Notch signalling is essential for animal development. It integrates multiple pathways controlling cell fate and specification. The paper reports the genetic characterization of Gliolectin, presumably a lectin, a cytoplasmic protein, significantly enriched in Golgi bodies. Its expression overlaps with regions where Notch is activated. Loss of gliolectin function results in ectopic veins, while gain of its function causes loss of wing veins. It positively regulates Enhancer of split mβ, a target of Notch signalling. These observations suggest that it is a positive regulator of Notch signalling during wing development in Drosophila (Prasad, 2015).

Gliolectin is a carbohydrate-binding protein (lectin) that mediates cell adhesion in vitro and is expressed by midline glial cells in the Drosophila embryo. Gliolectin expression is maximal during early pathfinding of commissural axons across the midline (stages 12-13), a process that requires extensive signaling and cell-cell interactions between the midline glia and extending axons. Deletion of the gliolectin locus disrupts the formation of commissural pathways and also delays the completion of longitudinal pathfinding. The disruption in commissure formation is accompanied by reduced axon-glial contact, such that extending axons grow on other axons and form a tightly fasciculated bundle that arches over the midline. By contrast, pioneering commissural axons normally cross the midline as a distributed array of fibers that interdigitate among the midline glia, maximizing contact and, therefore, communication between axon and glia. Restoration of Gliolectin protein expression in the midline glia rescues the observed pathfinding defects of null mutants in a dose-dependent manner. Hypomorphic alleles generated by ethylmethanesulfonate mutagenesis exhibit a similar phenotype in combination with a deletion and these defects are also rescued by transgenic expression of Gliolectin protein. The observed phenotypes indicate that carbohydrate-lectin interactions at the Drosophila midline provide the necessary surface contact to capture extending axons, thereby ensuring that combinatorial codes of positive and negative growth signals are interpreted appropriately (Sharrow, 2001).

An anti-Gliolectin monoclonal antibody, designated 1B7, detects Gliolectin expression at the midline of the embryonic ventral nerve cord from stage 12/3 through to early stage 15. Staining is particularly robust in the midline glial cells, MGA and MGM, from stages 12/1 through mid-13, when extending axons pioneer commissural and longitudinal pathways. Double-staining with 1B7 and the monoclonal antibody BP102, which recognizes an epitope shared by many CNS axons, demonstrates the intimate association between midline glial cells and commissural fibers as these axons cross the midline during late stage 12 and early stage 13. Embryos homozygous for a chromosomal deletion designated Delta3013 (breakpoints 93C6-94A1), lack the gliolectin locus and are not stained by monoclonal antibody 1B7 (Sharrow, 2001).

In wild-type embryos at late stage 12, neurons that pioneer commissural pathways extend processes that interdigitate among the midline glia. By mid stage 13, coordinated movements of axons and glial cells segregate the commissural mass into distinct anterior and posterior bundles. By contrast, Delta3013 homozygous embryos at stage 12/1 and early 13 form a commissural projection with a distinctively fused or monofilamentous appearance. The fused commissure phenotype persists later into stage 13 in the deletion homozygote when a pronounced absence of axons in the longitudinal pathways also becomes apparent. Quantitation of the fused commissure phenotype demonstrates that all deletion homozygote embryos are phenotypically abnormal with, on average, defects in greater than one-half of their segments (Sharrow, 2001).

The arched appearance of the fused commissures in late stage 12, Delta3013/Delta3013 embryo dissections implies that axons must follow aberrant routes across the midline. Thus, transverse sections through the ventral nerve cord reveal that commissural axons form a dense bundle at the dorsal boundary of the nerve cord in deletion homozygotes. By contrast, commissural axons interdigitate amongst the midline glia in wild-type embryos. Furthermore, commissural bundles appear thicker and the longitudinally oriented fibers are sparse in deletion embryos, indicating that commissural accumulation is at the expense of longitudinal mass. Although not quantitated, the width of the axon scaffold, as labeled by mAb BP102, is consistently greater in Delta3013 homozygotes than in age-matched wild-type embryos at late stage 12 through mid-stage 13. Segmental width is also altered in another mutation affecting axon-glial interactions at the Drosophila midline, suggesting that the integrity of segmental CNS architecture builds upon appropriate commissure formation (Sharrow, 2001).

EMS mutagenesis yielded ten homozygous lethal lines that failed to complement the lethality of Delta3013. When embryos collected from balanced, non-complementing EMS mutants were stained with BP102, axon pathfinding defects were not apparent. However, when crossed to the Delta3013 stock, two EMS lines, glec^m24 and glec^m98, both demonstrated fused and disrupted commissural architecture although less frequently than in the deletion homozygote. Therefore, the EMS-mutagenized chromosomes, glec^m24 and glec^m98, fail to rescue the loss-of-function phenotype observed in Delta3013/Delta3013 embryos (Sharrow, 2001).

The two EMS mutants present slightly different phenotypes. In particular, glec^m98 is characterized by greater commissural fusion and more severe loss of longitudinal mass than in glec^m24. Although embryos homozygous for either EMS mutation display no axonal phenotype, the two mutant chromosomes do affect the axon scaffold when combined. Embryos carrying both mutations exhibit commissural distortion, fusion and thinning as well as minimal loss of longitudinal mass. Since embryo collections from balanced EMS lines are normal and since the penetrance of the scored phenotypes in the sensitized background (glec^m24/Delta3013 or glec^m98/Delta3013) is intermediate between wild-type and Delta3013/Delta3013, both EMS alleles were classified as hypomorphs. However, it is unlikely that the effect of either mutation results solely from decreased gene product, because Gliolectin protein is detectable in both EMS mutants, even when placed over the Delta3013 chromosome. In addition, the ability of each EMS mutant to enhance the phenotype of the other suggests a synthetic interaction between altered molecular forms rather than a simple dose response (Sharrow, 2001).

The formation of longitudinal pathways is pioneered, in part, by the extension of the pCC and vMP2 axons from each segment towards the next anterior segment. Longitudinal extension of these axons is guided by growth cone repulsion from the midline, by positive interactions with laterally positioned intermediate targets (SP1 neurons and longitudinal glia) and by specific fasciculation with posteriorly-extending longitudinal processes (MP1 and dMP2). Several of the cells involved in longitudinal pathfinding are visualized with mAb 1D4 (anti-Fasciclin II), including pCC, vMP2, dMP2, MP1 and SP1. Early in longitudinal pathfinding, the pCC axon does not extend as far anteriorly in Delta3013 homozygotes or in glec^m98/Delta3013 embryos as in wild type; rather, it appears stalled after minimal outgrowth. Later (stage early 13), while the pCC/vMP2 process in wild type has completed its extension to the next segment, it frequently remains stalled and is often in intimate contact with 1D4-positive midline cells (MP1 and dMP2) in deletion embryos. In glec^m98/Delta3013 embryos, formation of the pCC/MP1 pathway is severely hampered with few pCC cells showing extension beyond the segment of origin. In slightly older Delta3013 homozygous embryos (stage late 13), many segments exhibit near normal extension of pCC to the next anterior segment. However, glec^m98/Delta3013 embryos continue to lack a formed pCC/MP1 pathway at this stage. Almost all Delta3013/Delta3013 embryos (95%) exhibited delayed longitudinal growth in, on average, more than one-half of their segments. Longitudinal pathfinding defects were not seen in glec^m24/Delta3013 embryos. The segmental broadening observed with mAb BP102 was not apparent with mAb 1D4 (Sharrow, 2001).

By mid- to late-stage 14, homozygous deletion embryos manifest additional morphological abnormalities that result in significant ventral nerve cord disruptions. In particular, the formation of midgut constrictions is blocked, leading to the generation of a large, poorly organized gut. The appearance of the midgut is identical to that seen in null alleles of tinman and bagpipe, which lie within the Delta3013 interval. Especially in upper abdominal segments, the midgut mass displaces and fragments the nerve cord, thereby generating both commissural and longitudinal discontinuities. However, the axonal phenotypes of younger embryos reported here are distributed evenly across the entire length of the nerve cord and are not clustered in the abdominal segments where gut-induced disruptions are consistently observed by late stage 14. Therefore, since this analysis focuses on stages of development which coincide with the peak of Gliolectin expression (stages 12-13) and precede the disruption of gut development, midgut malformation is excluded as a cause for the scored axonal phenotypes. In addition, transgenic rescue of the stage 12-13 axonal phenotype by rhomboid-driven Gliolectin expression, rescues neither the midgut defect nor later stage nerve cord discontinuities. Thus, the effect of genes other than glec that are also removed by the Delta3013 interval can be dissected away from the Gliolectin null phenotype. For the EMS hypomorphic alleles glec^m24 and glec^m98, similar gut disruptions were not evident in embryos collected from balanced stocks or from crosses to generate either EMS allele in a Delta3013 background (Sharrow, 2001).

To establish that fused commissure and delayed longitudinal phenotypes are attributable to altered glec function, Gliolectin was transgenically expressed (UAS-glec) at the midline of Delta3013/Delta3013 embryos under indirect control of the rhomboid promoter (rho-Gal4). Since rho is normally activated in cells that also express Gliolectin, rho-induced Glec closely reconstitutes wild-type expression. Although introduction of one copy of the UAS-glec transgene does not rescue the embryonic lethality associated with Delta3013 homozygosity, it does result in significant improvement in both the commissural organization and longitudinal outgrowth phenotypes. Commissural separation is more distinct and the intersegmental extension of longitudinal pioneers approaches completion on schedule. However, neither phenotype is completely rescued with only one copy of the UAS-glec transgene in Delta3013/Delta3013 embryos. Decreased longitudinal mass appears more resistant to rescue than commissural fusion. A single copy of UAS-glec completely rescues the axonal phenotypes of both glec^m24/Delta3013 and glec^m98/Delta3013 and transgenic Gliolectin expression rescues the lethality of both EMS mutants in the Delta3013 background. Construction of Delta3013/Delta3013 embryos in which a single rho-Gal4 element drives expression of Gliolectin from two copies of UAS-glec yields nearly complete rescue of delayed longitudinal outgrowth, decreased longitudinal mass and commissural disorganization associated with the loss-of-function phenotype (Sharrow, 2001).

In the absence of Gliolectin, midline repulsion of longitudinal axons is not abolished. However, as demonstrated by the behavior of the pCC axon, the efficiency of the repulsive response is reduced. The pCC axon is delayed in its anterior course, frequently exhibiting extended interactions with what are usually transient substrates. Such aberrant behavior indicates that Gliolectin normally provides sufficient contact between midline glia and filipodial extensions to ensure efficient repulsive signal transmission. Therefore, the observed glec^– phenotype predicts that partially penetrant midline-crossing defects associated with robo hypomorphic alleles should be enhanced by a concomitant reduction in Gliolectin (Sharrow, 2001).

Since loss of selectin-mediated interactions abolishes leukocyte extravasation while loss of Gliolectin does not completely abolish normal axon sorting, the parallels between selectin-mediated whole cell capture and Gliolectin-mediated axon capture are not absolute. Selectins, however, operate under conditions of hydrodynamic flow where cells are quickly swept away if not captured. The environment in which Gliolectin operates is more static; extending axons reside in appropriate regions of the developing nerve cord long enough to heed available signals, despite the loss of intimate glial contact. Under these conditions, loss of Gliolectin affects only the efficiency not the direction of axon pathfinding. Nonetheless, the important characteristic shared by Gliolectin- and Selectin-mediated carbohydrate binding is that they constitute the initial step in a process that leads to subsequent cellular responses (Sharrow, 2001) .