roughest

DEVELOPMENTAL BIOLOGY

Embryonic

See the embryonic expression pattern of rst at the Berkeley Drosophila Genome Project Patterns of Gene Expression Site.

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).

Effects of Mutation or Deletion

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 rst^CT mutant. The rst^CT 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 rst^CT 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 rst^CT 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 rst^CT 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 rst^CT 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 ìantimorphicî 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, rst^D. In addition to showing evidence that rst^D 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 rst^D 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 rst^D 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 rst^D 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 rst^D pupae have persistent salivary glands more than 24 h after puparium formation. This phenotype is absent in rst^D 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 rst^D 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 rst^CT, 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 rst^D 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 rst^D 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 rst^D 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 rst^D 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 rst^D 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 rst^D rearrangement and, therefore, be responsible for at least some aspects of the rst^D 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 rst^D 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 rst^D 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 mbc^C1 genetic background. In mbc^C1 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)w^67k30 causes embryonic lethality and displays an almost complete lack of myoblast fusion. The genomic interval removed by Df(1)w^67k30 extends from white to kirre. As yet, there is no single embryonic lethal locus known within this region. Hence, the Df(1)w^67k30 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)w^67k30 phenotype. Therefore, removal of kirre is partly responsible for the Df(1)w^67k30 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)w^67k30 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)w^67k30 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 rst^irreC1 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)w^67k30 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).

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date revised: 20 September 2007

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