The non-stop expression pattern was examined in the target area as a first step toward assessing in which cells non-stop may function. In situ hybridization labeling of eye-brain complexes with non-stop antisense mRNA probes revealed weak expression in the optic lobe and higher expression in the lamina precursor cells. Since the resolution of in situ hybridization in the optic lobe is not high, protein expression was examined by placing a c-myc tag at the C terminus of the non-stop open reading frame in a genomic clone and the transgene was introduced into flies. Since this transgene rescues the projection phenotype and lethality associated with non-stop mutations and expression is similar in two independently derived transgenic lines, it is concluded that the transgene is expressed in a pattern similar to wild type. Anti-myc labeling reveals ubiquitous expression in the cytoplasm of cells in the optic lobe and central brain. Higher levels of expression, however, are observed in lamina precursor cells but not in differentiated lamina neurons. Non-stop is also expressed at higher levels in marginal, epithelial, and medulla glial cells adjacent to the lamina plexus (Poeck, 2001).
The simple cellular composition and array of distally pointing hairs has made the Drosophila wing a favored system for studying planar polarity and the coordination of cellular and tissue level morphogenesis. A gene expression screen was carried out to identify candidate genes that functioned in wing and wing hair morphogenesis. Pupal wing RNA was isolated from tissue prior to, during and after hair growth and used to probe Affymetrix Drosophila gene chips. 435 genes were identified whose expression changed at least 5 fold during this period and 1335 whose expression changed at least 2 fold. As a functional validation, 10 genes were chosen where genetic reagents existed but where there was little or no evidence for a wing phenotype. New phenotypes were found for 9 of these genes providing functional validation for the collection of identified genes. Among the phenotypes seen were a delay in hair initiation, defects in hair maturation, defects in cuticle formation and pigmentation and abnormal wing hair polarity. The collection of identified genes should be a valuable data set for future studies on hair and bristle morphogenesis, cuticle synthesis and planar polarity (Ren, 2005).
The expression of the non-stop (not) gene decreased 3.9 fold from 24 to 40 hrs. Mutations in not result in photoreceptor neurons projecting through the lamina instead of terminating there. The mutations also result in approximately 20% of ommatidia being misoriented -- a planar polarity phenotype. Strong alleles of not die as prepupae so not clones were examined in both adult and pupal wings. Large numbers of clones were induced. Perhaps 25% of wing cells are found in clones. All adult wings of this genotype had regions where there were cells that failed to form hairs or that had very small hairs. These were found only in proximal medial regions on the ventral wing surface. All such wings also had subtle polarity abnormalities; small groups of hairs with slightly abnormal polarity in all regions of the wing. Consistently finding such defects leads to the conclusion that these were due to not clones. Of 47 such wings examined 27 also contained multiple hair cells and a further 10 contained regions with planar polarity defects reminiscent of genes such as fz and dsh. When marked not clones were examined in pupal wings most, but not all, showed cells where hair differentiation was delayed or absent. Such clones were seen in all wing regions. It is suggested that all not clones have delayed hair formation. When the clones are located in wing regions where hairs normally form first (distal or peripheral regions) the hairs form later than normal but still have enough time to reach a relatively normal length. In contrast, when clones are located in regions where hair formation is normally late (proximal and medial regions on the ventral wing surface) not enough time remains prior to cuticle deposition to produce a normal hair. The not gene encodes a ubiquitin carboxyterminal hydrolase likely to function in the removal of ubiquitin from proteins during protein degradation (Ren, 2005).
Biochemical and genetic studies support a role for Non-stop acting as a ubiquitin-specific protease. Three ubiquitinated proteins of 29, 55, and 200 kDa accumulated in P-element insert mutations not1 and not2 extracts of third instar larval tissue, as assessed on Western blots probed with an anti-Ubiquitin antibody. Both the non-stop targeting defect and prepupal lethality are dominantly enhanced by loss of one copy of a proteasome subunit encoded by l(3)73Ai. This suggests that Non-stop functions to regulate the degradation of a substrate via a ubiquitin-dependent pathway (Poeck, 2001).
It was necessary to show that non-stop is not required in the eye to regulate R cell target selection. To examine the genetic requirement of non-stop in targeting, the FLP/FRT system was used to induce eye-specific mitotic recombination and the marker Rh1-taulacZ was used to assess R1-R6 projections in adult cryostat sections. The eyeless-FLP (eyFLP) transgene was used to provide FLP recombinase activity in dividing cells of eye imaginal discs. To increase the size and number of clones in the eye, the FRT chromosome carried either a recessive cell lethal mutation (rfc) or a Minute mutation [M(3)RpS174]. Using an eye pigment marker, it was estimated that about 60% (with Rfc) to 80% (with RpS) of the cells in mosaic eyes were homozygous mutant. As in wild type, all axons of Rh1-taulacZ-expressing R cells in non-stop mutant eye tissue terminate in the lamina. Minor defects in ommatidial polarity and cell number were observed in non-stop mosaic eyes. However, because the projections are normal, these defects cannot account for the R1-R6 targeting errors. In further support of this finding, the projections of non-stop mutant R cell axons into a wild-type target in third instar larvae, as visualized by mAb24B10, were largely indistinguishable from wild type. Thus, it is concluded that non-stop function is required in the target but not in R cell axons for normal layer selection (Poeck, 2001).
If non-stop were to act in the target tissue, then it could do so in two different ways. It could be directly required for the production of a targeting signal. Alternatively, it could be required for the development of the cells expressing targeting signals. To distinguish between these possibilities, the development of lamina neurons and glial cells was examined in the target area in non-stop mutants. A subpopulation of neuroblasts in the outer proliferation center, adjacent to the developing lamina, generates lamina precursor cells (LPCs) that, in response to inductive signals from R cell axons, give rise to neurons. Older R cell axon bundles are associated with columns of lamina neurons and are shifted posteriorly as additional R cell axon bundles enter the target region. Lamina neuron development in non-stop mutants was assessed using the early neuronal differentiation marker Dachshund, the late neuronal differentiation marker Elav, and an antibody to a Brain-specific homeobox (Bsh) protein. In addition, the organization of lamina neurons into columns was examined. As in wild type, Dachshund is expressed in LPCs and was maintained in differentiating lamina neurons in non-stop mutants. Elav is expressed in mature lamina neurons L1-L5, and anti-Bsh is restricted to L5. Lamina neurons in non-stop mutants form columns largely as in wild type. Hence, non-stop is not required for lamina neuron differentiation (Poeck, 2001).
R cell axons encounter different glial cell types in the eye disc and in the optic stalk as they project into the optic lobe. Glial cells in the larval eye-brain complex can be visualized using the nuclear glial cell-specific marker anti-Repo. R cell axons grow past subretinal glial cells at the base of the optic stalk as well as satellite glial cells, which are interspersed among lamina neurons. R1-R6 growth cones terminate between rows of epithelial (distal) and marginal (proximal) glial cells. A third row of glial cells (medulla glial cells) lies beneath the marginal glial cells, demarcating the boundary between the developing lamina and medulla. Analysis of glial cell morphology using specific Gal4 drivers to express cytoplasmic ß-galactosidase has shown that epithelial and marginal glial cells have a unique morphology in comparison with the other glial cell types in the larval visual system. In contrast to the long ensheathing processes of eye disc, satellite, and medulla glial cells, epithelial and marginal glial cells assume a cuboidal shape and elaborate numerous fine processes, especially from the surface juxtaposing R1-R6 growth cones within the lamina plexus. The close contact between these glial cells and R1-R6 growth cones is consistent with a role as intermediate target cells (Poeck, 2001).
The development of glial cells in non-stop homozygous mutant eye-brain complexes was assessed using the marker anti-Repo. While glial cells found in the eye imaginal disc, optic stalk, and the entrance to the optic lobe appear normal in non-stop mutants, the rows of epithelial, marginal, and medulla glial cells are severely disrupted. The number of Repo-positive glial cells surrounding the lamina plexus is reduced in non-stop when compared to wild type. Whereas an average of 55 epithelial, marginal, and medulla glial cells is found in optical sections of third instar larval optic lobes in wild type, only about 20 glial cells are observed in non-stop mutants. The number of glial cells in wild type was determined using single optical sections, while the number of these cells in non-stop mutants was assessed using a merged image comprising between 4 and 11 1 µm thick optical sections. Hence, this difference is an underestimate (Poeck, 2001).
Glial cells in the target area originate from glial precursor cell areas that are located at the most dorsal and ventral edges of the R cell projection field on the surface of the optic lobe. Glial cells migrate to their characteristic positions along the lamina plexus. In wild type, a small number of migrating glial cells express Repo as they enter the R cell projection field. In non-stop mutants, however, a marked increase (57%; wild type, n = 13; non-stop, n = 31) is observed in the number of Repo-expressing glial cells in this region. This indicates that non-stop, either directly or indirectly, affects the migration of epithelial, marginal, and medulla glial cells into the target region (Poeck, 2001).
Martin, K. A., Poeck, B., Roth, H., Ebens, A. J., Conley Ballard, L. and Zipursky, S. L. (1995). Mutations disrupting neuronal connectivity in the Drosophila visual system. Neuron 14: 229-240. 7857635
Poeck, B., Fischer, S., Gunning, D., Zipursky, S. L. and Salecker, I. (2001). Glial cells mediate target layer selection of retinal axons in the developing visual system of Drosophila. Neuron 29: 99-113. 11182084
Ren, N., Zhu, C., Lee, H. and Adler, P. N. (2005). Gene expression during Drosophila wing morphogenesis and differentiation. Genetics 171(2): 625-38. 15998724
Rørth, P., Szabo, K., and Texido, G. (2000). The level of C/EBP protein is critical for cell migration during Drosophila oogenesis and is tightly controlled by regulated degradation. Mol. Cell 6: 23-30.
date revised: 5 November 2005
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