BIOLOGICAL OVERVIEW

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, (irreC^UB883 and In(1)irreC^1R34) 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 (1^o p.c.) show a slightly higher level of expression. After the pair of 1^o p.c. has surrounded the cone cells, the ommatidial lattice compacts. The protein now accumulates at the borders between 1^o 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/1^o 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 1^o p.c. In the primaries it retreats from the border with cone cells but initially remains present at the two contact sites between the 1^o 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 1^o 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 1^o 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 1^o 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 1^o 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 1^o 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/1^o 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 2^o 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/1^o 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 1^o 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 1^o 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 1^op.c, which are mediated by Rst. An additional mechanism that can resolve side by side doublings of IOC is the extension of the IOC/1^o 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/1^o p.c. border. In the absence of an attractive border, IOC would not be prevented from giving up contacts to 1^o 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 1^o p.c., suggesting that a heterophilic interaction is the underlying mechanism. Expression of a putative Rst ligand would be expected on the surface of 1^o 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).

GENE STRUCTURE

cDNA clone length - 4082 bp

Bases in 5' UTR - 204

Exons - 8

Bases in 3' UTR - 1585

PROTEIN STRUCTURE

Amino Acids - 1585

Structural Domains

Sequence alignment of the extracellular region shows Rst is composed of five repeats of a structural motif containing characteristic arrangements of cysteins and tryptophans, as well as other conserved amino acids typically found in members of the immunoglobulin superfamily. The length of the repeats varies from 82 to 100 amino acids, and the spacing between the two cysteins in each domain ranges from 60 residues, in the second and largest one, to 42, in the fourth and smallest one. The size and the conserved amino acids surrounding the cysteines make all five domains similar to the C2-type of immunoglobulin domains. Blast search shows the extracellular protein is related to a number of proteins of the IG superfamily, most notably a C. elegans predicted protein with 55% identity and three chicken axonal surface proteins (DM-GRASP, BEN and SCI) all three of which are likely to be encoded by the same gene. The Rst protein is also similar to human myelin-associated glycoprotein (MAG), human melanoma marker MUC18, human poliovirus receptor, chicken axonal protein SPF3, and Drosophila Neuroglian. None of these molecules aligns well through the whole length of the Rst protein. Although significant homology is found to the intracellular domain, a potentially significant finding is the presence in the Rst protein of the C-terminal pattern IYXXXYXRXXXXX[LM]LPXXXXX, characteristic of ligand-dependent tyrosine kinases. This pattern is contained at the putative site of autophosphorylation, which is highly conserved among the family of insulin receptor-like tyrosine kinases (Ramos, 1993).

date revised: 12 October 99

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