolated in the Fischbach laboratory in a study of disorders in the optic chiasms. These optic lobe abnormalities can be grouped into two classes, depending on whether they affect the outer or the inner chiasm. Lamina, medulla and lobula are, respectively, the outer, intermediate, and inner optic ganglia of the brain's optic lobe. In class I defects (outer chiasm), axonal bundles originating in the posterior lamina are misrouted on their way toward the anterior medulla. This defect is correlated with the misplacement of the optic lobe pioneer neurons that apparently establish the outer chiasm. The misrouted bundles form neuronal terminals in their retinotopic target area. In class II defects (inner chiasm), fiber tracts connecting the medulla to the lobula plate frequently cross the lobula neuropil, instead of running via the inner chiasm. In extreme cases, this may result in an apparent fusion of lobula and lobula plate. The two classes of phenotypic abnormalities are not epigenetically coupled; their penetrance and expressivity are variable and dependent on the particular allele being studied. Recombination and deficiency mapping place roughest and irreC alleles, affecting the eye and optic lobe respectively, at the same genetic location suggesting that they are closely associated genetic functions that define two functional aspects of the same genetic unit. Rst has been show to mediate homophilic cell adhesion in vitro (Schneider, 1995). The rst gene product is required in at least three independent places: in the developing retina and in different cell populations involved in the formation of the first and the second optic chiasms (Boschert, 1990). Characterization of the gene has shown that it codes for a single transmembrane domain protein with an Ig domain and a 208 amino acid cytoplasmic tail (Ramos, 1993 and references).

Roughest is also involved in terminal stages of the development of the regular pattern of ommatidia in the eye. The final step of pattern formation in the developing retina is the elimination of excess cells between the ommatidia and the differentiation of the remaining cells into secondary and tertiary pigment cells. Temporally and spatially highly regulated expression of the Roughest protein is essential for correct sorting of cell-cell contacts in the pupal retina; without such sorting, the normally ensuing wave of apoptosis would not occur. Roughest protein accumulates strongly at the borders between primary pigment and interommatidial cells. Mutant and misexpression analyses show that this accumulation of the Roughest protein is necessary for aligning interommatidial cells in a single row. This reorganization is a prerequisite for the identification of death candidates. Roughest function in retinal development demonstrates the importance of specific cell contacts for assignment of the apoptotic fate (Reiter, 1996).

Ommatidial formation is characterized by an accretive mode of growth. Since the last cells to develop have common borders with several ommatidia, such an accretive process cannot be continued to complete retinal development. Corrective measures are necessary to integrate the individually formed ommatidial elements into a regular lattice. Such measures are provided by the initial overproduction of cells and the wave of cell death that eliminates surplus interommatidial cells. To produce a regular lattice, generalized cell death is not sufficient; individual cells that do not fit into the pattern must be identified and specifically eliminated. This is made possible by a reorganization of cell contacts, constraining interommatidial cells into chains lying end to end. In a following step, surplus cells are eliminated. These steps are disturbed in roughest and echinus mutants (Wolff, 1991). rst disrupts the initial step of contact reorganization so that surplus cells lie side by side. ec allows the reorganization but cell death does not ensue. Thus, interommatidial apoptosis relies on at least two distinct, sequentially acting genetic functions prior to activation of the general apoptosis machinery in an individual cell. Both Rst and echinus are not general cell death genes but are specifically required for weeding out unnecessary interommatidials. The second aspect of retinal development mediated by apoptosis -- elimination of perimeter clusters -- occurs normally in the absence of both genes (Wolff and Ready, 1991). Suppression of the apoptosis machinery by retinal expression of the baculovirus P35 protein or of Drosophila IAP1/IAP2, does not interfere with cell sorting and causes an interommatidial phenotype similar to ec, but additionally rescues perimeter clusters (Reiter, 1996 and references).

Roughest expression in the retina is dynamically regulated throughout development. The protein is localized apically and initially outlines all cell profiles in the morphogenetic furrow (mf). The first structures emerging from the furrow are rosettes that develop to arcs and then form five-cell preclusters (R8, R2, R5, R3, R4) initially including 'mystery cells', which are later expelled at the anterior end. Rst is strongly concentrated at the posterior borders of arcs adjacent to surrounding unpatterned cells. However, cytoplasmic staining is strongest between the arcs and preclusters; the interiors of clusters that emerge from the furrow are not stained. Thus, Rst protein produced in undifferentiated cells selectively accumulates at the border abutting differentiating cells. Within the 5-cell precluster, staining is present where membrane contacts between R8, R2 and R5 cells occur. The future R1, R6 and R7 cells that are recruited from the posterior of the cluster show strongest expression at this stage. No immunoreactivity is detectable in the anterior part of the cluster, containing R3 and R4. Cone cells form in the sixth to seventh row behind the furrow; they also express Rst, while membrane contacts among R1, R6 and R7 lose immunoreactivity. R2, R8 and R5 retain expression and show specific accumulation of the protein at different membrane contacts. Twelve percent of the way through pupal development (p12%), all membrane contacts among unpatterned cells between ommatidia stain equally strongly. Membranes of developing primary pigment cells (1o p.c.) show a slightly higher level of expression. After the pair of 1o p.c. has surrounded the cone cells, the ommatidial lattice compacts. The protein now accumulates at the borders between 1o p.c. and interommatidial cells (IOC). This border, previously straight, develops an involuted contour. The interommatidial cells -- distributed randomly at first -- reorganize into chains lying end to end between the ommatidia from approx. p16% to p21%. Preferential accumulation of the protein at the IOC/1o p.c. border is retained throughout this stage. At approx. p23%, during IOC apoptosis, the pattern of Rst expression shows a striking change: it disappears from all borders among IOC, and is only detectable at the borders of these cells with the 1o p.c. In the primaries it retreats from the border with cone cells but initially remains present at the two contact sites between the 1o p.cs (visible until p33% as two opposed bars in each ommatidium). After elimination of surplus cells, bristle cells are recognizable by their weaker staining. Even after the downregulation of Rst in 1o p.c., borders among IOC remain almost devoid of staining (p35%), indicating the presence of a factor that forces accumulation of Rst at the border with 1o p.c.: in situ hybridizations at this stage show strong interommatidial RST mRNA expression and no detectable levels of mRNA in the primaries. Thus, there is no protein made in 1o p.c. that can contribute to the strong staining seen at their outer borders. Rst expression is retained until about 60% of the way through the pupal stage and shows a specific pattern of accumulation around the bristle cell complex in late stages (Reiter, 1996).

Analysis of mutant phenotypes demonstrates the necessity for intact Rst protein for apoptosis of interommatidial cells (Ready, 1991). Study of the expression pattern reveals a specific accumulation of the protein at the border between primary pigment and interommatidial cells, a contact zone where cellular geometry is rearranged immediately before apoptotsis. Further functional analysis of Rst has been attempted by specifically altering the expression pattern, using the Gal4/UAS system. If Rst acts strictly as a receptor for a cell death inducing signal, then extending its expression to additional cell types could yield two possible results. Either additional cell death could occur, if the targeted cells receive the proper signal, and if the apoptotic program is not otherwise inhibited in them, or, no additional cell death would be expected, if the targeted cells do not have contact with the signal, or the apoptotic program is inhibited by the differentiational state of the cells (Reiter, 1996).

Transformants expressing Gal4 under control of the sevenless enhancer were used to target misexpression of Rst to the retina in UAS-HB3 transformants. The sevenless enhancer is active in a subset of photoreceptors and the cone cells. Activity in cone cells persists into the pupal stage. Animals homozygous either for sev-Gal4 or UAS-HB3 have wild-type retinae. When Rst is misexpressed in sev-Gal4/UAS-HB3 transformants, a strong rough eye phenotype results. The general regularity of the ommatidial lattice is severely disturbed. Neighboring ommatidia occasionally fuse, forming a single lens. Semithin sectioning reveals that the number of photoreceptor neurons is normal; the distribution of mechanosensory bristles is randomized, but their number is not significantly influenced. Therefore, the disruption of the ordered lattice in misexpressing retinae does not reflect an effect on neural differentiation. The outside appearance of sev-Gal4/UAS-HB3 is similar to that of the null mutant for the Rst gene. Distribution of Rst immunoreactivity in interommatidial cells, which are not targeted by the misexpression, is strongly altered in sev-Gal4/UAS-HB3. While in the wild type, the border between 1o p.c. and IOC is highlighted by strong immunoreactivity there is no longer preferential accumulation of the protein at this border in sev-Gal4/UAS-HB3. Instead, the apical membrane domains of IOC and 1o p.c. show equal intensity of staining. The cone cells -- the only apical cell type targeted by misexpression in sev-Gal4/UAS-HB3 -- show the expected strong immunoreactivity. In sev-Gal4/UAS-HB3, IOC/1o p.c. borders are not involuted, spacing of ommatidia is irregular and no sorting of IOC into chains between primaries is apparent. This suggests that the accumulation of Rst protein at the border of primary pigment and interommatidial cells is responsible for the proper sorting of cell contacts, and that this accumulation can be influenced by the presence of Rst in cone cells. No effects of Rst misexpression on the differentiation of primary pigment and cone cells were apparent (Reiter, 1996).

The sev-Gal4/UAS-HB3 phenotype is not due to an induction of cell death in populations misexpressing the Rst protein, but arises as a result of cell death inhibition in interommatidial cells. Acridine orange staining of sev-Gal4/UAS-HB3 pupal retinae reveals few or no apoptotic fragments between the ommatidia, and many surplus IOC are visible in later pupal stages. mAb 24A5.1 staining of retinae at p42%, when sev-Gal4- driven expression is no longer present but IOC still express the normal Rst gene, shows that several 2o p.c. lie between ommatidia and are aligned side by side as in rst. Thus, the primary effect of misexpression is on the sorting of cells. Preferential accumulation of Rst protein at the IOC/1o p.c. border appears to be required for this sorting process (Reiter, 1996).

The sev enhancer is able to target expression to photoreceptors as well as to cone cells, beginning in the third instar larva. Which of these cell types causes the suppression of apoptosis by Rst misexpression? As a control, an elav-Gal4 construct was used to drive Rst expression. The elav promoter is active in all neurons, e.g. in photoreceptors, but not in cone cells. No retinal defects result; cell sorting proceeds normally, and the subcellular localization of the Rst protein in IOC is normal. Misexpressed Rst protein in photoreceptors is transported almost exclusively to the precursor structures of the rhabdomeres, which correspond to the apical domain of photoreceptors. This control experiment shows that Rst misexpression during retinal development does not generally influence the differentiation process of targeted cells or their neighbours. Therefore, expression in cone cells must be responsible for the sev-Gal4/UAS-HB3 phenotype. The intriguing conclusion is that misexpression of Rst in cone cells can exert a cell-death suppressing effect on interommatidial cells across the primary pigment cells, by way of disturbing the rearrangement of cellular geometry prior to apoptosis (Reiter, 1996).

How does the process of cell sorting in the pupal retina proceed to shift a side by side arrangement of IOC from one contact to 1o p.c. to an end-to-end arrangement with two or three contacts? A model is proposed in which undifferentiated cells explore cell contacts and tend to sustain any contact made to a primary pigment cell. Contacts to other interommatidial cells should not be especially attractive. It is obvious from morphological studies that unpatterned cells do not retain the same contacts throughout all of development. Given that different cell contacts are explored and that 1o p.c. provide an attractive border, cells would rearrange to achieve a greater number of contacts to primary pigment cells. It is proposed that this attractive border is provided by adhesional interactions between IOC and 1op.c, which are mediated by Rst. An additional mechanism that can resolve side by side doublings of IOC is the extension of the IOC/1o p.c. contact zone into interommatidial spaces. The ommatidial fusions found in sev-Gal4/UAS-HB3 and rst mutants are also explainable by loss of an adhesional interaction at the IOC/1o p.c. border. In the absence of an attractive border, IOC would not be prevented from giving up contacts to 1o p.c (Reiter, 1996).

The Rst protein mediates homophilic adhesion in transfected S2 cells and selectively accumulates at the membrane contacts of expressing cells (Schneider, 1995). A model based on this mode of binding, however, fails to explain the protein distribution seen in the retina. The arcs formed around assembling ommatidial clusters, for instance, are formed by several cells producing Rst. The protein accumulates only at their borders with the photoreceptor cluster, not at the borders among themselves, while initially the photoreceptor cluster shows no immunoreactivity. Similarly, homophilic adhesion cannot explain the dynamics of protein localization during pupal development. At a stage when interommatidial cells strongly express RST mRNA (Ramos, 1993), the protein does not accumulate at their common border; rather, it accumulates at the border with primary pigment cells. This accumulation persists beyond the downregulation of Rst in 1o p.c., suggesting that a heterophilic interaction is the underlying mechanism. Expression of a putative Rst ligand would be expected on the surface of 1o p.c (Reiter, 1996).

It is concluded that wild-type expression of Rst is essential for the lining up of interommatidial cells in single cell rows prior to the induction of apoptosis. This level of cellular order appears to be a prerequisite for correct assignment of the apoptotic fate to individual cells (Reiter, 1996).

The phosphoinositide phosphatase Sac1 regulates cell shape and microtubule stability in the developing Drosophila eye

Epithelial patterning in the developing Drosophila melanogaster eye requires the Neph1 homolog Roughest (Rst), an immunoglobulin family cell surface adhesion molecule expressed in interommatidial cells (IOCs). This study using a novel temperature-sensitive (ts) allele, showed that the phosphoinositide phosphatase Sac1 is also required for IOC patterning. Sac1ts mutants have rough eyes and retinal patterning defects that resemble rst mutants. Sac1ts retinas exhibit elevated levels of phosphatidylinositol 4-phosphate (PI4P), consistent with the role of Sac1 as a PI4P phosphatase. Indeed, genetic rescue and interaction experiments reveal that restriction of PI4P levels by Sac1 is crucial for normal eye development. Rst is delivered to the cell surface in Sac1ts mutants. However, Sac1ts mutant IOCs exhibit severe defects in microtubule organization, associated with accumulation of Rst and the exocyst subunit Sec8 in enlarged intracellular vesicles upon cold fixation ex vivo. Together, these data reveal a novel requirement for Sac1 in promoting microtubule stability and suggest that Rst trafficking occurs in a microtubule- and exocyst-dependent manner (Del Bel, 2018).

Undifferentiated epithelial cells are patterned and specified during development to yield highly ordered tissues composed of multiple cell types. Patterning and differentiation within an epithelium are driven in part by cell surface adhesion molecules that promote intercellular interactions. Defects in cell-cell adhesion lead to developmental abnormalities and contribute to disease progression. For example, the mammalian Irre cell recognition module (IRM) adhesion proteins NEPH1 (KIRREL) and nephrin (NPHS1) are required in the kidney for development and maintenance of the filtration barrier or slit diaphragm. Despite their importance in animal development and physiology, little is known about how trafficking and delivery of cell surface adhesion molecules such as IRM proteins is achieved (Del Bel, 2018).

The adult Drosophila eye contains ~750 individual units called ommatidia. Prior to pupariation, each ommatidium consists of eight photoreceptor (PR) cells and four cone cells, surrounded by undifferentiated interommatidial cells (IOCs). During the first half of pupal eye development, 0-42 h after puparium formation (APF), IOCs undergo dynamic morphogenetic changes to give rise to two primary pigment cells (1°pc), six secondary pigment cells (2°pc), three tertiary pigment cells (3°pc) and three bristles, arranged in a honeycomb lattice. These cells support and optically isolate individual ommatidia (Del Bel, 2018).

Specification and organization of Drosophila retinal cells requires IRM protein function. 1°pc express the nephrin homologs Hibris (Hbs) and Sticks and stones (Sns), whereas IOCs express the NEPH1 homologs Roughest (Rst) and Kin of Irre (Kirre, also called Dumbfounded). Between 24 and 30 h APF, all four proteins localize to the plasma membrane (PM) of these cells at the 1°pc:IOC border, and heterophilic binding of Rst and Kirre with Hbs and Sns is needed for specification and morphogenesis of 2°pc and 3°pc (2°/3°pc). Mutations that affect cell surface accumulation of Rst result in rough eyes and reduced levels of pigmentation as a result of defects in 2°/3°pc differentiation (Del Bel, 2018).

Phosphoinositides, or phosphatidylinositol (PI) phosphates (PIPs), regulate essential cellular processes such as membrane trafficking and actin cytoskeletal organization. In the canonical PIP pathway, PI is phosphorylated by PI 4-kinases (PI4Ks) to generate PI 4-phosphate (PI4P), which is a precursor for other PIPs, including PI 4,5-bisphosphate [PI(4,5)P2], and serves as a potent signaling molecule, for example by recruiting effectors to the Golgi body to promote membrane trafficking. Downregulation of PI4P in specific membrane compartments also drives cellular events. For example, in budding yeast (Saccharomyces cerevisiae), PI4P dephosphorylation is required for targeted delivery of cargo to the PM by the exocyst complex. Drosophila has three PI4Ks, including a single type II enzyme (PI4KII) that generates PI4P at the trans-Golgi network (TGN) and on endosomes. PI4P levels are kept in check by the phosphoinositide phosphatase Sac1, a conserved transmembrane protein present in endoplasmic reticulum and Golgi membranes (Del Bel, 2018).

Previous work has shown that Sac1 is required for normal eye development in Drosophila (Wei, 2003b). Sac1 is essential for viability in Drosophila (Wei, 2003a,b). Flies transheterozygous for a hypomorphic allele and a null allele exhibit rough eyes with necrotic patches and drown in the food soon after eclosing (Wei, 2003b). Using the hypomorphic allele, which is shown to be temperature sensitive (ts), it was demonstrated that Sac1 plays a crucial role in patterning the retinal epithelium. IOCs of Sac1ts flies cultured at 25°C exhibit a dramatic increase in PI4P levels and decreased levels of PM PI(4,5)P2. Although Rst is present at the cell surface of these IOCs, fixation on ice ex vivo results in re-distribution of Rst to enlarged structures containing the exocyst complex subunit Sec8, suggesting a microtubule defect. Indeed, Sac1ts IOCs contain sparse, disorganized microtubules (MTs) that disappear when fixed on ice. These results thus identify a novel link between Sac1, PIP levels and microtubule stability in the developing Drosophila eye. Because Sac1 is conserved, these findings suggest a possible role for MT regulation by Sac1 in human development and disease (Del Bel, 2018).

Conditional alleles are powerful tools for studying the functions of essential genes. Using a temperature-sensitive allele of Sac1, this study shows that proper phospholipid regulation by Sac1 is required for normal development of the Drosophila retinal epithelium (Del Bel, 2018).

The results indicate that retinal patterning is highly sensitive to elevated levels of PI4P. Loss of Sac1 leads to increased Golgi PI4P and IOC patterning defects that are rescued by expression of catalytically active Sac1. Deletion of PI4KII suppresses Sac1ts, whereas expression of a WT PI4KII transgene exacerbates the observed defects. Thus, Sac1 and PI4KII regulate a pool of PI4P that is required for retinal patterning. Loss of PI4KII itself results in mild patterning defects (i.e. occasional extra bristles and fused ommatidia), indicating that reducing PI4P levels also has consequences for retinal development. Although these data point to a clear role for PI4KII in signaling required for 2°/3°pc adhesion and patterning, the experiments do not rule out possible contributions of other PI4Ks to this process. Moreover, it is anticipated that, in addition to the Golgi, other pools of PI4P at the PM and elsewhere may be regulated by Sac1 (Del Bel, 2018).

Because PI4P is phosphorylated by PIP 5-kinases to produce PI(4,5)P2, Sac1ts mutants might be expected to show increased levels of PI(4,5)P2 as well as PI4P. Strikingly, however, this study found that Sac1ts mutants exhibit reduced levels of the PI(4,5)P2 marker PLCΔ-PH at the PM. Although this result is counterintuitive, similar observations have been reported in budding yeast, and the same same phenomenon in Drosophila is observed embryos homozygous for a lethal allele of Sac1. This observation is in contrast to other Drosophila tissues, where Sac1 loss was reported to have no effect on PI(4,5)P2 levels (Forrest, 2013; Yavari, 2010). It is possible that PIP 5-kinases lack access to the increased PI4P in Sac1 mutants, thereby preventing the expected increase in PI(4,5)P2, or perhaps increased PI4P triggers a feedback mechanism that activates PIP phosphatases or phospholipases, leading to PI(4,5)P2 breakdown. Thus, in addition to elevated PI4P levels, reduced levels of PI(4,5)P2 might contribute to the IOC patterning defects in Sac1ts. Indeed, in preliminary results a genetic interaction was observed between Sac1 and the PI4P 5-kinase skittles, which could stem from reduced PI(4,5)P2 or increased PI4P levels, or a combination of the two (Del Bel, 2018).

Drosophila Sac1ts mutants exhibit rough eyes, ommatidial mispatterning and abnormal 2°/3°pc morphology, phenotypes that resemble mutants for the IRM protein Rst. Maintenance of Rst at the 1°pc:IOC border is sensitive to ice fixation in Sac1ts mutant retinas. Because MTs are labile under cold conditions, this suggested a MT defect in Sac1ts. MTs are disorganized in Sac1ts mutant IOCs fixed at RT, and even more dramatically affected in mutant IOCs fixed on ice. Moreover, Sac1ts IOCs fixed on ice display enlarged Sec8-positive vesicles containing Rst, suggesting that Rst travels to the cell surface via an exocyst- and MT-associated pathway that is sensitive to cold fixation in Sac1ts (Del Bel, 2018).

The simplest model to explain these data is that Rst is constantly turned over at the PM and requires continuous replenishment from internal stores, as has been reported for mammalian IRM proteins. This turnover appears to occur in both WT and Sac1ts, as this study observed similar uptake of anti-Rst antibody in each. It remains possible that subtle defects in the activity or turnover of Rst (or other proteins) at the PM contribute to the observed defects in Sac1ts. Indeed, compromised expression of the differentiation marker BA12-lacZ was observed in Sac1ts IOCs. Defects in BA12-lacZ expression, as well as IOC number and specification, also occur in RstD mutants, which show a delay in Rst accumulation at the 1°pc:IOC border. Relevant to this point, it is noted that when quantification of Rst distribution after RT or cold fixation was independently validated at a later date, a slight but significant reduction was observed in Rst accumulation in Sac1ts following RT fixation. However, as this difference was not observed in earlier experiments, it is not possible to conclude that there is a reproducibly significant decrease in Rst accumulation in Sac1ts (Del Bel, 2018).

Sac1 was originally identified as a suppressor of yeast actin mutants. Thus, much of the literature has focused on how Sac1 loss affects the actin cytoskeleton. This observed that retinal cells in Drosophila Sac1ts mutants exhibit normal actin organization during early stages of pupal eye development (24-30 h APF), yet show defects in MT stability and organization. Interestingly, both increased levels of PI4P and reduced levels of PI(4,5)P2 have previously been associated with defects in MT organization. The molecular mechanisms involved remain unknown, and further investigation will be required to determine the link between Sac1 and MT stability. However, the current observations suggest that regulation of the MT cytoskeleton by Sac1 may be more important when moving from fungal to animal models (Del Bel, 2018).

In summary, this study has characterized a requirement for Sac1 in maintaining PI4P and PI(4,5)P2 levels and promoting normal development of the Drosophila retina. Additionally, it was demonstrated that Rst distribution in Sac1ts is sensitive to cold fixation, and that Sac1ts exhibits a microtubule defect at 24 h APF. The results indicate that Rst is delivered to the PM via exocyst- and microtubule-based trafficking (which is cold sensitive in Sac1ts) and is turned over at the PM, similar to its mammalian homologs. Further investigation will be required to unravel precisely how Sac1 regulates microtubules, and how this contributes to normal retinal development (Del Bel, 2018).


Sns, a member of the immunoglobulin superfamily that is essential for myoblast fusion and mediates heterotypic adhesion with Duf/Kirre and IrreC-rst-expressing cells

The body wall muscles in the Drosophila larva arise from interactions between Dumbfounded/Kirre and Irregular chiasm C-roughest (IrreC-rst)-expressing founder myoblasts and Sticks and stones (Sns)-expressing fusion competent myoblasts in the embryo. Sns, a member of the immunoglobulin superfamily that is essential for myoblast fusion (Bour, 2000), mediates heterotypic adhesion of S2 cells with Duf/Kirre and IrreC-rst-expressing S2 cells, and colocalizes with these proteins at points of cell contact. These properties are independent of their transmembrane and cytoplasmic domains, and are observed quite readily with GPI-anchored forms of the ectodomains. Heterotypic interactions between Duf/Kirre and Sns-expressing S2 cells occur more rapidly and to a greater extent than homotypic interactions with other Duf/Kirre-expressing cells. In addition, Duf/Kirre and Sns are present in an immunoprecipitable complex from S2 cells. In the embryo, Duf/Kirre and Sns are present at points of contact between founder and fusion competent cells. Moreover, Sns clustering on the cell surface is dependent on Duf/Kirre and/or IrreC-rst. Finally, although the cytoplasmic and transmembrane domains of Sns are expendable for interactions in culture, they are essential for fusion of embryonic myoblasts (Galletta, 2004).

The ability of Sns, Duf/Kirre and IrreC-rst to mediate cell–cell adhesion was examined using Drosophila S2 cells, which are predominantly non-adherent under normal conditions. As a prelude to examining the behavior of these molecules in combination, each was examined individually to evaluate their ability to direct homotypic aggregation. S2 cells were transiently transfected with Duf/Kirre, IrreC-rst, or Sns under the control of the copper inducible metallothionein promoter and allowed to aggregate. Following aggregation, the cells were fixed and examined by indirect immunofluorescence using anti-sera directed against specific domains or tags within each protein. As anticipated from previous studies (Dworak, 2001), Duf/Kirre-expressing S2 cells were frequently found in aggregates. Duf/Kirre protein accumulates at points of cell-cell contact in aggregates but is uniformly distributed on the surface of non-aggregated S2 cells. Similar to the behavior of Duf/Kirre, IrreC-rst mediates homotypic aggregation of S2 cells, and becomes enriched at points of cell-cell contact in the resulting cell clusters. Duf/Kirre and IrreC-rst enrichment is occasionally observed in regions where cell-cell contact is not apparent, possibly as a consequence of processes, visible by transmission electron microscopy, that extend around neighboring cells. In contrast to the behavior of Duf/Kirre or IrreC-rst, expression of Sns protein on the surface of S2 cells does not lead to homotypic cell adhesion (Dworak, 2001). A lower magnification view emphasizes the presence of many unassociated Sns-expressing cells. As anticipated, Sns is distributed uniformly on the surface in the absence of aggregation (Galletta, 2004).

To ensure that the Duf/Kirre and IrreC-rst clusters were the consequence of aggregation rather than cell division, the number of cells in aggregates of three cells or more were counted. Since cells should only divide at most once during the course of the experiment, clusters of three cells must represent those formed from adhesive events. In a survey of 4171 Duf/Kirre-expressing cells, 40% (1697) were found in aggregates of three or more. In a survey of 1002 IrreC-rst-expressing cells, 19% (192) were in aggregates of three or more cells. These results suggest that while Duf/Kirre may be more efficient in mediating homotypic aggregation than IrreC-rst, clearly both are capable of mediating such interactions. By contrast, a survey of 3275 Sns-expressing cells revealed only 26 cells in aggregates of three or more cells (Galletta, 2004).

While Sns-expressing cells do not aggregate homophilically, studies have indicated that these cells aggregate with cells expressing Duf/Kirre (Dworak, 2001). It was of interest to determine whether cells expressing Sns would interact with cells expressing IrreC-rst, and whether Sns and Duf/Kirre or IrreC-rst co-localize at points of cell-cell contact. To this end, S2 cells were independently, transiently transfected and the ability of Duf/Kirre and IrreC-rst-expressing cells to form aggregates and direct membrane co-localization of Sns in these aggregates was examined. All of these proteins were uniformly distributed on the cell surface in unaggregated cells. In contrast to their behavior in isolation, Sns-expressing cells readily associated in large clusters when combined with cells expressing Duf/Kirre. The Sns-expressing cells also associated with cells expressing IrreC-rst, with a similar efficiency. At least one of these IgSF members must be expressed on the cell surface for it to cluster, since no untransfected cells were observed in an analysis of 1109 small clusters of either Duf/Kirre:Sns or IrreC-rst:Sns-expressing cells. While the biological significance of such an interaction remains unclear, Duf/Kirre and IrreC-rst-expressing cells are capable of forming heterotypic aggregates with each other when expressed in S2 cells under similar conditions (Galletta, 2004).

Examination of individual proteins in small aggregates revealed clustering of Sns with either Duf/Kirre or IrreC-rst at points of cell contact. Thus, either Duf/Kirre or IrreC-rst can direct cells to associate with Sns-expressing cells, and co-localize with Sns at points of cell contact. Frequently much of the Sns protein in the cell accumulates at the points of cell-cell contact, leaving little if any protein on the rest of the cell surface. In rare cases, both proteins are observed in regions outside of obvious cell contacts. However, this pattern may reflect cell membranes that are extending around neighboring cells, mentioned earlier. Since Duf/Kirre and IrreC-rst serve redundant functions in the founder myoblasts, and behave similarly in the assays described above, subsequent experiments focused on Duf/Kirre (Galletta, 2004).

In some cases, the cytoplasmic domains of cell adhesion molecules play no role in their ability to direct cell interactions, while this domain can be critical in other cases. It was therefore of interest to determine whether these regions of Sns or Duf/Kirre were required for the S2 cell interactions. For these studies, the extracellular domains of Duf/Kirre and Sns were fused in frame to the GPI-anchor sequence of Fasciclin I. These constructs were separately, transiently transfected into S2 cells, and aggregation was examined. In the case of Duf/Kirre-GPI, the efficiency of homotypic aggregation was severely reduced compared to that of cells expressing full length Duf/Kirre. Since the relevance of Duf/Kirre homotypic aggregates in vivo is unclear, the role of the Duf/Kirre and Sns cytoplasmic and transmembrane domains in heterotypic aggregation was also examined. In an analysis similar to that done for Duf/Kirre homotypic aggregates, the ability of cells expressing the GPI-anchored or full length forms of Duf/Kirre to mediate heterotypic adhesion with cells expressing full-length or GPI-anchored forms of Sns was examined in pairwise comparisons. The influence of the Sns cytoplasmic and transmembrane domains was examined on adhesion with cells expressing full length Duf/Kirre. Within the limits of statistical significance, GPI-anchored Sns mediate aggregation at a level comparable to that of full length Sns. A similar analysis was carried out to examine the influence of the Sns cytoplasmic and transmembrane regions on adhesion with cells expressing Duf/Kirre-GPI. Again, Sns-GPI mediates adhesion with the Duf/Kirre-GPI-expressing cells at a level comparable to that of full length Sns. Thus, the Sns cytodomain and membrane spanning region appear to play no role in its ability to direct aggregation with Duf/Kirre-expressing cells (Galletta, 2004).

Since the Duf/Kirre-expressing cells were in excess in the above experiments, these data could not be used to determine the relative contribution of the Duf/Kirre cytodomain and transmembrane region. Therefore additional assays were carried out in which Sns or Sns-GPI-expressing cells were in a five-fold excess over either Duf/Kirre or Duf/Kirre-GPI-expressing cells to determine whether there was a requirement for the Duf/Kirre cytodomain or membrane spanning region in interactions with cells expressing Sns. These experiments were also set up as pairwise comparisons, and demonstrated that Duf/Kirre-GPI mediate aggregation at a level comparable to that of full length Duf/Kirre. These data suggest that there is no significant difference between the ability of Duf/Kirre or Duf/Kirre-GPI to aggregate with cells expressing full-length Sns. Lastly, the cytoplasmic and transmembrane domain of Duf/Kirre have a modest affect on its ability to direct aggregation with GPI-anchored Sns. However, the effect of the cytoplasmic or transmembrane domain on Duf/Kirre's ability to mediate heterotypic aggregation with Sns-GPI was not as great as its effect on the ability of Duf/Kirre to mediate homotypic cell adhesion. Of note, Duf/Kirre-GPI was enriched at points of cell-cell contact in both homotypic aggregates and in heterotypic aggregates with Sns and Sns-GPI. Thus, neither the cytoplasmic nor transmembrane domains of Sns or Duf/Kirre are essential for recruitment to cell-cell contacts (Galletta, 2004).

In summary, these results indicate that the cytoplasmic/transmembrane domains of Sns and Duf/Kirre do not influence the efficacy with which they direct heterotypic cell-cell adhesion. This observation is in contrast to that seen for Duf/Kirre, in which the cytodomain or membrane spanning region of Duf/Kirre plays a critical role in its ability to direct homotypic aggregation. One possible explanation for these results is that heterotypic association of Sns and Duf/Kirre is stronger, and does not require stabilization of the receptor through cytoplasmic or intramembrane interactions. Since the affinity of Duf/Kirre for homotypic versus heterotypic interactions could play a critical role in myoblast interactions in the embryo, the S2 cell aggregation assay was used to examine this preference (Galletta, 2004).

In the embryonic musculature, founder cells appear to fuse only with fusion competent myoblasts, and never fuse with each other. In principle, this directional fusion could be attributed to the differential expression of Duf/Kirre and Sns by these two cell types, and inability of these molecules to associate homotypically. However, results reported in this study and by Dworak (2001) demonstrate that Duf/Kirre-expressing cells do associate with each other in culture. It was therefore of interest to determine whether the affinity of Duf/Kirre-expressing cells for cells expressing Sns was greater than the affinity of Duf/Kirre-expressing cells for each other. To address this question, Duf/Kirre-expressing cells were aggregated in isolation or in the presence of an equal number of Sns-expressing cells. This analysis utilized stable cell lines in which approximately 30% of the corresponding population expressed Duf/Kirre and approximately 8% expressed Sns. Aggregation of Duf/Kirre-expressing cells was examined in three different conditions, all with the same total cell number. The goal was to ensure that any change in aggregation of Duf/Kirre cells was due to the specific addition of Sns-expressing cells rather than a consequence of doubling the number of adherent cells. For each time point, the number of Duf/Kirre-expressing cells free in solution was counted and the number that had been incorporated into aggregates. Duf/Kirre-expressing cells were incorporated into aggregates that included Sns-expressing cells at a faster rate and to a greater extent than those containing only Duf/Kirre-expressing cells. This behavior was not a simple consequence of the number of adherent cells present, since a two-fold increase in the number of Duf/Kirre-expressing cells did not have a dramatic effect on the rate or extent of aggregation. Thus, Duf/Kirre-expressing cells associate more readily into heterotypic aggregates with Sns-expressing cells than into homotypic aggregates with only Duf/Kirre-expressing cells (Galletta, 2004).

The striking colocalization of Duf/Kirre and Sns described earlier suggested the possibility that these proteins might physically associate in trans. To address this possibility, aggregates of stably transfected, Sns and Duf/Kirre-expressing cells were subjected to reversible protein cross-linking and lysed. HA-tagged Duf/Kirre was immunoprecipitated from the cell lysate using anti-HA resin, and the resulting immunoprecipitate examined by Western blot for the presence of Sns. HA-tagged Duf/Kirre was efficiently precipitated from both Duf/Kirre-only and Duf/Kirre-Sns mixed cell populations. As expected, Sns was not present in the anti-HA immunoprecipitate from cells expressing only Duf/Kirre or only Sns. However, it was clearly detected in immunoprecipitates from the mixed population of cells expressing Duf/Kirre-HA and Sns. Thus, Duf/Kirre and Sns are closely associated in an immunoprecipitable protein complex, possibly through a direct protein interaction (Galletta, 2004).

In the embryonic musculature, Duf/Kirre, IrreC-rst and Sns are necessary, either directly or indirectly, for the association of founder and fusion competent myoblasts. The striking co-localization of Sns with either Duf/Kirre or IrreC-rst in S2 cells prompted an examination of whether similar co-localization could be observed between embryonic myoblasts. First it was examined whether punctate clustering of Sns on the surface of embryonic myoblasts, previously described by Bour (2000), was dependent on the presence of Duf/Kirre or IrreC-rst. The distribution of Sns protein was examined in embryos deficient for both Duf/Kirre and IrreC-rst, and compared to that seen in wild-type embryos. As anticipated, Sns becomes localized to discrete sites in wild-type myoblasts (Bour, 2000). In contrast, Sns is distributed more uniformly on the myoblast surface in embryos lacking Duf/Kirre and IrreC-rst. Thus in embryos, as in S2 cells, the localization of Sns is dependent on the presence of Duf/Kirre or IrreC-rst (Galletta, 2004).

To determine whether Sns and Duf/Kirre co-localize in embryonic myoblasts in a manner similar to that observed in S2 cells, stage 13 embryos were examined by indirect immunofluorescence using polyclonal antisera directed against the Duf/Kirre and Sns proteins. As previously described for Sns (Bour, 2000), Duf/Kirre is expressed in a dynamic pattern that is restricted to discrete sites on the surface and in the cytoplasm of expressing cells. The pattern of Sns expression intersects that of Duf/Kirre, and is in close proximity to rP298-lacZ positive founder cell nuclei in the somatic mesoderm. Of note, punctate Sns expression is apparent at some sites in which Duf/Kirre expression is not detected. To address whether these sites might intersect points of IrreC-rst protein, which can interact with Sns-expressing cells and can substitute for Duf/Kirre in vivo, embryos were triple labeled with Duf/Kirre, IrreC-rst and Sns. IrreC-rst is readily detected at many sites of Sns enrichment that do not appear to colocalize with Duf/Kirre. In fact, examination of 204 discrete sites of Sns protein, derived from eight stage 13 embryos, revealed that 97% were colocalized with either Duf/Kirre and/or IrreC-rst (Galletta, 2004).

To determine whether sites of Duf/Kirre and Sns colocalization occur, as expected, on the cell surface, mesodermally expressed CD2 was used to visualize the cell membrane. CD2 staining revealed the surface of a growing myofiber and associated myoblasts. Duf/Kirre and Sns co-localize to points of contact between the fiber and a myoblast. Since the expression of both Duf/Kirre and Sns is dynamic and rapidly decreases upon fusion (Bour, 2000), co-localization of Duf/Kirre and Sns was examined in myoblast city (mbc) mutant embryos in which the myoblasts associate but remain unfused. By stage 14, the founder cells of these mutant embryos become morphologically distinct from the fusion competent cells, elongating and extending processes. As an apparent consequence of this fusion block, Duf/Kirre and Sns are stabilized at points of contact between the extended founder cell and several fusion competent cells. These data clearly show that Sns and Duf/Kirre co-localize in the embryo at critical contact points between founder cells and fusion competent myoblasts (Galletta, 2004).

IrreC/rst-mediated cell sorting during Drosophila pupal eye development depends on proper localisation of DE-cadherin

Remodelling of tissues depends on the coordinated regulation of multiple cellular processes, such as cell-cell communication, differential cell adhesion and programmed cell death. During pupal development, interommatidial cells (IOCs) of the Drosophila eye initially form two or three cell rows between individual ommatidia, but then rearrange into a single row of cells. The surplus cells are eliminated by programmed cell death, and the definitive hexagonal array of cells is formed, which is the basis for the regular pattern of ommatidia visible in the adult eye. This cell-sorting process depends on the presence of a continuous belt of the homophilic cell adhesion protein DE-cadherin at the apical end of the IOCs. Elimination of this adhesion belt by mutations in shotgun, which encodes DE-cadherin, or its disruption by overexpression of DE-cadherin, the intracellular domain of Crumbs, or by a dominant version of the monomeric GTPase Rho1, prevents localisation of the transmembrane protein IrreC-rst to the border between primary pigment cells and IOCs. As a consequence, the IOCs are not properly sorted and supernumerary cells survive. During the sorting process, Notch-mediated signalling in IOCs acts downstream of DE-cadherin to restrict IrreC-rst to this border. The data are discussed in relation to the roles of selective cell adhesion and cell signalling during tissue reorganisation (Grzeschik, 2005).

To summarise, IrreC-rst is colocalised with DE-cadherin in epithelial cells of pupal eye discs, and misdistribution of adherens junction components induces the mislocalisation of IrreC-rst, which then affects sorting of IOCs. However, although DE-cadherin forms a continuous belt in the apical regions of all cells (including all IOCs) in wild-type discs, IrreC-rst colocalises with DE-cadherin only at the border between 1° pigment cells and IOCs. What factor(s) might be responsible for the spatial restriction of IrreC-rst to this border? It has recently been shown that the removal of Notch or Delta function during cell-sorting results in the ubiquitous distribution of IrreC-rst to all plasma membranes and the prevention of programmed cell death. This study analysed whether this might be the result of defective DE-cadherin localisation. Antibody staining reveals no influence of Notch on the continuous apical localisation of DE-cadherin, but shows that IrreC-rst now colocalises with the latter on all plasma membranes of the IOCs. This suggests that Notch acts downstream of DE-cadherin in the control of IrreC-rst localisation. It is therefore tempting to speculate that it is the Notch pathway, which provides local signalling between the lattice cells to direct cell death, that prevents the accumulation of IrreC-rst at the borders between IOCs and thus restricts its localisation to the 1°/IOC cell boundary (Grzeschik, 2005).

Pattern formation in the Drosophila eye disc depends on a well-balanced system of signals that promote either the survival or the death of cells, mediated by the EGF and Notch receptor pathways, respectively. In addition, the morphogenetic events, which take place in a single-layered epithelium, crucially depend on factors that regulate the maintenance of cell polarity and cell shape, and modulate cell adhesion. Sorting of interommatidial cells (IOCs) during pupal development, which results in the conversion of several parallel rows of cells into a single ring, requires the weakening of pre-existing adhesive cell contacts and the establishment of new ones without interrupting the epithelial integrity of the tissue. During tissue morphogenesis, epithelial cells use different strategies to modify their adhesive contacts. One of these consists of regulating the amount and/or distribution of the homophilic cell-adhesion molecule E-cadherin, one of the central components of the adherens junctions. The first in vivo evidence for this kind of regulation came from the analysis of the Drosophila egg chamber. There, the localisation of the oocyte at the posterior pole depends on a higher level of expression of DE-cadherin in the oocyte and the posterior follicle cells, when compared with the nurse cells and other follicle cells. Differential adhesion can also be regulated by alterations in the composition or activity of intracellular binding partners, or by the integration of various other molecules into the adhesive complexes. No change in the distribution of the adherens junction components DE-cadherin and alpha-catenin could be detected in wild-type discs undergoing rearrangements of the IOCs. This behaviour contrasts with epithelial rearrangements during morphogenesis of the Drosophila tracheal system, which are associated with alterations in the amount of DE-cadherin, controlled by Drosophila Rac, another member of the Rho GTPase family. This in turn suggests that other cytoplasmic or transmembrane proteins are involved in the modulation of adhesion in IOCs. The adhesion protein IrreC-rst is involved in the control of the cell sorting process. Its predominant localisation at the border between primary pigment cells and IOCs (at the 1°/IOC border) has been suggested to provide an attractive interface that controls sorting. According to this proposal, IOCs tend to maximise their contacts with primary pigment cells. Failure to restrict IrreC-rst to this border results in the inability to sort the IOCs properly. Although IrreC-rst behaves as a homophilic adhesion molecule when expressed in cell culture, data from expression analysis argue for the presence of a different, as yet unknown, partner in the primary pigment cell (Grzeschik, 2005).

Of particular interest is the relationship between the localisation of DE-cadherin, a component of the zonula adherens (ZA) and IrreC-rst. In wild-type discs IrreC-rst colocalises with DE-cadherin at the 1°/IOC border in the apical ZA of the cell and removal of DE-cadherin completely abolishes IrreC-rst accumulation. Nothing is yet known about how IrreC-rst may integrate into the ZA at this border. In vertebrates, the Ca++-independent cell adhesion molecule nectin, a transmembrane protein of the immunoglobulin superfamily, has been implicated in the organisation of cadherin-based adherens junctions, tight junctions and synapses. It is recruited into cadherin-based adherens junctions through interactions with the PDZ domain of l-afadin, an F-actin-binding protein. Intriguingly, the C-terminal sequence of IrreC-rst (T-A-V) matches the consensus binding site for class I PDZ domains (S/T-X-V). Interestingly, the protein encoded by the mutant allele irreC-rstCT, which lacks the C-terminal 175 amino acids of the wild-type form, is no longer recruited into the ZA. It is, however, unlikely that IrreC-rst acts as a general adhesion molecule in IOCs of pupal eye discs, because the epithelial tissue structure is stable in the absence of irreC-rst function, as deduced from the formation of the continuous apical belt of DE-cadherin in irreC-rst mutants (Grzeschik, 2005).

The continuous belt of DE-cadherin can be disrupted by a number of different genetic conditions, such as overexpression of the membrane-bound intracellular domain of Crumbs, of DE-cadherin itself, or of a dominant-negative version of the monomeric GTPase Rho1. Overexpression of the membrane-bound intracellular domain of Crumbs in embryonic epithelia has been shown to lead to a redistribution of DE-cadherin throughout the plasma membrane and the formation of multilayered tissues. By contrast, IOCs overexpressing Crbintra exhibit a fragmented DE-cadherin belt, which remains localized in the apical zone of the cells, and apicobasal organisation and tissue integrity are not affected. This suggests that IOCs may contain additional adhesion components which are independent of, or less affected by, Crb. Support for this view comes from the phenotype of discs lacking crb function, in which the apical belt of DE-cadherin expression is fragmented, yet there is no major effect on polarity or adhesion of the epithelium: the cells undergo nearly normal sorting and IrreC-rst is still restricted to the membrane at the 1°/IOC border. Overexpression of CrbintraDeltaERLI (lacking four C-terminal amino acids [ERLI] of its short cytoplasmic domain, which serve to recruit a multiprotein complex that forms apical to the zonula adherens) does not interfere with sorting, suggesting that a protein complex similar to the one that controls apicobasal polarity in embryonic epithelia (which includes Stardust, DPATJ and D-Lin7) contributes to the development of the dominant phenotype (Grzeschik, 2005).

Overexpression of DE-cadherin similarly results in the fragmentation of the adhesion belt and defects in cell sorting. In various tissues, overexpression of full-length DE-cadherin can also reduce Wingless signalling by sequestering Armadillo from the cytoplasmic pool, thus making it unavailable to transduce the Wingless signal. However, the possibility that the defects in sorting are the result of a suppression of Wingless signalling can be excluded. Inactivation of components of the Wingless pathway in eye imaginal discs induces the initiation of ectopic morphogenetic furrows, and this phenotype was not observed upon overexpression of DE-cadherin. Overexpression of DE-cadherin in eye discs therefore seems to interfere with adhesion, rather than Wingless signalling (Grzeschik, 2005).

Rho GTPases play central roles in the organisation of the actin cytoskeleton and in cell adhesion. In mammals, inhibition of Rho activity results in the removal of cadherins from epithelial cell junctions, while increased Rho activity induces an invasive and metastatic phenotype. Members of the Rho GTPase family are recruited into the adherens junctions by direct interactions with junctional components. Thus, in Drosophila, Rho1 localises to the adherens junctions and interacts directly with alpha-catenin and p120ctn, a homologue of ß-catenin. As in pupal epithelia expressing a dominant-negative form of Rho1, Rho1 mutant embryos exhibit a diffuse distribution of components of the ZA, such as DE-cadherin and alpha- and ß-catenin. Rho1 may either act directly on the accumulation of cadherins at the junctions, or indirectly by recruiting accessory proteins, which then modulate the amounts or activity of junctional and/or cytoskeletal proteins. Rho1 plays a different role in tracheal epithelia insofar as its inactivation does not disrupt DE-cadherin localisation, but rather interferes with the formation of the apical surface and the tracheal lumen (Grzeschik, 2005).

Although it is evident that DE-cadherin plays a crucial role in the accumulation of IrreC-rst at the adherens junctions, other mechanisms are required to explain the asymmetric localisation and restriction of the latter to the 1°/IOC boundary. It has been speculated that an as yet unknown ligand expressed in the primary pigment cell may account for this restricted accumulation. As an alternative, but not mutually exclusive model, it is suggested that signalling between the IOCs, mediated by Notch, which is expressed in IOCs during pupal development, prevents the accumulation of IrreC-rst at their borders. Interplay between adhesion and signalling molecules also directs other processes in which cellular polarisation is involved in tissue remodelling. The growth of the wing imaginal disc along the proximodistal axis, for example, is the result of cell shape changes and cell rearrangements during pupal development, which are controlled by the atypical cadherins Fat and Dachsous, as well as Four-Jointed, which is assumed to be a secreted molecule. This process, in turn, is responsible for the asymmetric localisation of components that control planar polarity, such as Frizzled, Dishevelled or Strabismus, that serves to ensure that bristles and hairs adopt a common orientation. During germ band elongation in the Drosophila embryo, adherens junction remodelling in intercalating ectodermal cells is facilitated by the polarised expression of non-muscle myosin II at the anteroposterior and of Bazooka at the dorsoventral cell boundaries. Future experiments will demonstrate whether cell sorting in pupal eye discs makes use of any of the components known to be involved in these processes (Grzeschik, 2005).

Preferential adhesion mediated by Hibris and Roughest regulates morphogenesis and patterning in the Drosophila eye

Cell adhesion is essential for morphogenesis; however, the mechanisms by which cell adhesion coordinates precisely regulate morphogenesis are poorly understood. This study analyzes the morphogenetic processes that organize the interommatidial precursor cells (IPCs) of the Drosophila pupal eye. The Drosophila immunoglobulin superfamily members Hibris and Roughest are essential for IPC morphogenesis in the eye. The two loci are expressed in complementary cell types, and Hibris and Roughest proteins bind directly in vivo. Primary pigment cells employ Hibris to function as organizers in this process; IPCs minimize contacts with neighboring IPCs and utilize Roughest to maximize contacts with primaries. In addition, evidence is provided that interactions between Hibris and Roughest promote junction formation and that levels of Roughest in individual cells determine their capacity for competition. These results demonstrate that preferential adhesion mediated by heterophilic interacting cell-adhesion molecules can create a precise pattern by minimizing surface free energy (Bao, 2005).

To properly organize the ommatidia into a precise pattern, the interommatidial precursor cells (IPCs) undergo dynamic cell rearrangements between 18 and 42 hr after puparium formation (APF). These cells will eventually differentiate as secondary and tertiary pigment cells (2ºs, 3ºs) and mechanosensory bristles. Emergence of the interommatidial lattice was further analyzed with an antibody to the β-catenin ortholog Armadillo (Arm), a core component of the adherens junction. Based on this work, IPC and ommatidial patterning was classified into four stages (hours are based on the approximate center of the eye field), which are briefly described (Bao, 2005).

(1) Initial cell sorting (18-24 hr APF). Initially, IPCs are scattered between ommatidia with a relaxed apical profile. As development progresses, two cells emerge from the IPC pool to enwrap the cone cells and become 1ºs; the remaining IPCs simultaneously line up in single file to contact 1ºs from adjacent ommatidia. Concurrently, some cells are removed by apoptosis (Bao, 2005).

(2) Emergence of 3ºs (24-27 hr APF). Typically, three cells are initially positioned equally at a vertex. One cell reaches past the other two to contact a third 1º ; this cell will then physically “invade” the vertex and mature as a 3º (Bao, 2005).

(3) Selection of 2º s (27-36 hr APF). Cells that fail to become 3ºs either become 2ºs or are removed by programmed cell death. During this final cell-fate decision, cell-cell adhesion becomes visibly polarized as IPCs form detectable junctional contacts with 1º s but not with other IPCs. In addition, a 'scalloping' of membrane profiles is observed as 1ºs push between IPCs, further confirming that the adhesion between 1ºs and IPCs is greater than between neighboring IPCs. By 36 hr APF, the hexagonal pattern is essentially complete: it is composed of a single 2º at each side and a 3º or bristle organule at each vertex (Bao, 2005).

(4) Maturation (36-42 hr APF). Visible adherens junctions return to the interfaces between IPCs (now 2ºs and 3ºs). Contacts are now smoothed as the scalloping caused by invasive 1º contacts is now relaxed (Bao, 2005).

One particularly striking feature of this morphogenetic process is the dynamic nature of the cell junctions, which were visualized with the junctional protein Arm. For example, the level of Arm in the cone cells was constant but the levels of Arm in the IPCs decreased: this was seen by comparing the levels of Arm in the two cell groups. This drop in Arm levels is followed by its complete loss between IPCs after 3ºs emerge and eventual reemergence at the final maturation stage to levels similar to cone cells. Thus, junctions appear to be diminished during the period of maximal cell rearrangement, suggesting that IPCs are free to move during these stages (Bao, 2005).

Using laser ablation studies, it has been demonstrated that 1ºs are centrally important for the process of organizing IPCs into a correctly patterned interommatidial lattice. However, the mechanism by which one cell can provide such remarkably precise patterning information to a larger collection of uncommitted cells has not been not clear. The dynamic interactions between Hibris and Roughest provide such a mechanism (Bao, 2005).

The 'differential adhesion hypothesis' (DAH) proposes that sorting-out and segregation of cell populations are driven by differences in the intensities of cell adhesions. Given motile and cohesive cell populations, DAH predicts that weakly cohesive cells will tend to be displaced by more strongly cohesive ones; this process can direct cells to segregate away from unlike cell populations, and it can control tissue spreading during, for example, germ layer maturation in the embryo. DAH has been supported by several observations. For example, quantitative differences in the level of cadherin expression can lead two cell populations to be mutually immiscible: less cohesive cells will envelope more cohesive ones, creating a 'sphere within a sphere' configuration. Recently, the importance of differential adhesion for patterning developing tissue has been demonstrated in the pupal retina. Cone cells segregate from other cells and assemble into a simple pattern by minimizing surface area, as do soap bubbles. This assembly is mediated at least in part by E- and N-cadherins, and manipulating cadherin levels within the cone cells or their neighbors can alter the final cone cell pattern. These experiments illustrate that differential adhesion caused by differences in cadherin expression can mediate morphogenesis and pattern formation (Bao, 2005).

The current data indicate that IPC patterning follows a mechanism that shows unique aspects when compared with these classical DAH experiments. First, manipulating E-cadherin levels does not alter the morphogenesis or arrangement of IPCs. Even when two neighboring IPCs have higher levels of E-cadherin, adhesion between these two IPCs or their final patterning is not affected. More critically, IPCs do not aggregate together or segregate away from their neighbors. Rather, they separate away from each other to minimize IPC:IPC contacts, and aggregate with ommatidial cores to maximize 1º :IPC contacts. That is, the data indicate that IPCs have a preference for adherence to 1ºs. This preference can be seen most clearly at 27 hr APF: the junctions between IPCs and 1ºs are strong and elaborate; the junctions between IPCs are indistinct, and 1ºs are seen to push between IPCs to maximize contact and create a scalloping effect. The result is the precise aggregation of two different cell populations (Bao, 2005).

Why do IPCs sort away from other IPCs and preferentially adhere to 1ºs? The data indicate that interactions between Hibris and Roughest provide the mechanism. The immunoglobulin-class proteins Roughest and Hibris are utilized by IPCs and 1ºs, respectively, to form heterophilic interactions. Several lines of evidence support this view: (1) both Hibris and Roughest are required for proper interommatidial lattice assembly; (2) hibris is expressed in 1ºs as well as in cone cells and roughest is expressed in IPCs at the time of IPC rearrangement in the eye; (3) expression of ectopic Hibris in either the 1º or IPC is sufficient to relocalize Roughest protein -- conversely, downregulation of Hibris in 1ºs leads to decreased levels of Roughest protein at the 1º :IPC interface; (4) Hibris and Roughest are capable of directly binding each other when isolated in tissue culture experiments (Bao, 2005).

After 1ºs are specified and start to express Hibris, levels of Roughest protein decrease between IPCs and increase in the borders between IPCs and 1ºs; for example, at 30 hr APF, Roughest protein is undetectable between IPCs. Furthermore, ectopic Hibris in 1ºs is sufficient to attract still more Roughest protein toward the 1º :IPC border; by contrast, ectopic Roughest in 1ºs does not attract additional Roughest. It is concluded that although Roughest can show homophilic interactions in S2 cells, it strongly prefers heterophilic interactions with Hibris in situ (Bao, 2005).

Ubiquitous Hibris expression greatly increases the levels of cell-junction proteins between IPCs. Similarly, individual IPCs that received ectopic Hibris form E-cadherin-rich borders with neighboring IPCs that are sharp, straight, and significantly enlarged (Bao, 2005).

The evidence indicates that 1ºs and IPCs prefer to adhere to each other based on their expression of Hibris and Roughest, respectively. One principle of thermodynamics states that the binding of two adherent molecules will lead to a reduction of free energy within the system, provided the equilibrium constant of association (ka) is greater than the equilibrium constant of dissociation (kd). The essential role of Hibris and Roughest in IPC morphogenesis prompts making an assumption: among the various molecules being displayed in the surfaces of 1ºs and IPCs, Hibris and Roughest play a major role in determining the flow of free energy. Roughest has a higher affinity for Hibris than itself, and therefore heterophilic binding between Roughest and Hibris leads to a greater reduction in free energy. As a result, contacts between IPCs and 1ºs contribute to a reduction of free energy and are favored, while contacts between IPCs and IPCs do not contribute to reduction of free energy and are disfavored (Bao, 2005).

Other features of the developing pupal eye provide important components to this patterning process. After 1ºs are specified, they establish cell junctions with each other and with cone cells. These cone cell/1º units are not free to move within the epithelial plane and form a functional patterning unit. Therefore, 1ºs function as the organizers in this context. In contrast, IPCs have reduced levels of junctional proteins and are free to move within the epithelium. Numerous filopodia from IPCs observed by SEM studies also point to their potential for high motility. Taken together, these data suggest that IPC morphogenesis follows a preferential adhesion model: IPCs exhibit preferential adhesion to 1ºs; 1ºs function as organizers for IPC morphogenesis, and IPC:1º contacts are free energy favored while IPC:IPC contacts are disfavored (Bao, 2005).

The ommatidial clusters are poorly organized until 18 hr APF, when the morphogenetic movements of the IPCs begin to organize clusters into a hexagonal array. Preferential adhesion of IPCs to 1ºs yields two major outcomes. (1) IPCs compete to adhere directly to the limited, Hibris-rich surface presented by the 1ºs. High motility of IPCs permits this competition to proceed and achieve a favored configuration. (2) Preferential adhesion can also lead to the removal of cells that fail to contact a 1º . Specifically, IPCs that adhere to 1ºs have an increased chance to survive since the Hibris:Roughest interactions provide a greater opportunity to establish a stable junction. By the same token, those cells that do not have access to 1ºs are disadvantaged and are commonly dropped from the apical surface; these cells are likely to be eventually removed by programmed cell death. As a result, each stage proceeds with a progressive reduction of the IPC:IPC contacting surfaces and an increase in IPC:1º contacting surfaces (Bao, 2005).

At the onset of IPC morphogenesis (18 hr APF), the average size of IPC:IPC contacts is not significantly different from the size of IPC:1º contacts. During the time cells in multiple layers are sorted into single file after the initial cell-sorting stage (24 hr APF), IPC:IPC contacts are significantly reduced. After emergence of 3ºs, this reduction in IPC:IPC contacts is particularly dramatic. The IPC:1º contacts are increased by a scalloped profile, a further demonstration that IPC:IPC contacts are disfavored. To complete this pattern, therefore, IPC:IPC contacts are further minimized by reducing the number of candidate 2ºs to one cell between each 3º and bristle. Thus, IPC morphogenesis reveals a mechanism by which pattern is determined through minimizing disfavored cell-cell contacts and maximizing preferred cell-cell contacts (Bao, 2005).

Finally, it is interesting to note how 2ºs are selected. After emergence of 3ºs, two IPCs are commonly found between a 3º and bristle. In many ways, these two IPCs are equal: each contacts two 1ºs and each establishes equally strong cell junctions; each forms a scalloped contour with two neighboring 1ºs, and each is exposed to the same molecular cues. However, evidence is provided that these two cells have a low affinity for each other, a situation that is not favored by minimum free energy principles. One cell will be removed. How is this cell chosen? Clues came from manipulating levels of Roughest, which altered each cell's capacity for competition. Artificially high levels of Roughest rendered a cell a supercompetitor: the targeted cell even replaced two cells to become both a 2º and a 3º. Presumably, high levels of Roughest promote a higher level of cell junctions, which makes a cell more competitive and determines the survivor. Conversely, low levels of Roughest put the targeted cell at a disadvantage during this competition. Therefore, during the selection of a 2º , differing levels of Roughest expressed by each cell may determine its fate: survival or death (Bao, 2005).

Neph1/Nephrin family members are required for the development of a wide array of tissues including axonal pathfinding and myoblast fusion in Drosophila and formation of the slit diaphragm in the developing mammalian kidney. The role observed for preferential adhesion in IPC morphogenesis and patterning in the Drosophila eye leads to the interesting possibility that similar mechanisms are utilized broadly in pattern formation (Bao, 2005).

Notch controls cell adhesion in the Drosophila eye

Sporadic evidence suggests Notch is involved in cell adhesion. However, the underlying mechanism is unknown. This study has investigated an epithelial remodeling process in the Drosophila eye in which two primary pigment cells (PPCs) with a characteristic 'kidney' shape enwrap and eventually isolate a group of cone cells from inter-ommatidial cells (IOCs). This paper shows that in the developing Drosophila eye the ligand Delta is transcribed in cone cells and Notch is activated in the adjacent PPC precursors. In the absence of Notch, emerging PPCs fail to enwrap cone cells, and hibris (hbs) and sns, two genes coding for adhesion molecules of the Nephrin group that mediate preferential adhesion, are not transcribed in PPC precursors. Conversely, activation of Notch in single IOCs leads to ectopic expression of hbs and sns. By contrast, in a single IOC that normally transcribes rst, a gene coding for an adhesion molecule of the Neph1 group that binds Hbs and Sns, activation of Notch leads to a loss of rst transcription. In addition, in a Notch mutant where two emerging PPCs fail to enwrap cone cells, expression of hbs in PPC precursors restores the ability of these cells to surround cone cells. Further, expression of hbs or rst in a single rst- or hbs-expressing cell, respectively, leads to removal of the counterpart from the membrane within the same cell through cis-interaction and forced expression of Rst in all hbs-expressing PPCs strongly disrupts the remodeling process. Finally, a loss of both hbs and sns in single PPC precursors leads to constriction of the apical surface that compromises the 'kidney' shape of PPCs. Taken together, these results indicate that cone cells utilize Notch signaling to instruct neighboring PPC precursors to surround them and Notch controls the remodeling process by differentially regulating four adhesion genes (Bao, 2014).

Recognition of pre- and postsynaptic neurons via nephrin/NEPH1 homologs is a basis for the formation of the Drosophila retinotopic map

Topographic maps, which maintain the spatial order of neurons in the order of their axonal connections, are found in many parts of the nervous system. This study focused on the communication between retinal axons and their postsynaptic partners, lamina neurons, in the first ganglion of the Drosophila visual system, as a model for the formation of topographic maps. Post-mitotic lamina precursor cells differentiate upon receiving Hedgehog signals delivered through newly arriving retinal axons and, before maturing to extend neurites, extend short processes toward retinal axons to create the lamina column. The lamina column provides the cellular basis for establishing stereotypic synapses between retinal axons and lamina neurons. This study identified two cell-adhesion molecules: Hibris, which is expressed in post-mitotic lamina precursor cells; and Roughest, which is expressed on retinal axons. Both proteins belong to the nephrin/NEPH1 family. Evidence is provided that recognition between post-mitotic lamina precursor cells and retinal axons is mediated by interactions between Hibris and Roughest. These findings revealed mechanisms by which axons of presynaptic neurons deliver signals to induce the development of postsynaptic partners at the target area. Postsynaptic partners then recognize the presynaptic axons to make ensembles, thus establishing a topographic map along the anterior/posterior axis (Sugie, 2010).

This study shows that cell recognition between pre- and postsynaptic neurons via the Hbs-Rst interaction is required for the establishment of precise retinotopic mapping. During the development of the Drosophila visual center, presynaptic photoreceptors extend their axons to the lamina layer. Postsynaptic lamina precursor cells (pLPCs) start to differentiate in response to Hh delivered through newly arriving R axons. They then express Hbs, which interacts with Rst on R axons (see Model for the specific interaction between R axons and pLPCs mediated by an interaction between Hbs and Rst). This Hbs-Rst interaction is required for lamina column assembly, which underlies the topographic connections of the synapses along the anteroposterior axis (Sugie, 2010).

The process of lamina column assembly is unique in that presynaptic neurons regulate the development of postsynaptic partners in the target area, and the somata of postsynaptic neurons recognize the presynaptic axons at the developing stage well before neurite formation. This mechanism appears to be an efficient and accurate way to make a topographic map along the anterior/posterior axis. In addition, unlike the well-known axon guidance process, in which growth cones search for their targets, postsynaptic cells actively contribute to the pre- and postsynaptic interactions via direct communication. The changes in the Hbs localization that are associated with rst mutation were not only observed in pLPCs adjacent to R axons, but also in pLPCs far from R axons. This finding could be ascribed to the fact that pLPCs that are distant from R axons can contact R axons through their protrusions. Hbs might be preferentially localized at the protrusions of pLPCs that interact with R axons. The behavior of pLPCs is analogous to that of developing muscle cells, which extend filopodia to the axonal targeting of innervating motoneurons (Sugie, 2010).

Tests were performed to see whether the cell-adhesion mechanism mediated by Hbs and Rst was sufficient to rescue the sim phenotypes. Induction of exogenous hbs in pLPCs did not rescue sim loss-of-function mutants. Consistent with this finding, overexpression of sim using the NP6099-Gal4 driver caused the premature incorporation of pLPCs into the assembling domain, but overexpression of hbs did not. These results suggest that other molecules under the control of sim must be required for lamina column assembly (Sugie, 2010).

hbs expressed in photoreceptor cells does not play an essential role in lamina column assembly. The reason that Hbs originating in R axons does not interfere with the Hbs-Rst association remains unknown. The intracellular interaction of the two proteins might be blocked in R axons as a result of alternative subcellular localization and/or steric hindrance, or additional intermediates might be required for Hbs function in pLPCs, but not in R axons (Sugie, 2010).

Nephrin and NEPH1 homolog proteins tend to be located on opposing cell membranes so that they are brought into close apposition. This arrangement underlies the amazingly similar patterns of immunoreactivity in the eye disc, wing disc and somatic muscle as well as in the pupal optic lobe. These proteins are located in opposing cell membranes in the lamina. Consistent with previous studies, Hbs and Sns proteins were expressed in pLPCs, whereas Rst and Kirre were expressed in R axons; however, Hbs was also expressed in R axons. Recent studies have demonstrated that proteins of the nephrin and NEPH subfamilies are also expressed in neighboring cell types in vertebrate nervous systems. These observations reveal the conservation of nephrin/NEPH1 expression patterns across tissues and species (Sugie, 2010).

Previous work has identified SYG-1, a homolog of Rst, Kirre and NEPH1, as well as SYG-2, a homolog of Hbs, Sns and nephrin, which are necessary for synaptic specificity in Caenorhabditis elegans. The first Ig domain of SYG-1 and the first five Ig domains of SYG-2 are necessary and sufficient for binding and synapse formation in vivo. Similarly, it was found that the extracellular domain of Hbs and the first Ig domain of Rst are important for the association of pLPCs with R axons. These observations show remarkable functional conservation of the restricted domains of Drosophila and C. elegans nephrin/NEPH1 homologs (Sugie, 2010).

Further study of the preferential cell adhesion between nephrin/NEPH1 homolog proteins may reveal a common mechanism underlying the interaction between pre- and postsynaptic neurons in both Drosophila and vertebrate brains (Sugie, 2010).

Mutations in sec15 cause defects in synaptic specificity, axon targeting and localization of axon guidance components

The exocyst is a complex of proteins originally identified in yeast that has been implicated in polarized exocytosis/secretion. Components of the exocyst have been implicated in neurite outgrowth, cell polarity, and cell viability. An exocyst component, sec15, has been isolated in a screen for genes required for synaptic specificity. Loss of sec15 causes a targeting defect of photoreceptors that coincides with mislocalization of specific cell adhesion and signaling molecules. Additionally, sec15 mutant neurons fail to localize other exocyst members like Sec5 and Sec8, but not Sec6, to neuronal terminals. However, loss of sec15 does not cause cell lethality in contrast to loss of sec5 or sec6. The data suggest a role for Sec15 in an exocyst-like subcomplex for the targeting and subcellular distribution of specific proteins. The data also show that functions of other exocyst components persist in the absence of sec15, suggesting that different exocyst components have separable functions (Mehta, 2005).

Elevated levels of chaoptin in photoreceptor terminals have been described for another vesicle-trafficking mutant, the vesicle-SNARE neuronal-synaptobrevin (n-syb). This mutant also exhibits neuronal targeting defects. This observation raises the possibility that vesicle-dependent trafficking of transmembrane or other signaling molecules might be responsible for the neuronal targeting defects of sec15 mutant photoreceptors. Recently, Zhang (2004) identified Rab11 as an interacting partner of Sec15 in mammalian cell culture and proposed that Sec15 is an effector for some but not all Rabs. Indeed, an accumulation or upregulation of Rab11 immunoreactivity was seen in sec15 mutant photoreceptors, consistent with Rab11-positive vesicles failing to fuse with their target sites. To further test this hypothesis, the localization of cell adhesion and signaling molecules was examined in mutant photoreceptor cell bodies as well as terminals during photoreceptor development, precisely when target selection and cartridge formation occur (between P + 5% to P + 40% referring to time after pupation). Proteins were examined that have either been shown to be required for photoreceptor target selection, such as Dlar, N-cadherin, flamingo, and IrreC-rst, or that are likely to be required, based on work in other systems, such as Armadillo, Chaoptin, and Fasciclin II (Mehta, 2005).

Fasciclin II (Fas2) localization was examined in sec15 mutant photoreceptors, since chaoptin upregulation coincides with elevated levels of Fas2 in n-syb mutant photoreceptors. Fas2 appears to be present in aggregates in sec15 mutant photoreceptor cell bodies at P + 20%, in contrast to wild-type photoreceptors. In addition, the neuronal connections of the cell bodies exhibit Fas2 aggregated along the length of the mutant axons. Similarly, overexpression of Fas2 in photoreceptors causes neuronal targeting defects between P + 20% and P + 40%. In contrast to n-syb, however, no elevated levels of Fas2 are observed later in development. Hence, the data suggest that an aberrant localization of Fas2 in a specific developmental time window may at least partially underlie the observed phenotypes (Mehta, 2005).

Similar mislocalization phenotypes in photoreceptor cell bodies were also observed for other cell adhesion molecules such as Dlar and IrreC-rst during the developmental time window of photoreceptor target selection. Dlar is normally restricted apically in developing wild-type photoreceptors, at the center of the ommatidial array. In sec15 mutant photoreceptors it appears much more randomly distributed, such that a basal optical section through the eye shows Dlar at higher levels in mutant ommatidia. Although these results show mislocalization of cell adhesion molecules in the correct cell at the time when they are known to be required for proper target selection, no obvious defects were detected in the localization of Dlar or IrreC-rst in the developing lamina. This leaves open the question of whether mislocalization of Dlar and IrreC-rst beyond the resolution limit of confocal microscopy additionally contributes to the observed targeting defects (Mehta, 2005).

In vertebrates, Lar is known to localize to adherens junctions. Hence, a possible explanation for the mislocalization of Fas2, IrreC-rst, and Dlar in mutant photoreceptor cell bodies is a defect of adherens junctions. The subcellular localization of the adherens junction markers N-cadherin and armadillo was examined in the cell bodies as well as the terminals of mutant photoreceptors, but no mislocalization of N-cadherin was detected in either compartment. However, armadillo displayed localization defects selectively in the developing lamina, but not the photoreceptor cell bodies. Several other cell adhesion and signaling molecules, including flamingo, Crumbs, and Bazooka, were examined, all of which did not display aberrant localization at the level of light microscopy. It is concluded conclude that a specific subset of proteins is mislocalized in sec15 mutants (Mehta, 2005).

Single-minded, Dmef2, Pointed, and Su(H) act on identified regulatory sequences of the roughest

Roughest (Rst) is a cell adhesion molecule of the immunoglobulin superfamily that has multiple and diverse functions during the development of Drosophila melanogaster. The pleiotropic action of Rst is reflected by its complex and dynamic expression during the development of Drosophila. By an enhancer detection screen, several cis-regulatory modules have been identified that mediate specific expression of the roughest gene in Drosophila developmental processes. To identify trans-regulators of rst expression, the Gal4/UAS system was used to screen for factors that were sufficient to activate Rst expression when ectopically expressed. By this method the transcription factors Single-minded, Pointed.P1, and Su(H)-VP16 were identified. Furthermore, these factors and, in addition, Dmef2 are able to ectopically activate rst expression via the previously described rst cis-regulatory modules. This fact and the use of mutant analysis allocates the action of the transcription factors to specific developmental contexts. In the case of Sim, it could be shown to regulate rst expression in the embryonic midline, but not in the optic lobes. Mutagenesis of Sim consensus binding sites in the regulatory module required for rst expression in the embryonic midline, abolishes rst expression; indicating that the regulation of rst by Sim is direct (Apitz, 2005).

Rst has complex and multifaceted functions throughout the development of the fly, which include myogenesis, eye development, as well as axonal pathfinding in the optic lobes. To gain a better understanding of these functions at the levels of gene regulation and signal transduction, a number of tests were designed to identify both the transcriptional activators and their respective targets surrounding the rst locus. In a preceding study (Apitz, 2004), a number of DNA segments upstream of rst were characterized and regulatory regions were discovered that mediate gene expression in myoblasts, midline, and eyes, respectively. In the present study these results were supplemented with an in vivo screen to identify regulators of rst expression using the Gal4/UAS system. Several factors were discovered that are able to induce ectopic Rst expression and to activate reporter gene expression via rst cis-regulatory sequences (Apitz, 2005).

The experimental route taken to identify protein factors involved in the regulation of the rst gene is based on the detection of their potential to induce ectopic Rst expression in vivo. The use of sca-Gal4 as a driver line in this experimental approach is based on the following criteria. sca-Gal4 mediates expression in neuroectodermal cells of the embryo. At embryonic stage 10, these cells can be examined for ectopic Rst expression because no endogenous Rst expression is found at this time in these cells; this allows operators to obtain clear-cut and unequivocal results. The use of alternative Gal4 driver lines did not prove suitable because of the dynamic expression of Rst during all developmental stages, and due to its subcellular localization. For example, when dll-Gal4 is used as a driver, it is difficult to distinguish between ectopic Rst expression induced in the apical tips of cells of the leg discs, and endogenous Rst expression in the overlaying ectodermal cells. Furthermore, the use of sca-Gal4 has the advantage that neuroectodermal cells are not fully differentiated cells. This may more closely resemble the developmental state of the cells in which Rst expression is normally induced endogenously, e.g., in undifferentiated cells of the developing eye disc. However, this approach generally fails to reveal transcription factors that need a coactivator for induction of rst expression, which is not present in the cells of the neuroectoderm at embryonic stage 10. This may explain the failure of Dmef2 to induce rst expression at this stage. It was shown, however, that Dmef2 is able to induce rst expression at later stages by the use of rst-lacZ constructs. The function of the different rst-lacZ constructs has been linked to specific developmental circumstances (Apitz, 2004) and their activation by corresponding factors is consistent with the known roles of these proteins in development (Apitz, 2005).

Single-minded (Sim) is a basic helix-loop-helix-PAS (bHLH-PAS) transcription factor that serves as a master regulatory gene of CNS midline development. The CNS midline is derived from two mesectodermal cell rows that intermingle at the ventral midline following gastrulation of the embryo. The ventral midline cells move into the interior of the embryo where they function (among other roles) in the patterning of the CNS by secreting axonal guidance cues. It has been shown that expression of more than 20 genes depends on sim. Because Rst expression in the embryonic midline is lost in sim mutant embryos, rst is added to this list (Apitz, 2005).

Tgo, the coactivator of Sim, is ubiquitously expressed in the embryo. Thus, ectopic expression of target genes can be induced by Sim in transactivation studies. The early onset of strong ectopic expression of Rst in neuroectodermal cells under the transcriptional control of ectopic Sim suggests that rst may be its direct target. Several lines of evidence support this hypothesis. The expression of rst in the embryonic midline sets in shortly after gastrulation in the ventral midline, following accumulation of Sim protein in these cells. In an expression profiling study, rst was assigned to a group of 37 genes that mimic the expression pattern of sim in the embryonic midline. These genes are therefore likely direct targets of Sim/Tgo heterodimers. Since Sim does not act on the F5 fragment in the transactivation assay, the Sim-responsive rst cis-regulatory sequence was mapped to a 2.5-kb region contained within the F6 fragment, i.e., F6d. F6d was shown to contain a regulatory module for expression in the embryonic midline (Apitz, 2004). Two putative central midline elements (CME) are located in this region and both are conserved between D. melanogaster and D. pseudoobscura. It has been shown that heterodimers of Drosophila Sim and human Tgo bind to the CME and that Sim activates midline gene transcription through CME. Site-directed mutagenesis studies described in this paper show that both CME in F6d are required for reporter gene expression in the embryonic midline. Significantly, analysis of polytene chromosomal binding sites of ectopically expressed Sim in salivary gland nuclei revealed that Sim binds to chromatin within chromosomal subdivision 3C, coinciding with the rst locus. These data indicate that rst expression in the embryonic midline is directly regulated by Sim (Apitz, 2005).

Unexpectedly, ectopic reporter gene expression was observed in cells outside the midline using the mutated rstF6d sequence. This finding indicates that by mutating the CME the affinity of this DNA sequence for other transcription factors may have been altered. It is conceivable that either a suppressor normally preventing expression outside the midline lost its affinity for the site, or an activator, which is active in the corresponding cells, gained affinity. Similar CME mutations were used in other studies, and no ectopic reporter gene expression was reported in these cases. The different outcome of these studies may be based on the different chromatin regions, in which the CME are embedded (Apitz, 2005).

The function of Rst in the embryonic midline is as yet unknown. The apical localization of Rst in cytoplasmic projections of midline cells that migrate in the interior of the embryo (Apitz, 2004), points to an adhesive function which may support the correct localization of the midline cells during migration. Since the cell adhesion molecule Hibris (Hbs) is the only known interaction partner of Rst that is also expressed in the midline, it will be interesting to examine the interactions of Rst and Hbs in CNS midline development (Apitz, 2005).

Sim is also involved in postembryonic optic lobe development. Although the expression pattern and axonal pathfinding defects of sim and rst point to a possible regulation of rst by Sim in the optic lobes, no evidence was found supporting this hypothesis using simts mutants. This result is corroborated by analysis of rst cis-regulatory elements (Apitz, 2004). F6d, the rst regulatory sequence that mediates reporter gene expression in the embryonic midline and which is activated by ectopic Sim, does not display a corresponding reporter gene expression in the optic lobes. These results are unexpected since they point to a differential set of Sim target genes in postembryonic stages as compared to the embryo. It will be interesting to examine whether other known target genes of Sim are expressed independently of sim in the optic lobes as well, and whether this differential regulation depends on the differential use of alternative cofactors in the embryo and in the optic lobes, respectively. Since Tgo colocalizes with Sim in the optic lobes, one may suspect the involvement of additional uncharacterized cofactors. The existence of tissue-specific cofactors that direct Sim/Tgo activity to the embryonic midline have already been postulated by other authors (Apitz, 2005).

Ectopic expression of a constitutively active Pnt variant (Pnt.P1) mediates strong activation of Rst expression in neuroectodermal cells. Since Pnt.P1 recognizes the same target sequences as its splice variant Pnt.P2, the nuclear effector of the Ras-MAPK pathway, ectopic activation of Rst expression by Pnt.P1 is consistent with a regulation by the Ras-MAPK pathway. Similarly, the ectoptic activation of Rst expression in neuroectodermal cells by Su(H)-VP16 points to a regulation of rst by the Notch pathway (Apitz, 2005).

The Ras-MAPK and the Notch pathways display significant crosstalk during developmental processes in Drosophila, e.g., in cell fate specification of the eye disc. It is difficult to elucidate a possible regulation of a candidate gene by mutant analysis if it is activated by both pathways. In mutants, for one of the pathways, the activity of the other pathway will ensure residual expression of the candidate gene under scrutiny. rst expression is activated by both pathways and single mutant analysis did not reveal a significant loss of expression. Both pathways converge on regulatory elements contained within F6 and not in F5. Furthermore, a regulatory module is present in the nonoverlapping part of F6 that is activated in IOC before apoptotic decisions are made in these cells (Apitz, 2004). This module is located within an approximately 600-bp sequence and is active during several apoptotic decisions (Apitz, 2004). Consensus binding sites for Su(H) and Pnt in this module are conserved between D. melanogaster and D. pseudoobscura. Both the Ras-MAPK and Notch pathways are involved in apoptotic processes of IOC cells. Together, these data suggest that rst transcription is regulated by these pathways in the context of apoptotic decisions (Apitz, 2005).

The transactivation screen shows that Dmef2 acts on rst cis-regulatory sequences: the overlapping reporter gene constructs rstF5 -lacZ and rstF6 -lacZ are both ectopically activated by Dmef2 in the scabrous expression domain. Regions F5 and F6 contain partially overlapping sequences and it is likely that Dmef2 acts on a sequence interval bracketed by this overlap. This region contains a regulatory module for expression in the mesoderm (Apitz, 2004). This sequence contains a putative Dmef2 binding site that is also conserved between D. melanogaster and D. pseudoobscura. The sequence of this putative element exactly matches a Dmef2 binding site found in the enhancer of β3 tubulin. Therefore, it is argued that rst may be a direct target of Dmef2. This is consistent with similar mesodermal expression patterns and mutant phenotypes of Dmef2 and rst. In contrast, Rst is expressed in the mesoderm of Dmef2 loss-of-function mutants. An explanation for this result is provided by analysis of rst cis-regulatory sequences. rst is regulated in the mesoderm by at least two independent regulatory modules that mediate a differential expression pattern (Apitz, 2004). One is contained in F6p, the other in F5p. This points to a mesodermal regulation of rst by at least two factors, one of which is still active in Dmef2 mutants (Apitz, 2005).

Anterograde Jelly belly and Alk receptor tyrosine kinase signaling mediates retinal axon targeting in Drosophila

Anaplastic lymphoma kinase (Alk) has been proposed to regulate neuronal development based on its expression pattern in vertebrates and invertebrates; however, its function in vivo is unknown. This study demonstrated that Alk and its ligand Jelly belly (Jeb) play a central role as an anterograde signaling pathway mediating neuronal circuit assembly in the Drosophila visual system. Alk is expressed and required in target neurons in the optic lobe, whereas Jeb is primarily generated by photoreceptor axons and functions in the eye to control target selection of R1-R6 axons in the lamina and R8 axons in the medulla. Impaired Jeb/Alk function affects layer-specific expression of three cell-adhesion molecules, Dumbfounded/Kirre, Roughest/IrreC, and Flamingo, in the medulla. Moreover, loss of flamingo in target neurons causes some R8-axon targeting errors observed in Jeb and Alk mosaic animals. Together, these findings suggest that Jeb/Alk signaling helps R-cell axons to shape their environment for target recognition (Bazigou, 2007).

These genetic studies in Drosophila provide functional evidence in vivo that Alk plays a crucial role in the developing central nervous system. This study shows that Alk and its cognate ligand Jeb form an anterograde signaling pathway in the fly visual system, which is required for target selection by R cell axons within the lamina and medulla. It is proposed that R cell axons release Jeb to activate Alk signaling in target neurons and, through direct or indirect regulation of downstream guidance molecules, contribute to creating the appropriate environment for target recognition (Bazigou, 2007).

In the visual system, R cell axons provide two known anterograde signals to the optic lobe to promote neuronal proliferation and differentiation of target neurons during the third instar larval stage. R cell-derived Hh induces mitotic divisions of lamina precursor cells (LPCs), as well as expression of the early neuronal marker Dachshund in both LPCs and postmitotic lamina neurons. Dachshund in turn is required to control the expression of the EGF receptor in lamina neurons, thus making them competent for the second anterograde R cell-derived signal Spitz that induces the next step of lamina neuron differentiation. A third so far unidentified signal controls glial cell development and migration in the optic lobe. The current findings show that R cell axons provide an unexpected fourth anterograde signal -- Jeb -- that is required to mediate target selection of R cell axons during pupal development. Unlike the Hh and Spitz signals, Jeb represents an anterograde signal delivered by R cell axons not only to the lamina but also to the medulla (Bazigou, 2007).

That Jeb and Alk form an anterograde signaling pathway in the visual system that is supported by three lines of evidence: first, Jeb and Alk are expressed in a largely complementary pattern from the third instar larval to midpupal stages. The ligand Jeb is produced in R cells, whereas the receptor Alk is specifically expressed by target neurons. Since the Jeb protein has been shown to be secreted in vitro, it is highly likely released from R cell growth cones. Second, jeb is genetically required in R cells, whereas Alk functions in target neurons. Third, in the converse experiment, removal of jeb function in the target or Alk in the eye does not produce any conspicuous targeting phenotypes (Bazigou, 2007).

It is proposed that Jeb/Alk signaling plays a role in regulating late events of target-neuron maturation to control R1-R6 axons in the lamina and R8 axons in the medulla. Consistent with this model, the data indicate that loss of Jeb/Alk signaling affects the expression of three guidance molecules, Duf/Kirre, Rst/IrreC, and Fmi, in the R8 recipient layer of the medulla, while Caps, LAR, PTP69D, and CadN appear normal at this level of resolution. Interestingly, animals lacking Jeb/Alk signaling display similar R8 projection defects as fmi and caps eye mosaics. It was further shown that loss of fmi in target neurons causes R8-targeting defects, which qualitatively resemble those observed in Jeb/Alk mosaics. As Jeb/Alk signaling acts upstream of multiple cell-adhesion molecules, loss of one factor likely results in milder targeting defects. In support of this notion, it was observed that phenotypes in jeb or Alk mosaics were more frequent in comparison to fmi knockdown or fmi ELF mosaics. Moreover, loss of fmi in the target appeared to cause one prevalent targeting defect, i.e., the fasciculation of R8 axons with processes in adjacent medulla columns. Notably, loss of sec15 in R cells, which encodes an exocyst component regulating the localization of cell-adhesion molecules to axon terminals, also causes distinct targeting errors. This is consistent with the model that regulating the precise expression of guidance molecules by Jeb/Alk signaling is indeed important for axon targeting in the visual system (Bazigou, 2007).

R cell-targeting defects occurred in both null and kinase domain mutant alleles of Alk, showing that tyrosine kinase activity is essential. Furthermore, studies of vertebrate Alk in vitro, as well as Drosophila Alk in vivo, demonstrate that this RTK drives an ERK/MAPK-mediated signaling pathway, suggesting that Alk may also act through this pathway in the visual system. There are three possible mechanisms as to how Jeb/Alk signaling could regulate downstream guidance molecules: (1) Jeb and Alk may directly regulate the expression of guidance molecules, (2) they could indirectly regulate the expression pattern of guidance molecules via the activation of transcriptional programs determining target neuron identities, or (3) they could separately control both the expression of guidance molecules and transcription factors. Such mechanisms would be analogous to what has been observed in the developing visceral mesoderm, where Jeb/Alk signaling induces the expression of both Duf/Kirre and Org-1, a transcription factor and mammalian Tbx1 homolog, to drive muscle fusion. At present, it cannot be excluded that Alk additionally modulates the activity of downstream targets (Bazigou, 2007).

Anterograde Jeb/Alk signaling would make it possible to coordinate the timing of R cell growth-cone extension with local expression of guidance factors in the target. These in turn could directly regulate afferent axon targeting. Alternatively, guidance factors may be required to shape dendritic and axonal arbors of target neurons and to mediate R cell-targeting decisions. Fmi could indeed take part in both processes, as it can control dendrite development, as well as axon guidance by afferent/afferent and afferent/target interactions. Similar to CadN or LAR eye mosaics, some R1-R6 axons lacking jeb function failed to extend from their original bundle. Extension and cartridge assembly phenotypes were also detected in jeb eye or Alk target mosaics, which qualitatively resembled those described for fmi eye mosaics. Future studies will require the identification and validation of (other) downstream guidance molecules, as well as the isolation of transcriptional regulators controlling target neuron subtype specificity in both the lamina and medulla to provide further insights into the mechanisms underlying Jeb/Alk function (Bazigou, 2007).

It was observed that ectopic expression of Jeb in the visual system strongly reduces the number of activated Caspase 3-positive cells in the medulla at 24 hr APF, when many postmitotic medulla neurons normally undergo apoptosis in wild-type. Thus, Jeb/Alk signaling may also mediate cell survival in parallel to neuronal maturation. The mechanism could be similar to the pleiotropic function of EGF-receptor signaling, which, depending on low or high level of activation regulates cell-cycle withdrawal, mitosis, cell survival, and differentiation in the developing eye imaginal disc of Drosophila (Bazigou, 2007).

Although Jeb shares some sequence similarity with proteins such as the secreted bovine glycoprotein Sco-Spondin , no Jeb homolog has been isolated so far in vertebrates. However, the growth factors Pleiotrophin and Midkine have been reported to act as ligands for Alk in vertebrates, and both have been linked to neuronal development and neurodegenerative diseases. Therefore, Alk may work with different ligands in the vertebrate nervous system. The C. elegans homolog of Alk is localized presynaptically at the neuromuscular junction and has been proposed to mediate synapse stabilization. Also, the vertebrate homologs of Alk are strongly expressed in the developing and adult nervous systems. This includes motor-neuron columns in the spinal cord and, intriguingly, also the superior colliculus, a higher-order processing center for visual information in the brain. That Alk may play a role in neuronal development in vertebrates is further supported by in vitro studies indicating that activated Alk can promote neuronal differentiation and neurite outgrowth in specific cell line. These observations suggest that the function of Alk in regulating specific aspects of neuronal development may be conserved (Bazigou, 2007).

rst transcriptional activity influences kirre mRNA concentration in the Drosophila pupal retina during the final steps of ommatidial patterning

Drosophila retinal architecture is laid down between 24-48 hours after puparium formation, when some of the still uncommitted interommatidial cells (IOCs) are recruited to become secondary and tertiary pigment cells while the remaining ones undergo apoptosis. This choice between survival and death requires the product of the roughest (rst) gene, an immunoglobulin superfamily transmembrane glycoprotein involved in a wide range of developmental processes. Both temporal misexpression of Rst and truncation of the protein intracytoplasmic domain, lead to severe defects in which IOCs either remain mostly undifferentiated and die late and erratically or, instead, differentiate into extra pigment cells. Intriguingly, mutants not expressing wild type protein often have normal or very mild rough eyes. By using quantitative real time PCR to examine rst transcriptional dynamics in the pupal retina, both in wild type and mutant alleles, it was shown that tightly regulated temporal changes in rst transcriptional rate underlie its proper function during the final steps of eye patterning. Furthermore it was demonstrated that the unexpected wild type eye phenotype of mutants with low or no rst expression correlates with an upregulation in the mRNA levels of the rst paralogue kin-of-irre (kirre), which seems able to substitute for rst function in this process, similarly to their role in myoblast fusion. This compensatory upregulation of kirre mRNA levels could be directly induced in wild type pupa upon RNAi-mediated silencing of rst, indicating that expression of both genes is also coordinately regulated in physiological conditions. These findings suggest a general mechanism by which rst and kirre expression could be fine tuned to optimize their redundant roles during development and provide a clearer picture of how the specification of survival and apoptotic fates by differential cell adhesion during the final steps of retinal morphogenesis in insects are controlled at the transcriptional level (Machado, 2011).

The ability of interommatidial cells to reorganize their apical contacts such as to maximize their membrane interactions with primary pigment cells, around 24% AFP, is a key step for generating the highly precise geometrical pattern of the adult compound eye, but it is also needed to create differences in intercellular adhesion and signaling that allow the final cell fate specification decision in the pupal retina - that between survival and programmed cell death - to correctly take place. A central involvement of Rst cell adhesion molecule in the IOC reorganization event has been inferred not only from the observation that this latter process paralleled Rst protein redistribution in IOC membranes but also from two independent sets of evidence. first, it was observed that the complete absence or truncation of rst protein product often blocked IOC sorting and led to an 'all to one' switch in cell fate, with surplus pigment cells appearing in the adult eye as consequence of lack of cell death. Second, when Rst redistribution was delayed, so was IOC sorting, producing an 'all to none' response in which the survival versus death choice is either not correctly made or not properly implemented, Thus lead to surplus cells dying later and erratically while the remaining ones failed to differentiate into proper pigment cells. These data emphasized the need for rst function not only at the right place but also at the right time, and implied very precise spatial and temporal controls of its expression. Unraveling the details of these control mechanisms is therefore an essential prerequisite for a fuller understanding of the nature and dynamics of the signals to which cells must respond to die or to differentiate in the final steps of ommatidial patterning. The quantitative analysis presented in this study showed a striking correlation between rst mRNA concentration over time and the qualitative dynamics of Rst protein localization in IOC membrane, both in wild type and in the regulatory rstD mutant, thus implicating the temporal control of rst transcription, rather than a reshuffling of Rst protein molecules previously present in the membrane, as a main factor responsible for the critical changes in cell adhesion specificity that allow IOC sorting to take place. These findings help to shed light on a little studied and, so far, underestimated aspect of eye development, adding a new dimension to the complex process of ommatidial patterning and differentiation. Besides, the results demonstrate functionally the ability of kirre to rescue rst function in IOC sorting. Although the possibility of redundant roles for rst and kirre in retinal development had been previously suggested, supporting evidence was mainly indirect and obtained from either protein co-localization or overexpression experiments. Bao (2010) has provided convincing evidence, based on the analysis of retinal patterning at the time of IOC sorting, that Rst and Kirre function redundantly to maintain the spacing between developing ommatidial groups. This study has extended these observations by directly showing the ability of high levels of kirre expression in mutants with no or very little rst mRNA to bypass the need for rst function, including its influence in cell fate choice. The molecular mechanism underlying this redundancy, however, remains to be elucidated. Rst and Kirre have highly conserved extracellular domains, co-localize in IOCs and both appear to bind Hbs at the border with primary pigment cells (although in the case of Kirre direct evidence for this latter assertion is missing), seeming to imply that the two proteins are fully interchangeable in their interactions with other extracellular and intracellular molecules during retinal development. However their intracellular domains show very little similarity suggesting that they might have few, if any, common cytoplasmic binding partners. Genetic and biochemical studies aiming at identifying possible intracellular interactors of Kirre and Rst have been performed in different tissues, uncovering sets of directly binding proteins and potential signaling pathways that are distinct for each gene product. Since the intracellular domain of Rst is required for its function in the pupal retina, probably by interacting with actin cytoskeleton, it would be important to ascertain whether rst/kirre functional redundancy in this context results from their ability to act through the same intracellular pathways or it is a consequence of their interaction with different ensembles of intracellular targets that can nevertheless lead to the same end result (Machado, 2011).

Perhaps the most interesting finding presented in this study is the evidence for compensatory co-regulation of rst and kirre mRNA concentrations and its asymmetrical nature. A possible, although unlikely explanation for this asymmetry could reside in the much higher absolute levels of rst mRNA present during the temporal period examined, making small changes in relative concentration easier to detect with confidence. Also neither the effect of overexpression of one gene on the concentration of the other was investiged, nor whether similar coordination in mRNA expression is taking places in other developmental contexts where complementary or redundant functioning of rst and kirre seem to be required, such as myoblast fusion, salivary gland and optic lobe development. In this latter context it is worth mentioning that rst revertant RTW6, but neither RTW8 nor rstD, shows axonal pathfinding defects in the optic lobe suggesting that at least in some developmental contexts rst and kirre might not be fully redundant (Machado, 2011).

Finally, an intriguing aspect of the compensatory response reported in this study is that it implies some kind of sensor mechanism capable to post-transcriptionally read rst expression levels and adjust those of kirre accordingly. Here again mRNA, rather than protein concentration, may play the main role, since in rstCT, which carries a small deletion in the coding region of an otherwise normal mRNA, low levels of rst activity caused by the truncated intracellular domain and destabilization of the protein from the membrane leads only to a minimal, if any, increase of kirre mRNA levels at 24% APF. Also it is conceivable that rather than upregulating kirre transcription, a decrease in degradation could be taking place, leading to an accumulation of kirre mRNA molecules. Experiments designed to further test this possibility as well as to map rst mRNA sequences that might be relevant for this putative regulatory feedback loop between rst and kirre are currently underway. Whatever the case, these findings suggest a general mechanism by which the expression both genes could be fine tuned to optimize their redundant roles during development (Machado, 2011).

Protein Interactions

The adaptor protein X11Lalpha/Dmint1 interacts with the PDZ-binding domain of the cell recognition protein Rst in Drosophila

The Drosophila cell adhesion molecule Roughest (Rst) plays key roles during the development of the embryonic musculature, spacing of ommatidia in the compound eye and of sensory organs on the antenna, as well as in the neuronal wiring of the optic lobe. In rstCT mutants lacking the cytoplasmic domain of the Rst protein, cell sorting and apoptosis in the eye are affected, suggesting a requirement of this domain for Rst function. To identify potential interacting proteins, yeast two-hybrid screens were performed using as baits the cytoplasmic domains of Rst and its paralogue Kirre. Among several putative interactors, two paralogous Drosophila PDZ motif proteins related to X11/Mint were identified. X11/Mint family members in C. elegans (LIN-10) and vertebrates are believed to function as adaptor proteins and to regulate the assembly of multi-subunit complexes at the synapse, thereby linking the vesicle cycle to cell adhesion. Using genetic, cell biological, and biochemical approaches, the interaction of Rst with X11La has been shown to be of biological significance. The proteins interact, for example, in the context of cell sorting in the pupal retina (Vishnu, 2006).

The X11/Mint family comprises proteins with several distinct protein-protein interaction domains, including a phosphotyrosine-binding (PTB) domain, commonly found among signalling molecules, and two PDZ domains. These domains are specialized for binding to the C-terminus of transmembrane proteins and play a role in polarized protein targeting. PDZ proteins are most abundant in the cytoplasm and are known to link transmembrane proteins to the underlying cytoskeleton and cytosolic signalling proteins (Vishnu, 2006).

This analysis focused on the Rst-X11Lα interaction since several additional lines of evidence consistently pointed to a biologically meaningful interaction. Both proteins can be co-immunoprecipitated in vivo, and the association is mediated by the carboxyterminal three amino acids of Rst comprising the PDZ binding motif, and the PDZ domain of X11Lα. Conversely, this interaction is abolished in rstCT flies lacking the C-terminal intracellular domain, confirming the importance of the PDZ binding domain for a functional interaction for these proteins in vivo. Flies heterozygous for rstCT are sensitized with regard to loss of X11Lα. Reduction of X11Lα protein levels by X11Lα-targeted RNAi strongly interfered with cell sorting on this genetic background. Both proteins are not only co-expressed during eye development, but also during maturation of a specific layer of the distal medulla neuropile, which seems to indicate the importance of this interaction during the process of synaptogenesis (Vishnu, 2006).

In mammals, three distinct X11/Mint paralogue groups exist with partially overlapping functions. It has been argued that the roles of the carboxyterminal PTB and PDZ domains are fully redundant, whereas the highly divergent N-terminal domains confer isoform-specific protein interactions. For example, the aminoterminal domain of mammalian Mint1 can bind to CASK, whereas those of Mint2 and Mint3 cannot. Conversely, both mammalian Mint1 and Mint2 bind to Munc-18, a protein essential for synaptic vesicle exocytosis (Vishnu, 2006).

Phylogenetic reconstructions based on the divergence of the PTB and PDZ domains revealed that the two Drosophila X11/Mint homologs cannot be assigned to any of the three paralogue groups defined by the vertebrate X11/Mint families but rather form a separate and distinct clade along with other known invertebrate homologs. This may reflect the extended separate evolutionary history but it may also imply specialized functions of the proteins in both clades. It appears, however, that X11/Mint-coding genes have been repeatedly and independently duplicated after the split of the deuterostomes from the protostomes, and some of the paralogues have subsequently been lost. Significantly, Drosophila melanogaster and the mosquito Anopheles appear to be the only invertebrates with two X11L copies, whereas the sister species D. pseudoobscura only seems to have a single copy that represents an orthologue of X11Lα. Available sequence information does not permit an unequivocal assignment of the two near-identical Anopheles X11L homologues. However, these analyses indicate that both correspond to the X11β paralogue group, and that they may be the result of a duplication that followed the split of the two dipteran families. Although the Drosophila members of the family display distinguishing signature sequences that allow their categorization, both paralogues are sufficiently similar in sequence pattern and genomic organization to suspect that they were also the result of a more recent duplication event (Vishnu, 2006).

In the pupal eye disc X11Lα is expressed in the cytoplasm of interommatidial precursor cells and of primary pigment cells, and it accumulates at contact sites of these cells where it colocalizes with Rst. In addition, X11Lα is strongly expressed in primary pigment cells in a belt near the outer apical membrane which contacts interommatidial cells (Vishnu, 2006).

When the level of X11Lα protein is reduced in IOPs by driving one copy of X11Lα-targeted RNAi in IOPs mild cell sorting defects can still be observed at stage P40 and the numbers of interommatidial cells is moderately increased relative to controls since some supernumerary cells are not eliminated by apoptosis. Two copies of X11Lα-targeted RNAi have an even stronger effect on cell sorting and cell survival. The single copy X11Lα-targeted RNAi genetic background was therefore considered a sensitized system to detect putative synergistic effects of removal of one copy of Rst. The rstCT/+; GMR–Gal4; X11Lα-targeted RNAi/+ pupae displayed strong sorting defects, thus supporting the proposed interaction of Rst and X11Lα at the genetic level. It has been postulated that cell sorting in the retina depends on an interaction of Rst in IOPs with a heterophilic ligand in primary pigment cells (identified as Hibris). It has been furthermore hypothesized that the strength of this selective interaction should be the driving force of cell sorting. As a result of the curent findings a modified model is now proposed. X11Lα does not seem to be required for the correct positioning of Rst in the membrane of IOPs. Its effect on cell sorting may therefore be better explained by Rst-mediated signalling following recognition of Hibris on primary pigment cells. The result of Rst-mediated signalling via X11Lα in IOPs could be to maximize their contacts to primary pigment cells. This could be achieved by stabilizing the DE-cadherin mediated cell adhesion (Vishnu, 2006).

Misexpression of full-length Rst in the eye imaginal disc by the Mz1369 driver leads to severe sorting defects as well as to occasional reduction of cone cells and primary pigment cells. The cell sorting defects are due to an interaction of the extracellular domain of ectopic Rst in cone cells with Hibris expressed by primary pigment cells. Misexpression of Rst-DD1 does not show this extracellular interaction, but the truncated molecule still seems to be active in signalling since it mediates differentiation defects, which are similar to those seen after misexpression of X11Lα. It was already reported by Hase (2002) that misexpression of X11Lα interferes with eye development. Increased levels of apoptosis were found in the eye disc of the third Instar larva. The data confirm that X11Lα misexpression severely disturbs the general regularity of the ommatidial lattice. The reduction in cone cell number is a highly penetrant feature of these eye discs. Most ommatidia contain only two or even fewer cone cells. This could explain the partial loss of primary pigment cells, since primary pigment cell formation is triggered by induction from the cone cells. The decrease in number of cone cells per ommatidium may also contribute to the deep rhabdomere phenotype. The expanded tips of cone cell processes participate in the formation of the fenestrated membrane, which contains the exit portals for the axons of retinula cells in an ommatidium. The disturbance of the portals could well play a role in the deep rhabdomere phenotype of X11Lα misexpressing flies. The phenocopy obtained with Rst-DD1 misexpression suggests that Rst is signalling via the X11Lα pathway (Vishnu, 2006).

Loss of X11Lα by RNAi knockdown methodology causes the reduction of rhabdomeres and retinula cells in adult flies. This is not due to a failure to form retinula cells, since retinula cells are specified and start differentiating during larval development. Their axons project into the brain. In addition, the initiation of rhabdomere formation can be observed as well in pupal stages. Mutants causing retinula cell degeneration at the adult stage, have compound eyes of normal depth, whereas loss of X11Lα leads to compound eyes with severely reduced thickness. It is therefore concluded that this is the consequence of cell degeneration in the late eye imaginal disc (Vishnu, 2006).

Loss of X11Lα by RNAi knockdown has much more severe consequences than any of the known Rst mutations. This could well be due to the redundancy between Rst and Kirre. However, it also cannot be ruled out that X11Lα has Rst and Kirre independent functions (Vishnu, 2006).

Is there a role for Rst-X11Lα interaction in synapse formation? Rst and X11Lα display a distinct overlapping expression pattern in a layer of the pupal distal medulla neuropil. This finding deserves special consideration, since it may indicate that both proteins and their interaction may play a specific role during synapse formation. In support of this hypothesis, it was shown at the neuromuscular junction of Drosophila, that downstream of the IgCAM Fasciclin II, X11Lα functions in bouton formation by interacting with the amyloid precursor protein, indicating a more general role for X11Lα in synaptogenesis (Vishnu, 2006).

IgCAM proteins of the Rst-family also function in the process of synapse formation. The Rst-like SYG-1 protein in C. elegans has been reported to play a role in initiating the assembly of presynaptic sites. Clustering of SYG-1 in HSNL neurons opposite the epithelial SNS-like SYG-2 signal determines the localization of the presynaptic transmitter release machinery in these neurons. So far, the chain of events leading from SYG-1 to the recruitment of presynaptic vesicles is not yet known. The Rst-X11Lα interaction in Drosophila suggests a putative mechanism. In vertebrates, X11/Mints functions in transmitter release and its role in the coupling of the vesicle cycle to cell adhesion has been convincingly shown. Mint1 knockout mice exhibit impaired GABAergic synaptic transmission. The overlap of X11Lα and Rst in the distal medulla of Drosophila in just one unique neuropil layer could well indicate a similar function here and will prompt further work (Vishnu, 2006).

In conclusion, the physical interaction between the X11Lα and Rst proteins has been demonstrated and it has been shown that they are involved in the same pathway that regulates cell sorting in the pupal retina. The involvement of this interaction in other processes requiring cell recognition is likely (Vishnu, 2006).

The Drosophila CD2AP/CIN85 orthologue Cindr regulates junctions and cytoskeleton dynamics during tissue patterning

Developing tissues require cells to undergo intricate processes to shift into appropriate niches. This requires a functional connection between adhesion-mediating events at the cell surface and a cytoskeletal reorganization to permit directed movement. A small number of proteins are proposed to link these processes. This study identifies one candidate, Cindr, the sole Drosophila melanogaster member of the CD2AP/CIN85 family (this family has been previously implicated in a variety of processes). Using Drosophila retina, it was demonstrated that Cindr links cell surface junctions (E-cadherin) and adhesion (Roughest) with multiple components of the actin cytoskeleton. Reducing cindr activity leads to defects in local cell movement and, consequently, tissue patterning and cell death. Cindr activity is required for normal localization of Drosophila E-cadherin and Roughest, and this study shows additional physical and functional links to multiple components of the actin cytoskeleton, including the actin-capping proteins capping protein alpha and capping protein beta. Together, these data demonstrate that Cindr is involved in dynamic cell rearrangement in an emerging epithelium (Johnson, 2008).

By recruiting proteins into complexes, adaptor proteins create nodes of regulation and activity. The founding member of the CD2AP/CIN85 family of adaptor proteins was initially isolated in a yeast interaction screen as a binding partner of the T cell receptor CD2, independently from the kidney (MET-1) and as a ligand for p130Cas. The homologue CIN85 was identified as a partner of the E3 ubiquitin ligase Cbl and separately as SETA and Ruk. Many roles have been ascribed to the CD2AP/CIN85 family but its function in situ remains poorly understood. This study examine the sole Drosophila melanogaster CD2AP/CIN85 orthologue, cindr (Johnson, 2008 and references therein).

The phenotype of CD2AP knockout mice is chiefly one of tissue degeneration: cardiac hypertrophy, splenic and thymic atrophy, glomerular sclerosis, and a loss of podocyte foot processes. The CD2AP/CIN85 family is primarily proposed to function in endocytosis to down-regulate receptor tyrosine kinase activity. This model arises from coimmunoprecipitation and interaction experiments that have identified a wealth of CD2AP/CIN85 interactors, colocalization studies performed in culture or tissue, and in vitro assays. CIN85 constitutively associates with endophilin and, on growth factor stimulation, complexes with Cbl to mediate receptor down-regulation. Furthermore, interactions between CD2AP/CIN85 and other trafficking proteins have been described including AP-2, Dab2, Rab4, PAK2, Scraps) at the actin-rich cleavage furrow. CD2AP activity is required for migration of rat gastric mucosal cells and polarization of the cytoskeleton during T cell receptor activation. The role and mechanism by which CD2AP/CIN85 regulates cytoskeletal dynamics within an epithelium in situ remains unclear (Johnson, 2008).

Furthermore, the CD2AP/CIN85 family has also been reported to bind the adhesion molecules E-cadherin and nephrin. Nephrin and NEPH-1 form the backbone of the slit diaphragm, a specialized junction that traverses podocyte foot processes in the mammalian kidney. Direct interactions between CD2AP, nephrin, and podocin and between CD2AP and the podocyte-specific actin-bundling protein synaptopodin are essential for slit diaphragm integrity. In addition, a protein complex containing nephrin, cadherin, p120-catenin, ZO-1, and CD2AP has been isolated from Madin-Darby canine kidney cells and mouse glomerular lysates. Collectively, these data suggest that CD2AP may have a role in anchoring junctions to the cytoskeleton or in regulating actin dynamics at this important intersection (Johnson, 2008).

The challenge remains to understand how different roles of CD2AP/CIN85 are integrated in the organism, which interactors are recruited into CD2AP/CIN85 complexes, and how these are regulated. This study shows that targeted reduction of cindr in the pupal fly eye resulted in defects in overall patterning due to aberrant local cell movements. These defects were linked to misregulation of actin dynamics and mislocalization of Drosophila E-Cadherin (DE-Cad) and the fly NEPH-1 orthologue Roughest (Rst) which, with its binding partner Hibris (Hbs; a Drosophila Nephrin orthologue), is a central mediator of cell-cell adhesion in the pupal retina. Cindr was lined functionally and physically to orthologues of capping protein alpha (Cpa) and capping protein beta (Cpb), and this study further explored the role of Cindr in modulating the actin cytoskeleton during eye maturation. The data support a primary role for cindr in linking junction and actin regulation and help account for many of the phenotypes ascribed to mutations in mammalian CD2AP/CIN85 (Johnson, 2008).

The evidence indicates that Cindr provides a functional link between dynamically regulated surface adhesion and the cytoskeletal changes required for normal pupal eye patterning. Loss of cindr activity leads to misplacement of retinal support cells, which adopt shapes uncharacteristic for their niche in the retinal field. The reasons for this became apparent when cell behavior was examined in live tissue. Reducing cindr prevents 1° cells from maintaining enwrapment of the cone cells; instead, cindr 1° cells are unable to firmly establish this niche and frequently retract, allowing neighboring interommatidial precursor cells (IPCs) to have direct contact with the cone cells. Similar instability was observed in the remaining IPCs fated to establish the 2°/3° hexagonal lattice. Histology demonstrated further changes both to AJ components and the actin cytoskeleton. Although additional roles for Cindr such as regulation of endocytosis cannot be ruled out, no evidence was observed for such a role during cell rearrangements (Johnson, 2008).

In wild-type tissue, Hbs and Rst are localized exclusively to 1°-IPC interfaces during IPC patterning; heterophilic interactions between these molecules are thought to direct the rearrangement of IPCs into single rows around each ommatidium. In cindr-IR tissue (an RNAi inverted repeat knockout), a mislocalization was observed of Rst to the entire IPC circumference. Such mislocalization would impede the generation of a preferential adhesive force, disrupting the direction or flow of cell movement and subsequent patterning. Irregularities were detected in the localization of DE-Cad around the circumference of retinal cells when Cindr was reduced. This presumably resulted in uneven or unreliable junctional stability that further destabilized the dynamic switching of cell positions. Genetic interactions with the loci for rst, hbs, and shg confirmed that these loci cooperate with cindr during patterning (Johnson, 2008).

It was also demonstrated that the actin cytoskeleton is dynamically remodeled during pupal eye patterning and that reducing cindr activity leads to a change in the details of cytoskeletal dynamics. In wild-type tissue, polymerized actin was initially detected almost exclusively at AJs of pre-1° cells and IPCs. These actin rings intensify as cells became rearranged and then, remarkably, once patterning is established, membrane-associated F-actin strongly diminishes. The functional significance of these changes is likely linked to concurrent modification of Rst- and AJ-mediated adhesion. For example, the levels of both DE-Cad and Armadillo (β-catenin) decrease between IPCs as they are rearranged, which would serve to facilitate Rst-mediated IPC movements toward 1° cells. Data indicate that the actin cytoskeleton is coordinately reinforced at AJs as adhesion is weakened. This may serve to maintain the surface integrity of the retinal cells while junctional strength decreases and the tissue is remodeled. Once patterning is achieved, the BMP receptor Thickvein then acts to restrengthen AJs; the reduction of F-actin may reflect the reduced need for a dense actin ring. Throughout this process, Cindr acts as a pivotal regulator to coordinate AJ modification and actin polymerization (Johnson, 2008).

Several data presented in this paper suggest that Cindr acts directly to regulate actin. First, the intensity and distribution of cytoplasmic Cindr puncta in retinal cells tracks that of F-actin in IPCs during development. Second, the striking dynamics of F-actin polymerization are lost, presumably helping account for their abnormal cell movements. Third, strong genetic interactions were observed between cindr-IR and multiple components of the actin regulatory machinery. Fourth, the two capping protein subunits Cpa and Cpb coimmunoprecipitated together with Cindr from Drosophila embryos. And, finally, several phenotypes are shared between tissue mutant for cindr-IR and tissue mutant for cpa or cpb: pupal eye mispatterning, gaps in the distribution of DE-Cad around the circumference of retinal cells, bristle malformation, and tissue degeneration (Johnson, 2008).

These results emphasize the important role of the actin cytoskeleton in regulating or maintaining AJ integrity. However, the data also argue that Cindr regulates the localization of transmembrane adhesion proteins at least in part independently of the cytoskeleton: reducing the genetic component of actin regulators enhances the patterning defects of cindr-IR but not the disruption to DE-Cad, and ectopic Arp66B rescues cindr-IR mispatterning but does not rescue aberrant localization of Rst. In the absence of Cindr, miscoordination of the actin machinery together with aberrant localization of junctional complexes is likely the underlying cause of tissue mispatterning during development. Similarly, deregulation of actin and junction instability is apparently cell lethal in more mature tissue, eventually leading to degeneration of mutant tissue. This may be analogous to the degeneration of mammalian podocytes that has been associated with mutations in CD2AP. How Cindr itself is regulated during development remains an open question (Johnson, 2008).

The cell adhesion molecule Roughest depends on βHeavy-spectrin during eye morphogenesis in Drosophila

Cell junctions have both structural and morphogenetic roles, and contain complex mixtures of proteins whose interdependencies are still largely unknown. Junctions are also major signaling centers that signify correct integration into a tissue, and modulate cell survival. During Drosophila eye development, the activity of the immunoglobulin cell adhesion molecule Roughest (also known as Irregular chiasm C-roughest protein) mediates interommatidial cell (IOC) reorganization, leading to an apoptotic event that refines the retinal lattice. Roughest and the cadherin-based zonula adherens (ZA) are interdependent and both are modulated by the apical polarity determinant, Crumbs. This study describes a novel relationship between the Crumbs partner βHeavy-spectrin (βH), the ZA and Roughest. Ectopic expression of the C-terminal segment 33 of βH (βH33) induces defects in retinal morphogenesis, resulting the preferential loss of IOC. This effect is associated with ZA disruption and Roughest displacement. In addition, loss-of-function karst and roughest mutations interact to cause a synergistic and catastrophic effect on retinal development. Finally, this study shows that βH coimmunoprecipitates with Roughest and that the distribution of Roughest protein is disrupted in karst mutant tissue. These results suggest that the apical spectrin membrane skeleton helps to coordinate the Cadherin-based ZA with Roughest-based morphogenesis (Lee, 2010).

Investigation of the overexpression of βH segment 33 (βH33) in the eye disc has shown that the phenotype it induces is closely associated with a morphogenetic event that is required for a normal round of apoptosis that refines the retinal lattice. This disruption is correlated with disruption of the ZA and the normal distribution of the Roughest protein, which mediates this morphogenesis. Further experiments demonstrated a strong genetic interaction between loss-of-function karst and rst alleles that appears to result in reduced adhesion between IOCs and ommatidia. A physical association between βH and the Roughest protein was demonstrated by coimmunoprecipitation, and the distribution of Roughest is significantly disrupted in karst mutant cells (Lee, 2010).

As part of a genetic study to probe the function of the βH C-terminal domain (βH33), an overexpression approach was taken to generate a dominant phenotype. It was found that the overexpression of this domain disrupts development, is associated with acridine orange accumulation and can be ameliorated by coexpression of the baculovirus p35 caspase inhibitor, revealing it to be at least in part apoptotic. This is a specific effect of the apical βH isoform, because overexpression of the C-terminus of the related basolateral β-spectrin produces no such phenotype (Lee, 2010).

βH33 contains a PH domain, which is required to induce this phenotype, indicating a requirement for lipid binding or overlapping protein binding. Sequestration of phospholipids is an obvious possible cause of the βH33-induced phenotype and in particular apoptosis. As a class, β-spectrin PH domains appear to use phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] binding as a major mechanism for membrane association. Furthermore, modulation of PtdIns(4,5)P2 levels regulates spectrin association with the Golgi membrane and secretory vesicles. This study demonstrates that the β-spectrin and βH PH domain regions have nearly identical phospholipid specificities, and that they both exhibit the distinct preference for PtdIns(4,5)P2 over PtdIns(3,4,5)P3 seen by Das (2008) for β-spectrin. The only difference is a conspicuous affinity of β-spectrin for phosphatidic acid that is not detectable with βH. This difference probably arises from a second lipid-binding site outside the PH domain, and does not readily account for the lack of phenotype during β-spectrin PH domain expression. The requirement for phospholipid binding in this case might therefore represent only a membrane anchoring mechanism for the ectopic βH33 domain. Finally, these results strongly indicate that bulk differences in phospholipid content are unlikely to be a driving force in the apical-basal polarization of βH and β-spectrin in Drosophila (Lee, 2010).

In the fly eye, a round of apoptosis during pupation eliminates excess IOCs to refine the retinal epithelium. During this period IOCs compete for contact with the primary cells and rearrange into a single row of pigment and bristle cells surrounding each ommatidium, whereas apoptosis culls their numbers to produce an almost crystalline lattice. IOC morphogenesis is mediated by Roughest, which is expressed only in the IOCs at this time where it binds to Hibris expressed on the surface of neighboring primary cells. Roughest colocalizes with the cadherin-based ZA and is both dependent upon the ZA and in turn modulates the properties of the ZA. The results of this study tighten the association between these two adhesion systems, providing a potential common mediator for their activities (Lee, 2010).

During eye development βH is recruited to the membrane by a domain in the Crumbs protein, which specifically regulates ZA stability but not polarity. As a consequence, mutants that lack wild-type βH exhibit a mild and variable disruption of the ZA, but maintain normal apicobasal polarity. This study shows that βH also has a physical association with Roughest during embryonic development; however, it is not yet possible to say whether this is via direct binding or is an indirect association via another protein, as with Crumbs. Physical association with spectrin can result in protein stabilization at the plasma membrane, and so the disruption of Roughest distribution in karst mutant discs strongly suggests that this physical association holds true in the eye. Thus, it is proposed that Crumbs-driven assembly of the apical spectrin-based membrane skeleton provides a means to coordinate the ZA and Roughest/Hibris adhesion systems. If binding of Roughest to βH mediates this coordination at the plasma membrane it must presumably occur when βH is at the ZA before or during cell movement (Lee, 2010).

Emerging data suggest a role for βH in protein trafficking: βH co-isolates with the Golgi-resident protein Lava lamp, and a dramatic reduction in the surface expression level of the apical V-type H+-ATPase in the gut is seen when βH-dependent protein recycling is disrupted in karst mutants. It is therefore possible that βH could play a similar role for Roughest in the eye. The increased levels of Roughest protein in cytoplasmic puncta in karst mutant discs are consistent with this notion. In this context, it is interesting to note that deletion of the Roughest cytoplasmic domain in the rstCT allele, which would be predicted to uncouple it from βH, also leads to elevated levels of vesicular Roughest protein. Because RstCT protein is unlikely to have any residual association with βH, it is suggested that the strong synergism of the rstCT-karst interaction arises from the simultaneous reduction of Roughest and ZA function below a threshold where adhesion is insufficient for IOC-primary cell adhesion (Lee, 2010).

βH33 and karst both disrupt Roughest distribution, but with different consequences: βH33 primarily affects IOC, whereas karst causes selective falling of ommatidia from the retina. It is speculated that the prominent accumulation of Roughest and βH at the IOC-primary cell boundary late in development is necessary to hold the ommatidia in place. Thus, simultaneous reduction in the function of both proteins selectively weakens this interface. By contrast, βH33 would appear to be having its effect earlier during morphogenesis. Given the lack of an obvious direct stimulation of any apoptotic pathway by βH33 expression, coupled with the novel association of βH with Roughest and its known role in cell survival decisions, it is suggested that the apoptotic effects of this domain are an indirect result of the disruption of apicolateral cell junctions that in turn regulate cell survival. It is speculated that the preferential loss of IOCs upon βH33 expression arises because the cell death or survival decisions that are made at this time arise from small differences in cell adhesion in amongst the IOCs that are 'predisposed' to die if a lower threshold is reached: reducing junction function at this time thus greatly, and selectively, increases the number of IOCs that die (Lee, 2010).

Conventional β-spectrins are well known to associate with Ig-CAMs of the L1 subfamily via the adapter molecule Ankyrin. Roughest belongs to a different subfamily that includes Nephrin and has a completely divergent cytoplasmic domain that lacks the conventional Ankryin binding site. Similarly, βH lacks the canonical Ankyrin binding domain and does not colocalize with Ankyrin in vivo. This raises an interesting evolutionary question as to whether a proto-spectrin was bound to a common proto-Ig-CAM and that divergence of the spectrins and Ig-CAMs was accompanied by the acquisition or loss of Ankyrin as an adaptor in the conventional or heavy β-spectrin lineage, or whether the spectrin -- Ig-CAM association came later and this is an example of convergence. Because all of these proteins appear to have emerged at around the time that multicellular animals evolved, this cannot be readily answered at present. Moreover, the separation of conventional and heavy β-spectrins occurred during a period of dynamic concerted evolution that might have erased the evidence within the spectrins themselves. It will be interesting to see if the βH-Ig-CAM association extends to other members of this subfamily such as Hibris, the Roughest ligand (Lee, 2010).



In situ hybridizations to embryonic whole mounts reveal that embryonic RST mRNA expression is temporally and spatially regulated, that is, in late embryonic stage 11, hybridization signals are detected in lateral mesodermal clusters, in cells of the nervous system, in cell clusters in the head (mandibular, maxillary and labial buds, and in the clypeolabrum) (Ramos, 1993).

Larval and Pupal

In situ hybridizations to first, second, and third instar larvae show only weak expression. However, in late third instar larvae strong signals in wild-type imaginal discs and in the outer optic anlagen can be observed. Expression in the eye imaginal disc starts just in front of the morphogenetic furrow. The central brain and ventral ganglia are only weakly labeled. In the pupal central brain (36 hr after puparium formation) no transcripts are detected, whereas the intensity of expression is very high and stays so until approximately 72 hours in the lamina and in subpopulations of medullar cells. At 36 hours strong expression in the retina is restricted to cells between the ommatidial clusters (i.e., to presumptive seconday and tertiary pigment cells and to cells of the bristle complex. Gene expression is then gradually down-regulated in the retina and no more transcripts can be detected 72 hours after puparium formation (Ramos, 1993).

Postembryonic expression of the Rst protein starts with the differentiation of the imaginal sensory organs and the outgrowth of sensory axons in the third larval instar. It then appears in subsets of visual fibers that form the neuropils of lamina, medulla and lobula and in imaginal discs of the wing, haltere, leg, eye and antenna. In cortex areas, cell bodies show localization of the protein in vesicular bodies; no membranes of cell bodies or cell body fibers are stained. Rst protein is present on young fibers of both optic chiasms and down-regulated on older fibers. Two distinct Rst postive layers lying proximally and distally (referring to the adult position of the medulla) are recognized in the older parts of the larval medulla neuropil. The distal medulla layer shows clear columnar organization. The staining is due to long visual fibers as well as ingrowing lamina monopolar axons, as judged by the vesicular immunostaining of the lamina cortex (and mRNA in situ data). The proximal layer is thinner at this developmental stage and homogenously structured. Immunoreactivity in the proximal medulla develops independent of retinal innervation, whereas the presence of immunoreactivity in the distal medulla is dependent on and proportional to retinal innervation. Both Rst positive layers are well separated in the oldest, basal part of the developing medulla (corresponding to the anterior medulla of the adult), but they are confluent in newly developing areas. Rst expression appears to shift from pre- to post-synaptic sites in the lamina. A similar phenomenon might explain the changes taking place at the level of the medulla. Down regulation of protein begins at about 56% of pupal development, first in the two outer medulla layers. Rst expression in the optic lobe decreases below detection threshhold by 74% of pupal development (Schneider, 1995).

To investigate a possible involvement of synaptic machinery in Drosophila visual system development, a study was made of the effects of a loss of function of neuronal synaptobrevin, a protein required for synaptic vesicle release. Expression of tetanus toxin light chain (which cleaves neuronal synaptobrevin) and genetic mosaics was used to analyze neuropil pattern formation and levels of selected neural adhesion molecules in the optic lobe. Targeted toxin expression in the developing optic lobe results in disturbances of the columnar organization of visual neuropils and of photoreceptor terminal morphology. Roughest (Rst) immunoreactivity in neuropils is increased after widespread expression of toxin. In photoreceptors, targeted toxin expression results in increased Fasciclin II and Chaoptin but not Rst immunoreactivity. Axonal pathfinding and programmed cell death are not affected. In genetic mosaics, patches of photoreceptors that lack neuronal synaptobrevin exhibit the same phenotypes observed after photoreceptor-specific toxin expression. These results demonstrate the requirement of neuronal synaptobrevin for regulation of cell adhesion molecules and development of the fine structure of the optic lobe. The finding that Rst immunoreactivity remains unaltered in photoreceptors without functional n-syb but that it is increased in proximal neuropils after widespread TeTxLC expression can be interpreted in two different ways: either Rst protein is not present on photoreceptor terminals at the addressed time of pupation, or the n-syb-dependent CAM downregulation mechanism has a different molecular specificity in photoreceptors than in other optic lobe cells. During axonal pathfinding Rst is expressed on photoreceptors (Schneider, 1995 and Reiter, 1996). In pupal stages Rst is shown to be localized on rhabdomeres but not on axons and cell bodies of photoreceptors during the second half of pupation. Because rhabdomeres are unique to this cell type and seem to be a preferred localization for Rst in photoreceptors, a cell-specific distribution that excludes terminals appears more likely than a specific CAM regulation mechanism for photoreceptors (Hiesinger, 1999).


The regular, reiterated cellular pattern of the Drosophila compound eye makes it a sensitive amplifier of defects in cell death. Quantitative and histological methods reveal a phase of cell death between 35 and 50 h of development that removes between two and three surplus cells per ommatidium. The timing of this epoch is consistent with cell death as the last fate to be specified in the progressive sequence of cell fates that build the ommatidium. An ultrastructural survey of cell death suggests dying cells in the fly eye have similarities as well as differences with standard descriptions of programmed cell death. A failure of cell death to remove surplus cells disorganizes the retinal lattice. A screen of rough eye mutants identifies two genes, roughest and echinus, required for the normal elimination of cells from the retinal epithelium. The use of an enhancer trap as a cell lineage marker shows that the cone cells, like other retinal cells, are not clonally related to one another or to their neighbors (Wolff, 1991).

Irregular chiasm C (irreC) is an X-linked genetic function necessary for the correct projection of visual fibers in the optic chiasms of Drosophila optic ganglia. In addition to a severe disorganization of the inner optic chiasm, irreC mutants display a subtle phenotype in the outer optic chiasm, in which some bundles of axons that leave the posterior equatorial part of the lamina on their way to the anterior medulla take a long detour before eventually finding their specific targets in the medulla neuropile. Deletion and recombination mapping of two irreC alleles (one P-element induced, the other associated with an inversion) have yielded a precise cytogenetic location in 3C4-5. A complex complementation pattern between roughest (rst) and irreC alleles indicates that both genetic functions are structurally and/or functionally closely interrelated. Flies in which the irreC locus is completely deleted by overlapping deficiencies are viable and their defects in the optic chiasms are similar to those seen in the two alleles. The defects in the outer and inner optic chiasms are not epigenetically connected and mosaic analyses have shown them to be independent from the genotype of the compound eye. Although the larval visual nerve looks normal, in the optic lobes of irreC mutants a group of early differentiating larval neurons is misplaced, suggesting a pioneering function of these cells during organization of the outer optic chiasms (Boschert, 1990).

The 104 kDa irreC-rst protein, a member of the immunoglobulin superfamily, mediates homophilic adhesion in cell cultures. In larval optic chiasms, the protein is found on recently formed axon bundles, not on older ones. In developing visual neuropils, it is present in all columnar domains of specific layers. The number of irreC-rst-positive neuropil stratifications increases until the midpupal stage. Immunoreactivity fades thereafter. The functional importance of the restricted expression pattern is demonstrated by the severe projection errors of axons in the first and second optic chiasms in loss of function mutants and in transformants that express the irreC-rst protein globally. A phenotype visible in rst pupae is related to target/address selection. Terminal specializations are occasionally formed in the wrong neuropil layer. The penetrance of this phenotype per visual column is low. It is observed in a few columns of about 27% of mutant pupae at 20% of pupal development. Such termination errors have neve been seen in wild type. Their low penetrance suggests the existence of regulatory mechanisms during target/address selection that can compensate for quantitative alterations of a single adhesion molecules. Fibers exiting the posterior lamina are normally the first that project through the outer optic chiasm. They project erroneously in rst mutants, whereas the more anterior axon bundles take the normal route. Posterior lamina and long retinal fibers are neighbors of the Fasciclin II-positive cell body fibers of C&T cells (C&T refers to a posterior lamina layer containing C2, C3, T2 and T3 cell bodies). In the absence of the Rst protein, the retinal fibers project like C&T cell fibers into the inner optic chiasm along the inner face of the proximal medulla. From there, lamina and long retinal fibers traverse the medulla neuropil to approach their normal target region. Epigenesis of the phenotypes can be explained partially on the bases of homophilic irreC-rst interactions (Schneider, 1995).

The subcellular localization of Rst protein is altered in the rstCT mutant. The rstCT mutation truncates the intracellular domain of the Rst protein and causes a severe eye phenotype, but no axonal pathfinding defects in the optic lobe. This suggests a specific function of the intracellular domain in apoptosis during retinal development. Immunostaining of rstCT retinae reveals a drastic alteration of subcellular protein localization. No homogenous staining of the apical membranes is found; instead, the protein collects in small patches along those membrane domains where it accumulates in the wild type. Conspicuous vesicular bodies are found in the cytoplasm. Such immunoreactive vesicles are also present in smaller numbers in wild-type. Protein distribution in the optic lobe is indistinguishable from the wild type. Sequestering of the mutant rstCT protein in vesicular bodies occurs in all other epithelial tissues of imaginal discs expressing Rst (antennal, wing, haltere and leg discs). This indicates that the rstCT mutation selectively affects Rst protein localization in epithelial but not in neural tissue. These vesicles are probably the result of endocytosis from the membrane and not stages of synthesis and transport to the membrane. There is no indication from mRNA in situ hybridization or Western blot studies that expression in rstCT is regionally increased and so it is not obvious why more vesicles are observed in the mutant if they represent stages of synthesis. Also, the large size of these vesicles, easily detectable by light microscopy, is atypical of Golgi vesicles in non-secretory cells (Reiter, 1996).

Scutoid is a classical dominant gain-of-function mutation of Drosophila, causing a loss of bristles and roughening of the compound eye. Previous genetic and molecular analyses have shown that Scutoid is associated with a chromosomal transposition resulting in a fusion of no ocelli (located at 35A4), a Zn finger protein involved in the development of the embryonic brain and the adult ocellar structures, and snail (located at 35D2) genes. How this gene fusion event leads to the defects in neurogenesis has not been known until now. snail has been found to be ectopically expressed in the eye-antennal and wing imaginal discs in Scutoid larvae, and this expression is reduced in Scutoid revertants. The expressivity of Scutoid is enhanced by zeste mutations. snail and escargot encode evolutionarily conserved zinc-finger proteins involved in the development of mesoderm and limbs. Snail and Escargot proteins share a common target DNA sequence with the basic helix-loop-helix (bHLH) type proneural gene products. When expressed in the developing external sense organ precursors of the thorax and the eye, these proteins cause a loss of mechanosensory bristles in the thorax and perturbed the development of the compound eye. Such phenotypes resemble those associated with Scutoid. Furthermore, the effect of ectopic Escargot on bristle development is antagonized by coexpression of the bHLH gene asense. Thus, these results suggest that the Scutoid phenotype is due to an ectopic snail expression under the control of no ocelii enhancer, antagonizing neurogenesis through its inhibitory interaction with bHLH proteins (Fuse, 1999).

Prior studies have shown that the Sco phenotype is caused by the fusion of a chromosome fragment containing a part of noc and Adh genes placed with the region approx. 16 kb upstream of sna. Analyses of Sco revertants have demonstrated that both sna and noc portions of the fusion are necessary to cause the phenotype. It has been proposed that Sco is an “antimorphic” allele of noc, based on the sensitivity of the Sco phenotype to the copy number of the noc gene. One model suggests that the noc-sna fusion on the Sco chromosome produces a fusion gene product that antagonizes the wild-type noc gene product. However, molecular analyses of the noc locus have led to questions about the model. noc is divided into physically separable units [l(2)35Ba, nocA, and nocB] based on the mapping of aberrations affecting noc functions. Mutations in l(2)35Ba lead to embryonic- larval lethality with defects in the optic lobe. nocA and nocB mutations cause a loss of ocelli and their associated bristles, with mutations in the former region having a stronger effect. l(2)35Ba encodes the No ocelli protein that contains a zinc-finger motif and requires nocA function for its expression. Since no transcript derived from the nocA region was identified, it is very likely that the l(2)35Ba-encoded protein carries out noc function, and that nocA is a cis-regulatory region of l(2)35Ba. Similar phenotypes of nocA and nocB suggest that nocB is also another regulatory region of l(2)35Ba, although more molecular analysis is required before a final conclusion can be drawn. The Sco transposition breaks between nocA and nocB, and places nocB next to sna, leaving l(2)35Ba and nocA in their original position far apart from the noc-sna fusion point. This makes the presence of an antimorphic sna-noc fusion gene product impossible. Given the current results showing that Sco is associated with ectopic sna expression, and that misexpression of wild-type sna mimics Sco phenotype, it is proposed that Sco is a gain-of-function, neomorphic mutation of sna (Fuse, 1999).

The Sco chromosome pairs tightly with the wild-type homolog in the polytene chromosome, suggesting that Sco and noc are placed in physical proximity. zeste is a mediator of transvection, a proximity-dependent, sometimes interchromosomal, interaction between enhancers. In this scenario sna expression from the Sco chromosome is normally down-regulated by the wild-type copy of the noc enhancer, and zeste mutations disrupt this trans-repression to enhance the phenotype. A mutation in noc would also interfere with this trans-repression. Such a copy number dependent repression of transcription has been shown for pairing dependent repression, in which the reporter gene white linked to a fragment containing a polycomb response element is repressed when two copies of the gene are placed in proximity (Fuse, 1999).

Irregular chiasmC-roughest (IrreC-rst), an immunoglobulin (Ig) superfamily member, plays a role in patterning sense organs on the Drosophila antenna. IrreC-rst protein is initially expressed homogeneously on apical profiles of ectodermal cells in regions of the antennal disc. During specification of founder cells (FCs), the intracellular protein distribution changes and becomes concentrated in regions where specific intercellular contacts presumably occur. Loss of function mutations as well as misexpression of irreC-rst results in an altered arrangement of FCs within the disc have been compared to wildtype. Sense organ development occurs normally, although spacing is affected. Unlike its role in interommatidial spacing, irreC-rst does not affect apoptosis during antennal development. It is proposde that IrreC-rst affects the spatial relationship between sensory and ectodermal cells during FC delamination (Reddy, 1999).

The effect of IrreC-rst on spacing of FCs can be interpreted in two ways:
(1) The general mechanisms of lateral inhibition during specification of sense organ precursors from the antennal disc epithelium are not sufficient on their own to define the correct position of FC, although they do specify its fate. Additional factors, such as IrreC-rst, are necessary to guide the correct choice of FCs within proneural domains. This implies that irreC-rst function in some way interacts with genes involved in lateral signaling. Loss or gain in function results in a lack of elevated expression in specific intercellular contacts resulting in the 'wrong' cell in the proneural domain being selected to form the FC. A similar phenotype is observed in mutants in the sca gene where loss of function results in an altered spacing of FCs. Sca is a secreted protein with similarity to the fibrinogens; it is expressed in proneural domains and acts together with Notch in the selection of SOPs as well as the founding photoreceptor R8. No synergistic effect between sca and irreC-rst could be demonstrated in mutants.
(2) Sense organ precursors are specified normally in the absence of irreC-rst function. In the wildtype, specification of FCs results in a sequestration of IrreC-rst to regions on apical membranes of cells that abut the FC. The cell lying within the strongly stained arcs (presumably the FC) did not show IrreC-rst immunoreactivity in its cytoplasm. This implies that the interaction between the FC and its neighbors is through a heterophilic adhesion with IrreC-rst. This intercellular adhesion stabilizes the position of the FC during delamination and assures the presence of epidermal cells between one precursor and the next. In cases in which IrreC-rst levels are altered, this intercellular contact fails to occur, and the spatial relationship between the FC and the epidermal cells is lost, leading to defects in spacing (Reddy, 1999).

The view is favored that IrreC-rst affects FC position on the disc ectoderm rather than FC choice. It is unclear whether the specification of FCs are causal to the re-distribution of IrreC-rst onto specific cellular contacts, or whether FC selection is epistatic to irreC-rst function. In developing eye discs IrreC-rst expression has been shown to play a major role in the rearrangement of cell contacts. While initially several rows of interommatidial cells may separate neighbouring ommatidia, rearrangement of cell contacts leads to a single row of inter-ommatidial cells. This process and subsequent cell death of supernumerary inter-ommatidial cells shapes the compound eye and gives it its quasi-crystal-line structure. IrreC-rst is required for the apparent preference of inter-ommatidial cells to contact primary pigment cells, which form the outer boundaries of the ommatidium. While the IrreC-rst protein is produced in inter-ommatidial cells, it preferentially accumulates at contact sites with primary pigment cells. This has been interpreted as evidence for a heterophilic ligand in the latter. Thus in the eye imaginal disc, at least during final stages of eye development, IrreC-rst seems to provide a driving force for the rearrangement of cell contacts. Specifically, it transfers adhesiveness of inter-ommatidial cells to primary pigment cells, thereby assuring that ommatidia are always separated by inter-ommatidial cells. In the absence of IrreC-rst or in case of its overall misexpression, improper sorting of cells occurs and apoptosis cannot take place, leading to defects in retinal pattern. There are obvious parallels between IrreC-rst expression and function in the eye and antennal discs. FCs seem to behave as primary pigment cells: IrreC-rst accumulates at their borders. In the absence of normally regulated IrreC-rst expression ectodermal cells fail to reliably separate FCs and clustering of sense organs occurs. This indicates that similar mechanisms are involved, as is the case during eye development (Reddy, 1999 and references therein).

Programmed cell death (PCD) in the Drosophila retina requires activity of the irregular roughest gene. Loss-of-function mutations in rst block PCD during retinal development and lead to a rough eye phenotype in the adult. To identify genes that interact with rst and may be involved in PCD, a genetic screen was conducted for dominant enhancers and suppressors of the adult rough eye phenotype. 150,000 mutagenized flies were screened and 170 dominant modifiers were recovered that localize primarily to the second and third chromosomes. At least two allelic groups correspond to previously identified death regulators, Delta and Ras1. Examination of retinae from homozygous viable mutants indicates two major phenotypic classes. One class exhibits pleiotropic defects while the other class exhibits defects specific to the cell population that normally undergoes PCD (Tanenbaum, 2000).

Mutant lines with pleiotropic effects exhibit an aberrant number of cone cells. Alteration in the number of cone cells is often an indicator of earlier defects: for example, abnormal photoreceptor differentiation can lead to subsequent abnormal cone cell recruitment. Many mutant lines contain a variable number of cone cells within each ommatidium, often five and sometimes three; these were in addition to ectopic 2°/3° cells. Cone cells provide a signal that rescues 2°/3° precursors from death, and the additional 2°/3° cells may reflect the additional cones. Surprisingly, mutant lines with a consistently reduced number of cone cells, typically one to three, that also contained ectopic 2°/3°s were also identified. These genes are good candidates to regulate both the cone cell fate and, independently, the 2°/3° vs. PCD fate decision. Interestingly, some mutant lines exhibit defects primarily in cell arrangement. This supports the idea that decisions about cell placement and cell death may be related during Drosophila retinal development (Tanenbaum, 2000).

Perhaps of greatest interest are the lines exhibiting defects specific to the interommatidial lattice. Ommatidia from these lines often contain an additional cell, the cone-contact cell, positioned between their two 1°s. Cone-contact cells have been observed also in retinae overexpressing the caspase inhibitor p35. This observation suggests that a block in cell death alone can cause this phenotype and that the phenotype is not the result of an earlier defect, e.g., in cone cell or 1° development. In general, the mutant phenotypes in the lattice-specific class are weak. This observation is not surprising given that the mutations analyzed were all homozygous viable and thus may represent weak alleles. It is likely that stronger alleles of the same genes may be associated with lethality, particularly if they are involved in PCD in other tissues and developmental stages (Tanenbaum, 2000).

Requirement of the roughest gene for differentiation and time of death of interommatidial cells during pupal stages of Drosophila compound eye development

The roughest locus encodes a transmembrane protein of the immunoglobulin superfamily required for several developmental processes, including axonal pathfinding in the developing optic lobe, mechanosensory bristle differentiation and myogenesis. In the compound eye, rst is required for establishing the correct number and spacing of secondary and tertiary pigment cells during the final steps of ommatidial assembly. Its function in the developing pupal retina was further investigated by performing a developmental and molecular analysis of a novel dominant rst allele, rstD. In addition to showing evidence that rstD is a regulatory mutant, the results strongly suggest a previously unnoticed role of the rst gene in the differentiation of secondary/tertiary pigment cell fate as well as establishing the correct timing of surplus cell removal by programmed cell death in the compound eye (Araujoa, 2003).

Most of the steps leading to the emergence of the adult ommatidial pattern occur in the first third of pupal life and are associated with three clearly discernible morphological events: (1) the reorganization of cell contacts between undifferentiated interommatidial cells (IOCs) and the two already recruited primary pigment cells (pc1); (2) a wave of programmed cell death, which removes 2.5 cells, on average, per ommatidial cluster, and (3) the positioning and initial differentiation of secondary and tertiary pigment cells (pc2/3). Since secondary and tertiary pigment cell specification is critically dependent on the correct unfolding of this sequence of events, the establishment of the exact causal relationship between them is essential for a precise understanding of the mechanisms controlling pattern formation in the compound eye (Araujoa, 2003).

The wild type product of the rst locus is required for cell death in the pupal retina, although not necessarily by directly controlling apoptosis. In fact, evidence suggests that the primary function of Rst in the pupal retina would be to direct the cell sorting events that organize IOC around ommatidia, without which no cell death could occur. However, recent data indicate that cell sorting does not appear to be an absolute requirement for triggering interommatidial cell death, since local laser ablation of cone cells and pc1 can induce massive interommatidial apoptosis without prior cell sorting. Those aspects of rst function have been further investigated by analyzing the developmental and molecular bases of the phenotype of a novel dominant allele in which a temporal shift in the expression of Rst protein is observed. In addition to showing that a delay in Rst expression can influence the onset of the apoptotic cell wave in the pupal retina, the results suggest that the correct temporal expression of rst might be needed for the implementation of the pc2/3 cell fate specification program or, at least, for the full differentiation of these cell types (Araujoa, 2003).

Although some spatial differences in Rst protein distribution between wild type and rstD seem to be present at very initial stages of eye disc morphogenesis, these are not, most likely, directly responsible for phenotypes seem in adult mutants, since both the number and organization of photoreceptors and cone cells appear normal. The most conspicuous feature observed in the ommatidial development of rstD mutants is a delay of 10%-12% p.d. in the onset of key morphological events, such as cell sorting and cell death, immediately preceding, and probably leading to, cell fate specification of IOC. These events depend on rst function during the first third of pupal development and for their correct inception and their delay in rstD correlates well with the time shift towards later stages observed in the dynamics of Rst protein expression pattern. It is important to emphasize that this delay is not a consequence of a general slow down in the overall development of these mutants, since the timing of other key developmental events in the pupa, such as head eversion and eclosion, is well within the range observed in wild type stocks. Besides, Rst expression in cone cells and in bristle precursors seems to follow a temporal course similar to wild type. It was found, however, that about 30%-35% of rstD pupae have persistent salivary glands more than 24 h after puparium formation. This phenotype is absent in rstD revertants but can be mimicked by overexpressing the extracellular portion of the Rst protein during the first 20 h of pupal development. These results, although preliminary, show some parallel to the data presented here and point towards a role of rst in the establishment of a 'dying competence' in the salivary gland, as well as in the pupal retina (Araujoa, 2003).

Despite the time shift, Rst immunoreactivity is present both in wild type and mutant IOC during most of the critical developmental period mentioned above, although with very different membrane distributions. For instance, whereas in wild type Rst immunoreactivity is already restricted to IOC/pc1 borders by 32% pupal development (p.d.) the same situation does not occur in rstD retinae before 43% p.d. This observation indicates that it is not the presence or absence of the Rst protein, but rather its different subcellular distribution at a given time point that seems to be critical for the mutant phenotype. The importance of Rst correct subcellular localization for its function in eye development has already been shown several times: it is altered in rstCT, for example, a mutant allele of rst having a severe rough eye phenotype and in which the intracellular domain of the Rst protein is truncated. Also, mutations affecting some components of the Notch pathway can interact phenotypically with rst loss-of-function alleles and this leads to an altered subcellular localization of Rst in pc2/3. However, the data presented here additionally show that the time when these changes in membrane distribution occur is equally important, since the same general expression dynamics of Rst can lead to a normal eye or to a severely disorganized one, depending simply on when it takes place (Araujoa, 2003).

The conclusion that pc2/3 do not differentiate properly in rstD mutants is based on three independent lines of evidence: (1) pigment production is abnormal in mutant pigment cells; (2) the basal actin cytoskeleton of pc2/3 as visualized by Rhodamine phalloidin is clearly disrupted, and (3) failure to express the pc2/3 and bristle marker BA12. These differentiation defects are only mildly improved when the elimination of surplus ommatidial cell is perturbed by the expression of the antiapoptotic baculoviral protein P35 and thus appear to be largely unrelated to programmed cell death. However, it has been recently shown that P35 does not block Dronc dependent apotosis, and therefore it cannot be completely excluded that some of the aforementioned defects could be due, at least partially, to the activation of the cell death pathway in IOC (Araujoa, 2003).

Differentiation of essentially all cell types in the developing retina depends on the Ras signaling pathway, through the activation of the Drosophila Egf-receptor by Spitz (and, in photoreceptor R7, also by Boss/Sevenless-kinase interactions) or its inhibition by Argos. According to this model, the reiterated activation of the Egfr/Ras-pathway in an increasingly larger ensemble of cells throughout retinal development results in the sequential recruitment of cells to specific cell fates and subsequent differentiation. In the case of pigment cells, a further refinement suggests that in the final stages of ommatidial pattern formation the differentiative signal transduced by the Egfr/Ras pathway is antagonized by the Notch pathway: this is necessary for programmed cell death to occur in the interommatidial lattice. It is important to note however that although the activation of the Egfr/Ras signaling pathway is necessary for differentiation, it does not directly specify cell fate. This choice seems to be determined primarily by the specific developmental time when this signaling pathway is activated in a given cell, with the implication that all cells in the retina pass through a sequence of 'competence states' as development proceeds, each defined by a unique combination of transcription factors. Further support for such a model of cell fate specification has been recently found, and its generality extended to other tissues (Araujoa, 2003).

Within this context, and given the importance of the correct temporal and spatial subcellular localization of Rst for its function, it is tempting to speculate that the initial, more or less homogeneous membrane distribution of the Rst protein in uncommitted interommatidial cells could be important to prevent them from responding prematurely to the general differentiation signal triggered by the activation of the Egfr/Ras pathway, thereby allowing a choice between the pc2/3 differentiation and cell death programs to be possible. This hypothesis is consistent with the observation that retinae lacking a fully functional Rst protein not only have extra cells due to the absence of programmed cell death but all these 'spared' cells are able to differentiate as pigment cells. The abnormalities in pc2/3 differentiation observed in rstD could then be explained if, because of the temporal shift in Rst expression, its redistribution to pc1/IOC membrane borders happens after the cells have lost their competence to respond to the differentiation signal or when the signal itself is not present anymore. Whether this proposed 'insulating' effect of Rst is a permissive one, caused simply by differences in adhesive properties of cells with different subcellular distributions of Rst or is a consequence of a direct interaction with the Egfr/ras transduction signal cascade is currently being investigated (Araujoa, 2003).

In this study a DNA rearrangement in rstD has been indentified, most likely an insertion, located about 18 kb upstream of the putative rst transcription initiation site, suggesting that the developmental abnormalities seen in this mutant are primarily due to defects in transcriptional regulation of the rst gene. The observed spatial and temporal differences of Rst expression in rstD are consistent with this assertion, but other interpretations are certainly possible. It cannot completely be excluded at this point that some of the phenotypic characteristics of rstD are not due to an interference with the expression pattern of neighboring genes. The regulatory regions of kirre, a rst paralog that seems to act redundantly with kirre to allow myoblast fusion during embryogenesis, is located not farther than 120 kb proximally to rst, on 3C6, and the two genes are transcribed from opposite strands, with their 5' ends toward one another. It is therefore conceivable, although unlikely, that kirre expression pattern could be affected by the rstD rearrangement and, therefore, be responsible for at least some aspects of the rstD phenotype. Also, alternative explanations based on RNA stability or translation efficiency are possible, since resolution of this analysis cannot exclude the existence of additional mutations affecting the rst coding region. However, the apparent absence of structural differences between the Rst protein in wild type and in rstD as well as other phenotypic characteristics of the mutant not examined here such as high reversion rate, make these possibilities not very likely either, thus raising the question of how the highly dynamic temporal changes in membrane localization of Rst seen during the final steps of ommatidial patterning could be directly influenced by a regulatory mutation. A simple explanation can be provided assuming that Rst molecules present at the pc1/IOC borders are more stable than for those localized at the IOC/IOC borders. This stability could be a consequence of the postulated heterophilic interaction between Rst and a ligand present in the pc1 membrane. Although speculative, such a model can satisfactorily reconcile the molecular nature of the rstD mutation with the main phenotypic features of the mutants carrying it, while providing a framework for future investigations on the role of the rst locus in the final steps of ommatidial pattern formation in Drosophila (Araujoa, 2003).

Function of roughest and its paralog kirre during muscle development

The polynucleate myotubes of vertebrates and invertebrates form by fusion of myoblasts. Drosophila Roughest (Rst) protein is a new membrane-spanning component in this process. Rst is strongly expressed in mesodermal tissues during embryogenesis, but rst null mutants display only subtle embryonic phenotypes. Evidence is presented that this is due to functional redundancy between Rst and its paralogue Kirre/Dumbfounded). Both are highly related single-pass transmembrane proteins with five extracellular immunoglobulin domains and three conserved motifs in the intracellular domain. The expression patterns of kirre and rst overlap during embryonic development in muscle founder cells. Simultaneous deletion of both genes causes an almost complete failure of fusion between muscle founder cells and fusion-competent myoblasts. This defect can be rescued by one copy of either gene. Moreover, Rst, like Kirre, is a myoblast attractant (Strünkelnberg, 2001).

The kirre locus maps cytogenetically to region 3C6 and lies 3 kb distal to Notch. The rst and kirre loci are separated by 127 kb and are transcribed from opposite strands with their 5' flanking regions towards each other. The kirre cDNA consists of 3295 residues and contains a single long open reading frame encoding a protein of 959 amino acids. A signal peptide sequence (amino acids 7-31) has been identified and one putative transmembrane region (amino acids 575-597) (Strünkelnberg, 2001).

The conceptual Kirre sequence shows an overall similarity of 45% to Rst. Like Rst, the predicted extracellular portion of the Kirre protein displays an array of five immunoglobulin (Ig) domains. Stretches of high conservation with Rst reside primarily in the region of the five Ig domains. Within these domains, the degree of conservation successively decreases from the N terminus to the transmembrane domain. Both proteins contain stretches of amino acids with short side chains at differing positions: Rst contains a stretch of glycines between the second and third immunoglobulin-domain and Kirre harbours an array of 18 serines interrupted by a single glycine residue at the N terminus (Strünkelnberg, 2001).

The intracellular domain of Kirre is considerably longer than that of Rst and displays only low overall homology with the intracellular domain of Rst. However, three highly conserved motifs have been detected: one is located close to the transmembrane domain consisting of the sequence PADVI. The second motif, R[Y/F]SAIYGNPYLR(S)[S/T]NSSLLPP, corresponds to the consensus sequence of autophosphorylation domains of receptor tyrosine kinases. The third motif, T[A/H]V, resides at the C terminus of both sequences and corresponds to the consensus sequence of the PDZ-binding motif ([T/S]XV). In addition to the site contained in the putative autophosphorylation domain, one putative tyrosine and one putative serine phosphorylation site are conserved between Rst and Kirre. A conspicuous difference between the Kirre and Rst proteins is the lack of the opa-like repeat of Rst in Kirre (Strünkelnberg, 2001).

Similarity searches using the BLAST algorithm have shown that the four N-terminal Ig domains of Kirre, Rst, Sticks and stones (Sns; Sticks and stones) and Hibris are closely related. Sns and Hibris (Bour, 2000) have been shown to be involved in muscle development (Strünkelnberg, 2001).

Expression of rst mRNA can be detected in embryonic stages 4 to 14. During stage 12, the rst transcript is detected in the majority of mesodermal cells. During stages 13 to 14 mesodermal expression of rst is detected close to the epidermis at positions where muscle founder cells reside, as well as immediately interior of the founder cells where fusion-competent myoblasts can be found. Unlike for kirre, individual muscle precursors could not be detected based on rst labelling (Strünkelnberg, 2001).

In comparison with rst, the expression of kirre is more restricted and switched on later during development. The kirre mRNA is detected from stage 11 through to stage 16. During stages 12-13, the kirre probe labels segmental clusters of mesodermal cells close to the epidermis. Based on position and morphology, this suggests that kirre is expressed in muscle founder cells. During stages 13 to 14, kirre labelled outgrowing founder cells and muscle precursors (Strünkelnberg, 2001).

A monoclonal antibody against Rst was used to address protein expression in more detail. To determine the myogenic cell types expressing Rst, the muscle founder cell-specific enhancer trap line rP298-lacZ was used. During embryonic stages 13 to 14, all cells expressing ß-galactosidase also showed Rst staining in their periphery, indicating that Rst is expressed by muscle founder cells. As predicted by in situ hybridization, Rst was also detected in mesodermal cells that did not express ß-galactosidase. Morphology and position of these cells suggest that they are fusion-competent myoblasts. The localization of Rst within the membranes of myogenic cells is restricted to discrete spots (Strünkelnberg, 2001).

In rP298-lacZ embryos, fusion-competent myoblasts that have started to fuse with founder cells begin to express ß-galactosidase. This complicates the distinction between the two cell types. To determine whether Rst is expressed in isolated founder cells, rP298-lacZ was crossed into a mbcC1 genetic background. In mbcC1 embryos, myoblast fusion is almost completely blocked and by stage 16 these embryos display a pattern of isolated, globular, fusion-competent myoblasts and stretched out, fibrous muscle founder cells. By stages 13 to 14, antibody staining for ß-galactosidase and Rst reveals a pattern comparable with staining in a wild-type background. However, during stages 15 to 16, Rst expression on fusion-competent myoblasts almost completely disappears, while labelling is pronounced on the cytoplasmic extensions of founder cells. Moreover, since rP298lacZ mirrors kirre expression, it follows that the expression patterns of rst and kirre overlap (Strünkelnberg, 2001).

Muscles attach at specific sites in the epidermis, the apodemes. Rst is also expressed in the apodemes, as shown by immunodetection of Rst in embryos of the apodeme-specific lacZ-reporter Wß1HI-lacZ (Strünkelnberg, 2001).

The deficiency Df(1)w67k30 causes embryonic lethality and displays an almost complete lack of myoblast fusion. The genomic interval removed by Df(1)w67k30 extends from white to kirre. As yet, there is no single embryonic lethal locus known within this region. Hence, the Df(1)w67k30 phenotype could be caused by the removal of two or several loci. Kirre has been shown to be a myoblast attractant expressed on founder cells and reintroduction of kirre can partially rescue the Df(1)w67k30 phenotype. Therefore, removal of kirre is partly responsible for the Df(1)w67k30 phenotype. However embryos deficient for a smaller genomic region including kirre do not show a defect in myoblast fusion. Therefore, removal of kirre alone cannot be responsible for the Df(1)w67k30 phenotype. Since the situation for rst is similar -- rst is involved in but not essential for myoblast fusion -- it is concluded that the phenotype of Df(1)w67k30 is caused by the simultaneous removal of the rst and kirre loci (Strünkelnberg, 2001).

Although the rst gene is not essential for muscle fusion, small defects, such as thinner and missing muscles can be detected in rst6 and rstirreC1 individuals, indicating the involvement of rst in muscle development. Overexpression of a secretable, extracellular version of Rst during stages when myoblast fusion occurs (stages 12-15) leads to embryonic lethality and defects in myoblast fusion. Mechanistically, the extracellular part of the protein may compete with endogenous Rst for an as yet unknown extracellular ligand or, since the Rst protein has been shown to mediate homophilic cell adhesion, the extracellular domain could also bind to endogenous Rst and thereby disturb its function (Strünkelnberg, 2001).

Ubiquitous overexpression of the full-length Rst protein also causes embryonic lethality and a severe muscle fusion phenotype. Ectodermal overexpression of Rst does not cause defects in the muscle pattern but ectopic localization and prolonged occurrence of myoblasts at sites of ectopic Rst expression. Mesodermal expression does not induce any detectable phenotype. The reason why global misexpression of Rst differs from misexpression in the mesoderm alone (in most of which Rst is expressed anyway) and from misexpression in the ectoderm alone appears to be the increase of Rst expressing sticky surfaces: the withdrawal of fusion-competent myoblasts from recruiting founders and precursors may considerably lower the probability for these cell types to contact each other (Strünkelnberg, 2001).

Some of the defects observed in rst mutants concern muscles in ectopic positions. Even though Rst is expressed in the apodemes, the data do not point to an essential role for kirre and/or rst in myotube guidance or attachment: analysis of the subcellular localization shows accumulation of Rst primarily around the apical borders of the apodemes, rather than basally, where outgrowing muscles would be expected to make contact. Moreover, apodeme specification is also not blocked in individuals lacking ectodermal Rst and Kirre, as judged by the muscle pattern. Hence, a putative function of Rst in apodeme specification would be redundantly safeguarded by additional as yet unknown factors. Apodeme specification is also not disrupted in da-Gal4/+;UAS-rst/+ embryos, as revealed by antibody staining against the signalosome component Alien. This clearly rules out the possibility that the strong muscle phenotype observed in these embryos is due to defects in specification of the muscle attachment sites, and argues that restricted expression of Rst is not essential for normal apodeme specification to occur. This is underlined by the fact that 69B-Gal4/+;UAS-rst/+ embryos that express Rst only in the ectoderm do not show attachment defects (Strünkelnberg, 2001).

Given the overlapping mesodermal expression patterns of rst and kirre, and the significant structural similarity between the two proteins, it is concluded that rst and kirre have at least partially redundant functions during muscle development. Rst expression in fusion-competent myoblasts is not essential for their attraction towards ectopic Kirre or Rst: myoblasts can be attracted to ectopic sites in a Df(1)w67k30 background, where Rst is only present at ectopic sites and not in fusion-competent myoblasts -- this strongly suggests a heterophilic trans-interaction. However, as Rst has been shown to mediate homophilic cell adhesion, a homophilic trans-interaction of Rst may also contribute to the fusion process (Strünkelnberg, 2001).

At present, the data do not allow a prediction of the molecular mechanisms in which Rst and Kirre take part; however, it is conceivable that they include the related cell adhesion molecules Sns and Hbs that are expressed on fusion-competent myoblasts. A model of the fusion machinery may include assembly of adhesion molecules within heteromeric complexes with differing compositions on the side of the fusion-competent myoblasts (including Sns, Hbs and Rst) and on the founder cells (including Kirre and Rst). These complexes may still function after loss of single components. It will need further analysis and binding assays to elucidate how these membrane proteins play together and how they are connected to the other components of the fusion machinery (Strünkelnberg, 2001).

Dynamic decapentaplegic signaling regulates patterning and adhesion in the Drosophila pupal retina; Rst activity opposes DE-cadherin-mediated cell adhesion

The correct organization of cells within an epithelium is essential for proper tissue and organ morphogenesis. The role of Decapentaplegic/Bone morphogenetic protein (Dpp/BMP) signaling in cellular morphogenesis during epithelial development is poorly understood. In this paper, the developing Drosophila pupal retina -- looking specifically at the reorganization of glial-like support cells that lie between the retinal ommatidia -- was used to better understand the role of Dpp signaling during epithelial patterning. The results indicate that Dpp pathway activity is tightly regulated across time in the pupal retina and that epithelial cells in this tissue require Dpp signaling to achieve their correct shape and position within the ommatidial hexagon. These results point to the Dpp pathway as a third component and functional link between two adhesion systems, Hibris-Roughest and DE-cadherin. A balanced interplay between these three systems is essential for epithelial patterning during morphogenesis of the pupal retina. Importantly, a similar functional connection has been identified between Dpp activity and DE-cadherin and Rho1 during cell fate determination in the wing, suggesting a broader link between Dpp function and junctional integrity during epithelial development (Cordero, 2007).

Loss of Dpp pathway activity results in a loss of epithelial integrity, but the function of Dpp signaling during maturation of developing epithelia is not fully understood. This study shows that reducing the activity of components of the Dpp pathway leads to abnormal Interommatidial precursor cells (IPC) shape and organization within the ommatidial hexagonal pattern. This activity is linked to fine regulation of apical junction components and is required to maintain stable cell-cell contacts during cell movements within the epithelium. The expression of Dpp in primary pigment cells and the segregation of its receptors to the neighboring IPCs suggest a model in which Dpp acts in the primaries to organize local IPCs through the dynamic control of apical junctions. This view is supported by the dynamic changes in p-Mad activity in the neighboring IPCs, which is highest during the stage (20-26 hours APF) when IPCs rearrangements are maximal (Cordero, 2007).

The role of Dpp in cellular morphogenesis during epithelial development is poorly understood. Therefore, advantage was taken of the unique stereotyped pattern of the pupal retina to study cell behavior as morphogenesis progresses, focusing on events at the single-cell level. In situ visualization experiments suggest that IPCs with reduced Tkv activity are incapable of maintaining their cell-cell contacts and are subject to aberrant changes in their cell shape. Further emphasizing the link with cellular adhesion, this function of Dpp signaling involves DE-cadherin and Rho1, which are essential regulators of cell adhesion and cell shape (Cordero, 2007).

Several lines of evidence are provided indicating that Rst is a negative regulator of Dpp signaling. Previous work has demonstrated that Rst directs IPC movements through selective cell adhesion: IPCs seek to maximize their Rst-mediated contacts with primaries while decreasing contacts with their neighbors. Additionally, reducing Rst activity leads to a failure of initial cell movement. Consistent with these results, Rst activity opposes DE-cadherin-mediated cell adhesion. One model to account for these observations is that cells require a balance between cell movement provided by Hibris-Rst and the stability of cell-cell contacts provided by Dpp signaling. Live visualization supports the view that reducing Dpp activity leaves cells with an imbalance, as IPCs move toward their proper positions but fail to stabilize cell-cell contacts or lock stably into their final positions. Furthermore, downregulation of Dpp signaling leads to unstable DE-cadherin IPC-IPC junctions. Conversely, loss of rst results in loss of cell movements, which can be compensated by either reducing cell adhesion or Dpp signaling activity, again supporting the importance of maintaining a balance between the Hbs-Rst and the Dpp-DE-cadherin systems. Perhaps Dpp (and, by extension, BMP) activity is utilized in the adult for similar functions -- for example, as a 'proof-reading' mechanism to remove aberrant cells from an epithelium (Cordero, 2007).

The results in the wing raise the interesting possibility that regulation of DE-cadherin and Rho1-dependent cell shape and cell adhesion might be a characteristic of Dpp pathway activity common to other biological systems. Similar to the pupal retina, epithelial cells in the wing disc with reduced Dpp signaling displayed abnormal morphologies and were unable to maintain their positions. In the case of the wing, these defects were manifested as viable cysts of mutant cells that were basally excluded from the epithelium. The mechanisms involved in such cell behaviors remain unknown. The results suggest that the role of Dpp signaling during wing patterning also involves DE-cadherin and Rho1. The experiments do not distinguish whether the defects in wing cell fates are a direct or a secondary effect of altered cell adhesion, although altering DE-cadherin activity by itself was not sufficient to cause such defects. Cell adhesion and cell fate have been related previously: for example, Rho-dependent cell shape changes can influence fate decisions in stem cells. Despite the commonalities observed, tissue-specific factors are likely to regulate Dpp-dependent epithelial patterning: for example, Rst does not appear to have a role in wing development, and no changes in retinal Tubulin distribution reported has been reported for the wing (Cordero, 2007).

Dpp is the closest ortholog of vertebrate BMP2/4, and it appears to be active during cellular morphogenesis in a number of contexts including the developing vertebrate eye. Interestingly, and similar to observations for IPCs, fiber cells in the developing vertebrate lens show high levels of p-SMAD activity during the period of cell elongation. Loss of the Type I receptor ALK3 (also known as BMPR1A) or expression of the inhibitor noggin led to abnormal morphogenesis of these fiber cells including mispositioning and failure to elongate; requirements for E-cadherin (also known as cadherin 1) and RHOA function have not been explored (Cordero, 2007).

Finally, Rst does regulate developmental processes other than IPC patterning. For example, Rst is expressed in retinal axons and is required for correct targeting of those axons into the larval brain lobes. Interestingly, Dpp signaling also has a role in this process. Genetic interactions between rst3 and members of the Dpp pathway in the arrangement of these descending axons, raising the intriguing possibility that the two systems act together in axon targeting as well (Cordero, 2007).

These results provide evidence to support a model in which the Dpp pathway acts as an intermediary between the Rst and DE-cadherin adhesion systems. A balanced interplay between these three systems is essential to regulate epithelial cell movements, cell shape and cell-cell contacts during morphogenesis of the pupal retina. Several questions emerge from this study. For example, the data suggest that Rst acts on Dpp signaling by regulating surface-associated Tkv. Immunoprecipitation experiments failed to identify a physical interaction between Rst and Tkv, suggesting intermediate steps remain to be identified. Also, the transcription factor Mad is required to regulate IPC patterning, but the transcriptional targets that link Dpp signaling to DE-cadherin and Rho1 are unknown. A better understanding of the links between these three pathways should help shed light on the mechanisms that regulate the fine cellular events required during patterning of developing epithelia (Cordero, 2007).

Polychaetoid controls patterning by modulating adhesion in the Drosophila pupal retina

Correct cellular patterning is central to tissue morphogenesis, but the role of epithelial junctions in this process is not well-understood. The Drosophila pupal eye provides a sensitive and accessible model for testing the role of junction-associated proteins in cells that undergo dynamic and coordinated movements during development. Mutations in polychaetoid (pyd), the Drosophila homologue of Zonula Occludens-1, are characterized by two phenotypes visible in the adult fly: increased sensory bristle number and the formation of a rough eye produced by poorly arranged ommatidia. It was found that Pyd is localized to the adherens junction in cells of the developing pupal retina. Reducing Pyd function in the pupal eye results in mis-patterning of the interommatidial cells and a failure to consistently switch cone cell contacts from an anterior-posterior to an equatorial-polar orientation. Levels of Roughest, DE-Cadherin and several other adherens junction-associated proteins are increased at the membrane when Pyd protein is reduced. Further, both over-expression and mutations in several junction-associated proteins greatly enhances the patterning defects caused by reduction of Pyd. These results suggest that Pyd modulates adherens junction strength and Roughest-mediated preferential cell adhesion (Seppa, 2008).

The data demonstrate that Pyd is an AJ-associated protein that is required for patterning of the pupal lattice cells. Live imaging of the developing eye indicates that Pyd is necessary for the directed movements of interommatidial precursor cells (IPCs) that allow cell sorting into defined niches. Membrane contacts are dynamically exchanged in the pupal eye: each shift in the position of a cell requires the removal of previous contacts and the establishment of new ones. Pyd regulates patterning at least in part through modulating levels of the AJ-associated proteins DE-Cadherin, β-Catenin, and α-Catenin. Other studies have suggested that cell adhesion is necessary both to facilitate and restrict cell movement within the eye epithelium; the interplay between these two processes requires tight regulation of the levels of both cell adhesion molecules and junctional proteins. The data indicate that removal of Pyd from the AJ compromises this tightly-regulated system and biases the cells toward poorly-directed movements, perhaps because of dysregulation of the timing or function of the mechanisms that control the stability of AJ proteins. This failure in precise regulation of adhesion was also highlighted in the inability of cone cells to exchange their membrane contacts: the apical interfaces of pyd-RNAi expressing cone cells were locked in place. Ectopic DE-Cadherin further increased the percentage of ommatidia affected, again emphasizing the link between pyd activity and the AJ (Seppa, 2008).

The localization of Pyd to the AJ in the pupal eye is dependent on both DE-Cadherin and α-Catenin. However, it was found that ectopic expression of either junctional protein is not sufficient to alter the localization of Pyd. Taken together, these data indicate that DE-Cadherin and α-Catenin are necessary to build or maintain the AJ and to localize Pyd but that, in excess, they are not sufficient to attract ectopic Pyd. This suggests that either Pyd protein levels are not easily altered or that Pyd may be binding to proteins other than the core AJ constituents. Recent work demonstrated that E-Cadherin was necessary for the initial steps of AJ formation while α-Catenin was essential for both the establishment and maintenance of the junction; only when α-Catenin was reduced was ZO-1 lost from established junctions. The results suggest that in dynamically restructured tissues such as the eye, both E-Cadherin and α-Catenin are necessary for the localization of AJ-associated proteins (Seppa, 2008).

The immunoglobulin superfamily member Roughest is necessary for appropriate sorting of IPCs during pupal eye development. Reducing Pyd increased Roughest protein levels specifically at the AJ. Roughest is the Drosophila orthologue of Neph1, a cell adhesion molecule necessary for the structure and function of the glomerular slit diaphragm in the mammalian kidney. The slit diaphragm is the main size-selective barrier in the filtration apparatus of the kidney and retains many characteristics of both the tight and AJ complexes from which it was derived. The Hibris orthologue Nephrin also forms part of the physical structure of the slit diaphragm and both cell adhesion molecules have been reported to bind to each other as well as to ZO-1. Perhaps ZO-1, as with Pyd, has a role in regulating the localization or levels of cell adhesion molecules such as Neph1 and Nephrin (Seppa, 2008).

The Dpp pathway has emerged as a major contributor to patterning of the Drosophila pupal eye. Its role requires functional connections to both DE-Cadherin and Roughest. For example, mutations in shotgun (the locus that encodes DE-Cadherin) suppresses the roughest eye phenotype but enhances Dpp pathway-dependent phenotypes in the eye and wing. Together, these data suggest a model in which (1) Roughest acts to promote the stability of membrane contacts to drive directed cell movements and (2) the Dpp pathway and Pyd act to destabilize the adherens junction complex and local cell contacts to allow for proper IPC sorting. Consistent with this view, it was observed that reducing pyd enhances the effects of reduced Dpp pathway activity in the eye and wing. Thus, Pyd appears to act in concert with the Dpp pathway to regulate select core components of the AJ during development (Seppa, 2008).

This study has shown that Pyd is required specifically for patterning the interommatidial cells of the Drosophila pupal eye. Pyd appears to regulate both cell shape and cell positioning by controlling the levels of AJ proteins such as DE-Cadherin and adhesion proteins such as Roughest. Thus, Pyd provides a link between adhesion and junction formation; a further understanding of its role in the pupal eye will shed light on how these processes are coordinated to generate precise cellular movements during epithelial patterning (Seppa, 2008).

Cellular behavior in the developing Drosophila pupal retina

Correct patterning of cells within an epithelium is key to establishing their normal function. However, the precise mechanisms by which individual cells arrive at their final developmental niche remains poorly understood. An optimized system was developed for imaging the developing Drosophila retina, an ideal tissue for the study of cell positioning. Using this technique, the cellular dynamics of developing wild-type pupal retinas were characterized. Two mutants affecting eye patterning were analyzed and it was demonstrated that cells mutant for Notch or Roughest signaling were aberrantly dynamic in their cell movements. A role for the adherens junction regulator P120-Catenin in retinal patterning was establised through its regulation of normal adherens junction integrity. The results indicate a requirement for P120-Catenin in the developing retina, the first reported developmental function of this protein in the epithelia of lower metazoa. Based upon live visualization of the P120-Catenin mutant as well as genetic data, it is concluded that P120-Catenin is acting to stabilize E-cadherin and adherens junction integrity during eye development (Larson, 2008).

The precise spatial arrangement of the cells within tissues is essential for their function. In some tissues, spatial restriction of cell fate is sufficient to generate the final pattern. In other tissues, such as the developing mammalian brain, the vertebrate retina, and the intestinal epithelium, cells migrate from their original position to their final niche. These migrations can occur across significant distances but in most examples likely reflect more subtle cellular movements within the epithelium. Although some of the molecules that mediate these migrations are known, most tissues do not provide the needed accessibility to dissect individual cell movements in detail (Larson, 2008).

The Drosophila pupal retina is an ideal system in which to study cell positioning during development. The fully patterned retinal epithelium consists of a regular array of identical unit eyes. These 'ommatidia' are initially crudely arrayed within the larval eye field and are separated by a loose collection of 'interommatidial precursor cells' (IPCs). In the pupa, a precisely regulated combination of cell movements, death, and differentiation corrals these IPCs into their final positions, yielding a honeycomb pattern that re-organizes the ommatidia into a hexagonal array (Larson, 2008 and references therein).

These patterning steps are dependent on both signaling and adhesion. Cell-cell signaling regulates the number of cells within developing ommatidia as well as the specification of each cell type. For example, Notch pathway activity is required for differentiation of each of the 20 cell fates within the eye field. In addition, Notch activity is required within the IPCs for proper cell number as well as cell sorting. However, while the role of Notch in directing cell fate is well-established, less is understood of whether Notch also regulates cell morphogenesis (Larson, 2008).

The adhesion molecules Roughest and Hibris also play an essential role in patterning the retina as these two molecules are required to refine the IPC lattice to a hexagon. Single cell expression experiments with Roughest and Hibris indicate that both mediate the final positioning of cells within the hexagon through direct heterophilic adhesion and that both control adherens junction formation between IPCs. For example, as IPCs re-arrange into their final pattern they briefly reduce their adherens junctions; these junctions are then re-assembled as patterning is completed. Ectopic expression of Hibris in the developing hexagonal lattice resulted in the premature re-appearance of these junctions as well as mis-patterning. However, the mechanisms by which the adherens junctions are normally dynamically regulated are not known (Larson, 2008).

This study presents a method for visualizing development of the living pupal eye in situ. This method was use to extend previous observations on the cellular movements of the developing retina in wild-type and in two classical eye mutants that alter cellular positioning, one through cell signaling and a second through cell adhesion. Previous work has suggested that regulation of adherens junctions is important for patterning. To begin to address this issue, live visualization was used to demonstrate a role for P120-Catenin as a regulator of E-Cadherin as IPCs undergo the precise movements required to generate a hexagonal pattern within the eye field (Larson, 2008).

It has been proposed that adherens junctions play an important role in patterning the pupal eye. The Notch allele Notchfacet-glossy (Nfa-g), that specifically reduces Notch activity in the pupal eye and the truncation mutant rstCT did not exhibit disruption of α-Catenin-GFP, suggesting that the adherens junctions were correctly regulated in Notch and rst mutants. Indeed, mutations specifically affecting the adherens junctions but not the integrity of the retinal epithelium have not been reported. P120-Catenin, encoded by p120ctn, is an armadillo repeat domain-containing protein that binds to the juxtamembrane domains of classical cadherins to regulate adherens junction stability and activity. In mammals, it is essential for viability and modulates the levels and adhesive properties of cadherins. In Drosophila and C. elegans, deletion of the p120ctn locus enhanced mutations in cadherin but was non-essential for viability. Recent work, however, has found that p120ctn is required to regulate neuron morphology. In contrast, despite earlier reports to the contrary, recent studies ascribe no phenotype to p120ctn in Drosophila epithelia. This led to the suggestion that P120-Catenin solely plays a supporting role in cadherin-based adhesion (Larson, 2008 and references therein).

The surface phenotype of adult fly eyes homozygous for the null p120ctn allele p120ctn308 was wild-type in appearance. However, examination of the pupal retina indicated that genotypically p120ctn mutant eyes have ectopic lattice cells and a partially penetrant mis-patterning of the 3° niche. Pupae bearing the p120ctn308 chromosome in trans to a deficiency covering the region (Df(2R)244) exhibited a phenotype similar to homozygous p120ctn308 retinas, consistent with previous reports that p120ctn308 represents a null allele. The p120ctn308 mutation was generated by imprecise excision of a P-element found in the parent line KG01086. Retinas bearing a single copy of p120ctn308 in trans to KG01086 exhibited a wild-type phenotype indicating that the p120ctn308 phenotype was a direct result of the excision event. Lastly, ubiquitous expression of a full-length p120ctn-GFP transgene in a p120ctn null background completely rescued the p120ctn null phenotype. Taken as a whole this data indicates that the eye phenotype is a direct result of a loss of p120ctn and represents the first reported developmental requirement of p120ctn in an epithelium of lower metazoa (Larson, 2008).

To better understand the role of p120ctn in patterning the fly eye, p120ctn null mutant development was imaged from 24 to 29 h a.p.f. (four retinas total). While the final p120ctn308 phenotype was fairly subtle, live imaging revealed surprisingly dramatic differences with wild-type development. In particular, live imaging of p120ctn308 pupal eyes showed consistent, transient separation of IPCs accompanied by a loss of α-Catenin-GFP fluorescence from the membranes at their contact face. This apparent breakdown of coherent junctions occurred almost exclusively between IPC:IPC and IPC:1° junctions and presumably accounted for their ability to achieve or maintain stable positions. To quantitate this difference, the dynamics of 3° emergence was followed throughout the stage of IPC patterning. A clear difference was observed in the ability of local IPCs to achieve and - in particular - to retain a position in the 3° niche. This instability and ectopic movement presumably accounts for the errors in 3° patterning observed in the mid-pupa. Other parameters such as cell movements were on the whole indistinguishable from wild-type. This result indicates that p120ctn308 IPCs are capable of forming adherens junctions but are unable to maintain them during dynamic cell movements (Larson, 2008).

No clear genetic interactions -- either as trans heterozygotes or as dominant modifier activity -- were observed between p120ctn and Egfr, wingless, roughest, Notch, shotgun, α-Catenin, or the small GTPases (reducing Rho1 or Cdc42). However, a closer genetic analysis of the relationship between p120ctn, shotgun, and Rho1 yielded surprising results. Based on both cell culture and in vivo data, mammalian P120-Catenin has been proposed to regulate both RhoA and E-cadherin (Larson, 2008).

Interestingly, the phenotype observed with complete loss of p120ctn activity (using the null deletion allele p120ctn308) was further enhanced by removing a functional genomic copy of shotgun (shgR69) or Rho1 (Rho172O). Removal of Rho1 resulted in additional ectopic cells and an increase in the frequency of patterning errors. In the case of the shg interaction, the hexagonal IPC pattern was disrupted with extra cells present in double layers around bristle cells. The severity of this interaction prevented its quantification. Neither shgR69 nor Rho172O, both null alleles, gave a dominant phenotype on their own. The ability of mutations in shotgun or Rho1 to further enhance a null mutation in p120ctn indicates that both DE-Cadherin and Rho1 act, at least in part, through a pathway that is independent of P120-Catenin. However, a consistent difference was observed in E-cadherin localization. While full loss of p120ctn led to at most a slight decrease in Armadillo and E-cadherin, it was noted that E-cadherin protein was discontinuous at the membranes of p120ctn308 cells. This was best observed when comparing loss of P120-Catenin next to a rescue construct of P120-Catenin in neighboring clonal patches (Larson, 2008).

This study has further characterized the cell movements required to pattern the developing pupal retina. While many of these movements have been inferred from dissected tissue, it was observed that the developing retina was more dynamic than expected in both wild-type and mutant flies. For example, it has been speculated that the roughest phenotype was due to a loss of cell movement. However, rstCT IPCs were observed to actively exchange contacts and neighbors despite the fact that this exchange did not productively pattern the retina. In an earlier study, scanning electron micrographs showed that rstCT IPCs extend filopodia from their apical surface in a manner identical to wild-type. Combined with live visualization studies, this data indicates that rstCT cells have an active cytoskeleton and can participate in cell rearrangement but cannot functionally recognize 1°s. Alternatively, rstCT cells may fail to establish junctions that stabilize a final position; consistent with this latter possibility, the ability of ectopic Hibris to direct precocious adherens junctions has been reported. While SEM studies of Nfa-g remain to be conducted, the similarity of the movements of Nfa-g IPCs to rstCT IPCs is striking. In fact, Notch is required for localization of Roughest protein perhaps accounting for their phenotypic similarity (Larson, 2008).

Cell adhesion plays a key role in patterning the developing pupal retina. In normal patterning, DE-cadherin staining between IPCs decreased during later stages of IPC re-arrangements, only to increase a few hours later as patterning was completed and cell contacts were finalized. The loss of roughest resulted in uniform DE-cadherin staining during this time, suggesting that one method by which Roughest may affect retinal patterning is through modulation of E-cadherin levels. BMP family signaling was found to regulate retinal patterning, in part by positively regulating E-cadherin. Using live visualization, this study found that P120-Catenin positively regulated DE-cadherin-based junctions, further demonstrating a role for DE-cadherin regulation in fine cellular patterning within the eye (Larson, 2008).

The mechanism by which P120-Catenin regulates cadherin-based junctions in Drosophila remains unclear. In mammals, P120-Catenin has been shown to regulate cadherin by modulating its endocytosis and degradation. It is noted, however, that Drosophila cadherins lack the di-leucine motif that P120-Catenin masks to prevent endocytosis in mammals. Mammalian P120-Catenin also acts as an inhibitor of the small GTPase Rho by regulating RhoGAPs such as p190RhoGap or Rho itself. During Drosophila embryogenesis, however, p120ctn failed to show functional interactions with mutations in Rho1. In contrast, this study detected an interaction between p120ctn and Rho1 during eye development, but this interaction was also inconsistent with the mammalian data. If the p120ctn null phenotype was the result of a loss of Rho inhibition, then it would have been expected that removal of a functional genomic copy of Rho would suppress the p120ctn phenotype. Instead the results are consistent with a model in which P120-Catenin and Rho1 act in parallel pathways to regulate eye development (Larson, 2008).


Cloning, structure and expression of Roughest homologs

The cDNA corresponding to DM-GRASP, a 95 kd cell surface protein expressed on a restricted population of axons has been cloned and sequenced and found to be a new member of the immunoglobulin superfamily of adhesion molecules. Consequently it has been named DM-GRASP, since it is an immunoglobulin-like restricted axonal surface protein. Its expression begins early in chick embryogenesis, and within the spinal cord it is localized to axons in the dorsal funiculus, midline floorplate cells, and motoneurons. Antibodies to DM-GRASP impair neurite extension on axons, and purified DM-GRASP supports neurite extension from chick sensory neurons (Burns, 1991).

A 95- to 100-kDa cell surface glycoprotein, named BEN (for bursal epithelium and neurons), is widely expressed during chicken embryonic development. In the central nervous system, it is restricted to subsets of neurons including the motoneurons and the inferior olivary nucleus neurons (which provide the cerebellum with the climbing fibers) where its expression occurs during the phase of axonogenesis and synaptogenesis. BEN expression extends to a variety of tissues originating from the three embryonic germ layers. BEN immunopurified from neural, epithelial, and hemopoietic tissues is differently glycosylated and may or may not carry the HNK-1 epitope. A full-length cDNA encoding this protein has been cloned. Analysis of its sequence reveals that BEN is a member of the immunoglobulin superfamily. Two proteins with an identical cDNA sequence have recently been reported: DM-GRASP and SC1. Their pattern of expression and structural properties are consistent with those reported for BEN. Therefore BEN, DM-GRASP, and SC1 are likely to be the same protein of the immunoglobulin superfamily (Pourquie, 1992).

The mAb E 21 recognizes a cell surface glycoprotein selectively associated with fish retinal ganglion cell axons that are in a state of growth. All retinal axons and ganglion cells in goldfish embryos stain for E 21. However, in adult fish E 21 immunoreactivity exhibits a patterned distribution in ganglion cells in the marginal growth zone of the continuously enlarging fish retina and the new axons emerging from these cells in the retina, optic nerve, and optic tract. The E 21 antigen is absent from older axons, except the terminal arbor layer in the tectum (the Stratum fibrosum et griseum superficiale) where the antigen is uniformly distributed. Upon optic nerve transection, the previously unlabeled axons reacquire an E 21 immunoreactivity as they regenerate throughout their paths to the tectum. However, several months after ONS, E 21 staining disappears from the regenerated axons over most of their lengths but reappears, as in normal fish, in the terminal arbor layer. The immunoaffinity-purified E 21 antigen, called Neurolin, has an apparent molecular mass of 86 kD and contains the HNK1/L2 carbohydrate moiety, like several members of the class of cell adhesion proteins of the Ig superfamily. The NH2-terminal amino acid sequence has homologies to the cell adhesion proteins DM-Grasp recently described in the chicken. Thus, retinal ganglion cell axons express Neurolin during their development and are able to reexpress this candidate cell adhesion protein during axonal regeneration, suggesting that Neurolin is functionally important for fish retinal axon growth (Paschke, 1992).

A full-length zebrafish cDNA clone and a partial mouse cDNA clone similar to chick DM-GRASP were isolated and analyzed. The nucleotide sequence of the full-length zebrafish clone shares 54% identity, and predicts 39% amino acid identity, with chick DM-GRASP. The partial mouse clone shares 76% nucleotide identity, and predicts 76% amino acid identity, with chick DM-GRASP. The predicted proteins encoded by both of these clones exhibit conserved structural domains that are characteristic of the chick protein. These features may identify them as a distinct subfamily within the immunoglobulin superfamily of cell adhesion proteins. Expression of the zebrafish DM-GRASP protein is similar to chick DM-GRASP and is principally restricted to a small subset of developing sensory and motor neurons during axonogenesis. Zebrafish DM-GRASP expression was temporally regulated and limited to specific axon domains. This regional expression correlates with fasciculated axon domains. These results suggest that the zebrafish and mouse cDNA clones represent the respective fish and mammalian homologs of chick DM-GRASP. The highly selective expression of zebrafish DM-GRASP suggests that it is involved in the selective fasciculation and guidance of axons along their normal pathways (Kanki, 1994).

The polymerase chain reaction was used to isolate cDNAs coding for goldfish and zebrafish neurolin, a previously identified 86 kDa cell surface glycoprotein in the goldfish visual system. Sequence analysis has demonstrated that neurolin belongs to the immunoglobulin superfamily and is 51% similar to the chick cell adhesion protein DM-GRASP/SC-1/BEN. Northern analysis with a riboprobe coding for the C-terminus of neurolin detected two mRNAs of 3.7 kb and 3.3 kb in both embryonic and adult goldfish. Several monoclonal and polyclonal antibodies were generated against immunopurified goldfish neurolin and two have been shown to crossreact with zebrafish proteins. Both antibodies identify a zebrafish protein of the same molecular weight as goldfish neurolin on immunoblots. Immunohistochemical studies with these antibodies in the zebrafish retinotectal system demonstrate labeling on young ganglion cells and growing retinal axons in a pattern similar to that found in goldfish. The similarity of neurolin to a known cell adhesion proteins, its expression on developing retinal ganglion cells and axons in both embryos and adult fish, and its re-expression during retinal axon regeneration in the goldfish suggests that neurolin is important during axonal growth in the fish central nervous system (Laessing, 1994).

The expression of neurolin, the fish homolog of the cell adhesion protein DM-GRASP/BEN/SC-1, is dynamically regulated. The expression of neurolin correlates with early events of retinal ganglion cell (RGC) differentiation in zebrafish embryos. Neurolin mRNA first appears [28 h postfertilization, (PF)] in nasoventral cells, representing the first RGCs, then in dorsal, central (34 to 40 h PF) and temporal RGCs. After differentiation of RGCs in the central portion of the retina, RGCs exhibiting neurolin mRNA form rings. These rings move toward the retinal periphery and encompass older (central) RGCs. Thereafter, such as at 3.5 days PF, neurolin mRNA expressing RGCs are confined to the annular growth zone at the retinal peripheral margin. Two hours after onset of mRNA expression, RGCs acquire antineurolin immunoreactivity on the surface of their somata and on their axons as they extend to the tectum. The mRNA signal in RGCs decreases significantly within 20 h after its appearance, which correlates with the arrival of axons in the tectum. This is followed by weakening of neurolin immunoreactivity on RGCs and axons. This pattern of RGC differentiation in zebrafish revealed by the expression of neurolin, is unique among vertebrates. The spatiotemporal expression pattern of neurolin suggests a functional significance of this cell adhesion protein in RGC recognition and RGC axon growth (Laessing, 1996).

In contrast to the spinal sensory ganglia that reiterate a basic organizational and functional unit, each cranial ganglion mediates a distinct sensory modality and exhibits a characteristic pattern of peripheral and central neuronal connectivity. Protein molecules responsible for establishment and maintenance of the cranial ganglion-specific networks are not known. Hamster monoclonal antibody 802C11 strongly stains neurons and their processes of the VIIIth cranial ganglion (hearing and equilibrium), but not the Vth cranial (somatosensory) or spinal ganglia in the mouse embryo. The cellular staining pattern of positive neurons suggests that the antigen is associated with the cell membrane, and biochemical analyses of the antigen from adult mouse brain show the antigen to be a glycosylated intrinsic membrane protein of approximately 100 kDa. The antigen was purified, and based on the partial amino acid sequences, its entire cDNA was cloned. The deduced amino acid sequence reveals that the antigen belongs to the immunoglobulin superfamily with a significant homology (73.5% identity) to chicken SC1 protein. Chicken SC1 has been shown to be a cell-cell adhesion proteins in vitro with a proposed role in neurite extension of spinal motor neurons. These results suggest that murine SC1-related protein (MuSC) is involved in the pathfinding and/or fasciculation of specific cranial sensory nerve fibers (Sekine-Aizawa, 1998).

SC1 is a secreted glycoprotein with a high amino acid sequence similarity to SPARC (Secreted Protein, Acidic, Rich in Cysteine). SC1 transcripts are first detected in mouse embryos after day 8.5 post coitus in somites at the medial lip of the dermomyotome. Expression of SC1 transcripts by the progenitor cells continues as they begin involuting under the dermomyotome and during their migration along the lateral wall of the dermomyotome. After myotome migration is completed, SC1 mRNA expression is downregulated in the trunk region. The data indicate that SC1 expression is restricted to the initial stages of epaxial myotome differentiation and migration, undergoing rapid downregulation prior to myotome emigration from the somitic environment (Ringuette, 1998).

It is well established that the notochord influences the development of adjacent neural and mesodermal tissue. Involvement of the notochord in the differentiation of the dorsal pancreas has been demonstrated. However, knowledge of the signals involved in pancreatic development is still incomplete. In order to identify proteins potentially implicated during pancreatic differentiation, monoclonal antibodies against previously established embryonic pancreatic ductal epithelial cell lines (BUD and RED) were raised and characterized. Using the MAb 2117, the cell surface antigen 2117 (Ag 2117) was cloned. The predicted sequence for Ag 2117 is the rat homolog of BEN. Initially reported as a protein expressed on epithelial cells of the chicken bursa of Fabricius, BEN is expressed in a variety of tissues during development and described as a marker for the developing central and peripheral chicken nervous systems. A role has been suggested for BEN in the adhesion of stem cells and progenitor cells to the blood-forming tissue microenvironment. BEN, initially expressed exclusively in the notochord during the early development of rat, is implicated in pancreatic development. Ag 2117 regulates the pancreatic epithelial cell growth through the ras and Jun kinase pathways. In addition, Ag 2117 is able to regulate the expression of the transcription factor PDX1, required for insulin gene expression, in embryonic pancreas organ cultures (Stephan, 1999).

Neurolin is a growth-associated cell surface glycoprotein from goldfish and zebra fish that has been shown to be involved in axonal path-finding in the goldfish retina and suggested to function as a receptor for axon guidance positively proteins. Being a member of the immunoglobulin superfamily of cell adhesion proteins, neurolin consists of five N-terminal extracellular immunoglobulin (Ig)-like domains, a transmembrane and a short cytoplasmatic domain. Repeated injections of polyclonal Fab fragments against neurolin and of monoclonal antibodies against any of the Ig domains cause path-finding errors and disturbance of axonal fasciculation. In order to obtain a complete structural characterization and a molecular basis for structure-function determination, recombinant neurolin with the complete extracellular part but lacking the transmembrane and cytoplasmatic domain was expressed in Chinese hamster ovary (CHO) cells (CHO-neurolin). The isolation of CHO-neurolin was carried out by Ni-affinity chromatography and subsequent high-performance liquid chromatography (HPLC). An exact molecular mass determination was obtained by matrix-assisted laser desorption/ionization mass spectrometry (MALDI/MS) and 60.9 kDa were revealed, which suggests that approximately 10 kDa are due to glycosylation. The predicted molecular mass is 51.5 kDa, whereas sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) yields an apparent molecular mass of 72 kDa. Gel shift assays using SDS-PAGE and Western blot analysis with anti-neurolin antibodies provided consistent molecular mass data. The complete primary structure and N-glycosylation patterns were identified using specific lectin assays, MALDI/MS peptide mapping analysis by proteolytic and in-gel digestion, electrospray ionization MS and MALDI/MS in combination with specific glycosidase degradation. HPLC isolation of glycosylated peptide fragments and MS after selective deglycosylation has revealed heterogeneous glycosylations at all five N-glycosylation consensus sites. All attached N-glycans are of the complex type and show a mainly biantennary structure: they are fucosylated with alpha(2,3)-terminal neuraminic acid. These data serve as a first detailed model to characterize the molecular recognition structures exhibited by the extracellular domains (Denzinger, 1999).

In an effort to identify aberrantly expressed genes in v-rel-induced tumors, monoclonal antibodies were developed that react selectively with avian B-cell tumors. One antibody, HY78, immunoprecipitates a 120-kDa glycoprotein (p120) from cells that express v-rel. N-terminal amino acid sequencing of p120 has identified a 27-amino-acid sequence that is also present in DM-GRASP, an adhesion protein belonging to the immunoglobulin superfamily. Evidence from tissue distribution, immunological cross-reaction, PCR amplification, cDNA cloning, and DNA sequence shows that p120 is indeed DM-GRASP. Northern (RNA) analysis using a probe from the DM-GRASP gene has identified a 5.3-kb transcript in mRNA from bursa, thymus, and brain as well as from v-rel-induced B-cell lymphomas but not from bursal B cells. The induction of this protein by v-rel during the development of bursal B-cell lymphomas appears, therefore, to be ectopic in nature. Overexpression of v-rel or c-rel in chicken embryonic fibroblasts, B-cell lines, and spleen mononuclear cells induces the expression of DM-GRASP. The ratio of DM-GRASP to v-Rel is fivefold higher than that of DM-GRASP/c-Rel in a B-cell line, DT95. Interestingly, the presence of HY78 antibody inhibits the in vitro proliferation of v-rel-transformed cells but not cells that are immortalized by myc. These data suggest that DM-GRASP is one of the genes induced during v-rel-mediated tumor development and that DM-GRASP may be involved in the growth of v-rel tumor cells (Zhang, 1995).

Roughest homologs are adhesion proteins

DM-GRASP is an immunoglobulin superfamily cell adhesion protein that is expressed in both the developing nervous and immune systems. Specific populations of neurons that respond to DM-GRASP substrates appear to require homophilic interactions between DM-GRASP proteins. It was of interest to determine whether DM-GRASP interacts heterophilically with other ligands as well. Eleven proteins from embryonic chick brain membranes have been found to consistently bind to and elute from a DM-GRASP-Sepharose affinity column. One of these proteins is DM-GRASP itself, consistent with its known homophilic binding. Another protein, at 130 kD, is immunoreactive with monoclonal antibodies to NgCAM. Other neural cell adhesion proteins were not detected in the eluate. The DM-GRASP-Sepharose eluate also contains a potent neurite stimulating activity, which cannot be accounted for by either DM-GRASP or NgCAM. To investigate the interaction of DM-GRASP and NgCAM, antibodies against DM-GRASP were added to neuronal cultures extending neurites on an NgCAM substrate. The presence of antibodies to DM-GRASP decreases neurite extension on laminin, suggesting that the antibody is not toxic or generally an inhibitor of motility. Two possible models are presented for the DM-GRASP-NgCAM association and a hypothesis is presented for neural cell adhesion function that features the dimerization of cell adhesion proteins (DeBernardo, 1996).

Roughest homologs: axon pathfinding and innervation

Examination was carried out of the expression and function of a cell membrane protein in the developing chick retinotectal system identified by a monoclonal antibody (mAb 4H5) and the corresponding antiserum. The protein shares a series of properties, including the N-terminal amino acid sequence, with a cell adhesion protein termed DM-GRASP, SC1, BEN, and JC7. It can therefore be considered identical with this molecule and is referred to as SC1/DMGRASP. In early development of the retinotectal system, SC1/DMGRASP is exclusively expressed on growing, far-projecting, tract-forming axons. Expression begins at the onset of retina ganglion cell axogenesis and its maximum expression overlaps with the phase of maximal axon extension. Later in development, SC1/DMGRASP appears on distinct laminae within plexiform layers in spatiotemporal correlation with synaptogenesis. In an in vitro assay system designed to study the elongation of RGC axonal processes on preexisting RGC axons, addition of SC1/DMGRASP antiserum specifically reduces lengths of axonal processes. In contrast, axonal growth on laminin or basal lamina preparations is not SC1/DMGRASP-dependent. Taken together, the data provide evidence for a role of SC1/DMGRASP in axonal elongation of SC1/DMGRASP-positive axons on such axons, thereby possibly contributing to the pathway and target finding mechanisms of far-projecting, tract-forming central nervous system neurons (Pollerberg, 1994).

DM-GRASP (GRASP) is an integral membrane glycoprotein expressed on a restricted set of axons in the developing chick nervous system. Purified GRASP supports neurite extension from the subpopulation of neurons that express GRASP. Sensory, sympathetic, and ciliary neurons express GRASP and extend neurites on a GRASP substrate. In contrast, tectal, diencephalic, and retinal cells express GRASP at a very low level, if at all, and do not extend neurites on a GRASP substrate. Moreover, during the developmental period in which GRASP is downregulated on sensory neurons, the neurons lose the capacity to extend neurites on a GRASP substrate. Recombinant GRASP produced in a baculovirus expression system is biochemically and functionally identical to GRASP purified from embryonic chick brain. The finding that GRASP selectively supports neurite extension supports the hypothesis that it mediates selective fasciculation via a homophilic binding mechanism (DeBernardo, 1995).

Young axons of new retinal ganglion cells (RGCs) in the continuously growing goldfish retina fasciculate with one another and their immediate forerunners on their path toward the optic disk and along the optic nerve. They express the immunoglobulin superfamily cell adhesion proteins (CAMs) neurolin (DM-GRASP) and the L1-like E587 antigen. Repeated injections of Fab fragments from polyclonal antisera against neurolin (neurolin Fabs), approximately three to four cm in length, into the eyes of rapidly growing goldfish causes highly aberrant pathways for young RGC axon subfascicles in the dorsal retina. Many axons grow in circles and fail to reach the optic disk. In contrast, E587 Fabs, used in parallel experiments, disrupt the fascicles but do not interfere with the disk-directed growth. Neurolin Fabs also disturb axonal fasciculation in vivo as well as in vitro but less severely than E587 Fabs. Coinjections of both Fabs increases defasciculation of the dorsal axons in both aberrant and disk-directed routes. They also disrupt the order of young RGC axons in the optic nerve more severely than E587 Fabs alone. This demonstrates that the development of tight and orderly fascicles in the dorsal retina and in the optic nerve requires both E587 antigen and neurolin. More importantly, these results suggest an involvement of neurolin in RGC axonal guidance from the retinal periphery to the optic disk. Because disrupted fascicles and errant axon routes are found only in the dorsal retinal half, a cooperation with so-called positional markers is possible (Ott, 1998).

BEN/SC1/DM-GRASP is a cell adhesion protein belonging to the Ig superfamily that is transiently expressed during avian embryogenesis in a variety of cell types, including the motoneurons of the spinal cord. The pattern of BEN expression during neuromuscular development of the chick has been characterized. Both motoneurons and their target myoblasts express BEN during early embryonic development, and the protein becomes restricted at neuromuscular contacts as soon as postsynaptic acetylcholine receptor clusters are observed in muscle fibers. Muscle cells grown in vitro express and maintain BEN expression even when they fuse and give rise to mature myotubes. When embryos are deprived of innervation by neural tube ablation, BEN expression is observed in muscle fibers, whereas, in control, the protein is already restricted at neuromuscular synaptic sites. These results demonstrate that all myogenic cells intrinsically express BEN and maintain the protein in the absence of innervation. Conversely, when neurons are added to myogenic cultures, BEN is rapidly downregulated in muscle cells, demonstrating that innervation controls the restricted pattern of BEN expression seen in innervated muscles. After nerve section in postnatal muscles, BEN protein becomes again widely spread over muscle fibers. When denervated muscles are allowed to be reinnervated, the protein is reexpressed in regenerating motor axons, and reinnervation of synaptic sites leads to the concentration of BEN at neuromuscular junctions. These results suggest that the BEN cell adhesion protein acts both in the formation of neuromuscular contacts during development and in the events leading to muscle reinnervation (Fournier-Thibault, 1999).

The optic disk-directed growth of retinal ganglion cell axons is markedly disturbed in the presence of polyclonal antineurolin antibodies, which mildly affect fasciculation. New monoclonal antibodies (mAbs) against goldfish neurolin, an immunoglobulin (Ig) superfamily cell adhesion/recognition protein with five Ig domains, were generated to assign function (guidance versus fasciculation) to specific Ig domains. By their ability or failure to recognize Chinese hamster ovary cells expressing recombinant neurolin with deletions of defined Ig domains, different mAbs were identified as being directed against the different Ig domains 1, 2, or 3. Repeated intraocular injections of a mAb against Ig domain 2 disturb the disk-directed growth: axons grow in aberrant routes and fail to reach the optic disk, but remain fasciculated. mAbs against Ig domains 1 and 3 disturb the formation of tight fascicles. mAb against Ig domain 2 significantly increases the incidence of growth cone departure from the disk-oriented fascicle track, while mAbs against Ig domains 1 and 3 do not. Thus, Ig domain 2 of neurolin is apparently essential for growth cone guidance towards the disk, presumably by being part of a receptor (or complex) for an axon guidance component (Leppert, 1999).

Cell surface adhesion proteins are thought to play a necessary role in axon guidance and fasciculation in the developing nervous system. A potential adhesion protein has been studied using the zn-5 monoclonal antibody, which recognizes the surfaces of zebrafish spinal motoneurons. Zn-5 recognizes zebrafish DM-GRASP. DM-GRASP is a cell adhesion protein of the immunoglobulin superfamily that mediates homophilic adhesion and neurite outgrowth in vitro. In zebrafish, primary motoneurons pioneer the peripheral motor nerve pathways, and the axons of secondary motoneurons follow the routes established by the primary motoneuron axons. Of the two classes of zebrafish spinal motoneurons, only the later growing secondary motoneurons express DM-GRASP. The secondary motoneurons restrict DM-GRASP protein to their cell bodies and fasciculated segments of their axons. Expression of DM-GRASP is transient: the protein is present during the period of axonal growth and disappears after axons have reached their muscle targets. Thus, homophilic adhesion mediated by DM-GRASP may play a role in fasciculation of secondary motoneuron axons but not in pathfinding by the pioneer axons of the primary motoneurons or in guidance of secondary motoneuron axons to their targets (Fashena, 1999).

During nervous system development, neurons form reproducible synapses onto specific targets. The development of stereotyped synapses of the C. elegans HSNL neuron has been examined in vivo. Postsynaptic neurons and muscles are not required for accurate synaptic vesicle clustering in HSNL. Instead, vulval epithelial cells that contact HSNL act as synaptic guidepost cells that direct HSNL presynaptic vesicles to adjacent regions. The mutant syg-1(ky652) has defects in synapse formation that resemble those in animals that lack vulval epithelial cells: HSNL synaptic vesicles fail to accumulate at normal synaptic locations and form ectopic anterior clusters. syg-1 encodes an immunoglobulin superfamily protein that acts in the presynaptic HSNL axon. SYG-1 protein is localized to the site of future synapses, where it initiates synapse formation and localizes synaptic connections in response to the epithelial signal. SYG-1 is related to Drosophila IrreC and vertebrate NEPH1 proteins, which mediate cell-cell recognition in diverse developmental contexts (Shen, 2003).

SYG-1 is closely related to two Drosophila proteins, Rst/IrreC and Kirre/Duf. Although they have not been characterized in synapse formation, these proteins act in a variety of developmental events that involve specific cell recognition. Rst/IrreC mutants have defects in cell adhesion in the retina (the roughest phenotype), and in axon pathfinding at higher steps of visual processing (the irregular chiasm phenotype). During development, founder cells for each muscle attract and fuse with fusion-competent cells. Recognition between the muscle founder and fusion-competent cells is mediated by Rst/IrreC and Kirre on the founder cells, and Sticks and Stones (Sns) and Hibris (Hib) on fusion-competent cells. Muscle fusion occurs through a characteristic prefusion complex, a tight adhesion between cells associated with alignment of paired vesicles at the cell junctions. Several structural features of the prefusion complex are similar to features of synapses, including tight local asymmetric adhesions between cells and the recruitment of vesicles. Sns and Hib are transmembrane proteins with eight immunoglobulin domains that have heterophilic interactions with the Kirre/Duf protein. A predicted ORF in the C. elegans genome, C26G2.1, shows strong homology to SNS and Hibris, suggesting that similar heterophilic recognition mechanisms could also exist in C. elegans (Shen, 2003 and references therein).

SYG-1 bears similarity to many vertebrate immunoglobulin superfamily members, and strongest similarity to three proteins that are only partly characterized, NEPH1, KIAA1867, and hCP41052. One of these genes, NEPH1, is regionally expressed in the mammalian brain, but its function there is unknown. NEPH1 is required for kidney function and the development of the podocyte slit membrane in mice. A human ortholog of Sns/Hib, Nephrin, is also required for kidney function and development of the podocyte slit membrane, a tight epithelial cell adhesion complex that plays a role in glomerular filtration. The adhesion complexes of the mammalian podocyte slit membrane, a future C. elegans synapse, and a Drosophila muscle prefusion complex appear to be initiated by similar molecular interactions that are interpreted in cell-type specific contexts (Shen, 2003 and references therein).

Toward an understanding of divergent compound eye development in drones and workers of the honeybee (Apis mellifera L.): A correlative analysis of morphology and gene expression

Eye development in insects is best understood in Drosophila melanogaster, but little is known for other holometabolous insects. Combining a morphological with a gene expression analysis, this study investigated eye development in the honeybee, putting emphasis on the sex-specific differences in eye size. Optic lobe development starts from an optic lobe anlage in the larval brain, which sequentially gives rise to the lobula, medulla, and lamina. The lamina differentiates in the last larval instar, when it receives optic nerve projections from the developing retina. The expression analysis focused on seven genes important for Drosophila eye development: eyes absent, sine oculis, embryonic lethal abnormal vision, minibrain, small optic lobes, epidermal growth factor receptor, and roughest. All except small optic lobes were more highly expressed in third-instar drone larvae, but then, in the fourth and fifth instar, their expression was sex-specifically modulated, showing shifts in temporal dynamics. The clearest differences were seen for small optic lobes, which is highly expressed in the developing eye of workers, and minibrain and roughest, which showed a strong expression peak coinciding with retina differentiation. A microarray analysis for optic lobe/retina complexes revealed the differential expression of several metabolism-related genes, as well as of two micro-RNAs. While major morphological differences were not seen in the developing eye structures before the pupal stage, the expression differences observed for the seven candidate genes and in the transcriptional microarray profiles indicate that molecular signatures underlying sex-specific optic lobe and retina development become established throughout the larval stages (Marco Antonio, 2016).


Search PubMed for articles about Drosophila roughest

Apitz, H., Strunkelnberg, M., de Couet, H. G. and Fischbach, K. F. (2005). Single-minded, Dmef2, Pointed, and Su(H) act on identified regulatory sequences of the roughest gene in Drosophila melanogaster. Dev. Genes Evol. 215(9): 460-69. 16096801

Apitz, H., Kambacheld, M., Höhne, M., Ramos, R. G. P., Straube, A. and Fischbach, K. F. (2004). Identification of regulatory modules mediating specific expression of the roughest gene in Drosophila melanogaster. Dev. Genes Evol. 214: 453-459. 15278452

Araujoa, H., et al. (2003). Requirement of the roughest gene for differentiation and time of death of interommatidial cells during pupal stages of Drosophila compound eye development. Mech. Dev. 120: 537-547. 12782271

Bao, S. and Cagan, R. (2005). Preferential adhesion mediated by Hibris and Roughest regulates morphogenesis and patterning in the Drosophila eye. Dev. Cell 8(6): 925-35. 15935781

Bao, S., Fischbach, K.-F., Corbin, V. and Cagan, R. L. (2010). Preferential adhesion maintains separation of ommatidia in the Drosophila eye. Dev. Biol. 344: 948-956. PubMed Citation: 20599904

Bao, S. (2014). Notch controls cell adhesion in the Drosophila eye. PLoS Genet 10: e1004087. PubMed ID: 24415957

Bazigou, E., Apitz, H., Johansson, J., Loren, C. E., Hirst, E. M., Chen, P. L., Palmer, R. H. and Salecker, I. (2007). Anterograde Jelly belly and Alk receptor tyrosine kinase signaling mediates retinal axon targeting in Drosophila. Cell 128: 961-975. Pubmed: 17350579

Boschert, U., et al. (1990). Genetic and developmental analysis of irreC, a genetic function required for optic chiasm formation in Drosophila. J. Neurogenet. 6(3): 153-71. PubMed Citation: 2358965

Bour, B. A., et al. (2000). Drosophila SNS, a member of the immunoglobulin superfamily that is essential for myoblast fusion. Genes Dev. 14(12): 1498-1511. 10859168

Burns, F. R., et al. (1991). DM-GRASP, a novel immunoglobulin superfamily axonal surface protein that supports neurite extension. Neuron 7(2): 209-20.

Cordero, J. B., Larson, D. E., Craig, C. R., Hays, R. and Cagan, R. (2007). Dynamic decapentaplegic signaling regulates patterning and adhesion in the Drosophila pupal retina. Development 134(10): 1861-71. Medline abstract: 17428827

Das, A., Base, C., Manna, D., Cho, W. and Dubreuil, R. R. (2008). Unexpected complexity in the mechanisms that target assembly of the spectrin cytoskeleton. J. Biol. Chem. 283: 12643-12653. PubMed Citation: 18287096

DeBernardo, A. P. and Chang, S. (1995). Native and recombinant DM-GRASP selectively support neurite extension from neurons that express GRASP. Dev. Biol. 169(1): 65-75. PubMed Citation: 7750658

DeBernardo, A. P. and Chang, S. (1996). Heterophilic interactions of DM-GRASP: GRASP-NgCAM interactions involved in neurite extension. J. Cell Biol. 133(3): 657-66. PubMed Citation: 8636239

Del Bel, L. M., Griffiths, N., Wilk, R., Wei, H. C., Blagoveshchenskaya, A., Burgess, J., Polevoy, G., Price, J. V., Mayinger, P. and Brill, J. A. (2018). The phosphoinositide phosphatase Sac1 regulates cell shape and microtubule stability in the developing Drosophila eye. Development 145(11). PubMed ID: 29752385

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Fashena, D. and Westerfield, M. (1999). Secondary motoneuron axons localize DM-GRASP on their fasciculated segments. J. Comp. Neurol. 406(3): 415-24. PubMed Citation: 10102505

Forrest, S., Chai, A., Sanhueza, M., Marescotti, M., Parry, K., Georgiev, A., Sahota, V., Mendez-Castro, R. and Pennetta, G. (2013). Increased levels of phosphoinositides cause neurodegeneration in a Drosophila model of amyotrophic lateral sclerosis. Hum Mol Genet 22(13): 2689-2704. PubMed ID: 23492670

Fournier-Thibault, C., et al. (1999). BEN/SC1/DM-GRASP expression during neuromuscular development: a cell adhesion molecule regulated by innervation. J. Neurosci. 19(4): 1382-92. PubMed Citation: 9952415

Fuse, N., et al. (1999). Snail-type zinc finger proteins prevent neurogenesis in Scutoid and transgenic animals of Drosophila. Dev. Genes Evol. 209: 573-580. PubMed Citation: 10552298

Galletta, B. J., Chakravarti, M., Banerjee, R. and Abmayr, S. M. (2004). Sns: Adhesive properties, localization requirements and ectodomain dependence in S2 cells and embryonic myoblasts. Mech. Dev. 121(12): 1455-68. 15511638

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Laessing, U., et al. (1994). Molecular characterization of fish neurolin: a growth-associated cell surface protein and member of the immunoglobulin superfamily in the fish retinotectal system with similarities to chick protein DM-GRASP/SC-1/BEN. Differentiation 56(1-2): 21-9

Laessing, U. and Stuermer, C. A. (1996). Spatiotemporal pattern of retinal ganglion cell differentiation revealed by the expression of neurolin in embryonic zebrafish. J. Neurobiol. 29(1): 65-74

Larson, D. E., Liberman, Z. and Cagan, R. L. (2008). Cellular behavior in the developing Drosophila pupal retina. Mech Dev. 125(3-4): 223-32. PubMed Citation: 18166433

Lee, H. G., Zarnescu, D. C., MacIver, B. and Thomas, G. H. (2010). The cell adhesion molecule Roughest depends on βHeavy-spectrin during eye morphogenesis in Drosophila. J. Cell Sci. 123(Pt 2): 277-85. PubMed Citation: 20048344

Leppert, C. A., et al. (1999). Neurolin Ig domain 2 participates in retinal axon guidance and Ig domains 1 and 3 in fasciculation. J. Cell Biol. 144(2): 339-49

Machado, M. C., Octacilio-Silva, S., Costa, M. S. and Ramos, R. G. (2011). rst transcriptional activity influences kirre mRNA concentration in the Drosophila pupal retina during the final steps of ommatidial patterning. PLoS One 6(8): e22536. PubMed Citation: 21857931

Marco Antonio, D. S. and Hartfelder, K. (2016). Toward an understanding of divergent compound eye development in drones and workers of the honeybee (Apis mellifera L.): A correlative analysis of morphology and gene expression. J Exp Zool B Mol Dev Evol [Epub ahead of print]. PubMed ID: 27658924

Mehta, S. Q., et al. (2005). Mutations in Drosophila sec15 reveal a function in neuronal targeting for a subset of exocyst components. Neuron 46(2): 219-32. 15848801

Ott, H., Bastmeyer, M. and Stuermer, C. A. (1998). Neurolin, the goldfish homolog of DM-GRASP, is involved in retinal axon pathfinding to the optic disk. J. Neurosci. 18(9): 3363-72

Paschke, K. A., Lottspeich, F. and Stuermer, C. A. (1992). Neurolin, a cell surface glycoprotein on growing retinal axons in the goldfish visual system, is reexpressed during retinal axonal regeneration. J. Cell Biol. 117(4): 863-75

Pollerberg, G. E. and Mack, T. G. (1994). Cell adhesion molecule SC1/DMGRASP is expressed on growing axons of retina ganglion cells and is involved in mediating their extension on axons. Dev. Biol. 165(2): 670-87

Pourquie, O., et al. (1992). BEN, a surface glycoprotein of the immunoglobulin superfamily, is expressed in a variety of developing systems. Proc. Natl. Acad. Sci. 89(12): 5261-5

Ramos, R. G., et al. (1993). The irregular chiasm C-roughest locus of Drosophila, which affects axonal projections and programmed cell death, encodes a novel immunoglobulin-like protein. Genes Dev. 7(12B): 2533-47. PubMed Citation: 7503814

Reddy, G. V., et al. (1999). Irregular chiasm-C-roughest, a member of the immunoglobulin superfamily, affects sense organ spacing on the Drosophila antenna by influencing the positioning of founder cells on the disc ectoderm. Dev. Genes Evol. 209: 581-591. PubMed Citation: 10552299

Reiter, C., et al. (1996). Reorganization of membrane contacts prior to apoptosis in the Drosophila retina: the role of the IrreC-rst protein. Development 122(6): 1931-40. PubMed Citation: 8674431

Ringuette, M., et al. (1998). Expression of SC1 is associated with the migration of myotomes along the dermomyotome during somitogenesis in early mouse embryos. Dev. Genes Evol. 208(7): 403-6

Ruiz-Gomez, M., et al. (2000). Drosophila dumbfounded: a myoblast attractant essential for fusion. Cell 102: 189-198. 10943839

Schneider, T., et al. (1995). Restricted expression of the irreC-rst protein is required for normal axonal projections of columnar visual neurons. Neuron 15(2): 259-71

Shen, K. and Bargmann, C. I. (2003). The immunoglobulin superfamily protein SYG-1 determines the location of specific synapses in C. elegans. Cell 112: 619-630. 12628183

Sekine-Aizawa, Y., Omori, A. and Fujita, S. C. (1998). MuSC, a novel member of the immunoglobulin superfamily, is expressed in neurons of a subset of cranial sensory ganglia in the mouse embryo. Eur. J. Neurosci. 10(9): 2810-24. PubMed ID: 9758151

Seppa, M. J., Johnson, R. I., Bao, S. and Cagan, R. L. (2008). Polychaetoid controls patterning by modulating adhesion in the Drosophila pupal retina. Dev. Biol. 318(1): 1-16. PubMed Citation: 18423436

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Strünkelnberg, M., et al. (2001). rst and its paralogue kirre act redundantly during embryonic muscle development in Drosophila. Development 128: 4229-4239. 11684659

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date revised: 4 April 2022

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