Twinfilin is required for actin-dependent developmental processes in Drosophila.

The actin cytoskeleton is essential for cellular remodeling and many developmental and morphological processes. Twinfilin is a ubiquitous actin monomer-binding protein whose biological function has remained unclear. We discovered and cloned the Drosophila twinfilin homologue, and show that this protein is ubiquitously expressed in different tissues and developmental stages. A mutation in the twf gene leads to a number of developmental defects, including aberrant bristle morphology. This results from uncontrolled polymerization of actin filaments and misorientation of actin bundles in developing bristles. In wild-type bristles, twinfilin localizes diffusively to cytoplasm and to the ends of actin bundles, and may therefore be involved in localization of actin monomers in cells. We also show that twinfilin and the ADF/cofilin encoding gene twinstar interact genetically in bristle morphogenesis. These results demonstrate that the accurate regulation of size and dynamics of the actin monomer pool by twinfilin is essential for a number of actin-dependent developmental processes in multicellular eukaryotes.

The Drosophila VEGF receptor homolog is expressed in hemocytes.

Several signalling pathways have been defined by studies of genes originally characterised in Drosophila. However, some mammalian signalling systems have so far escaped discovery in the fly. Here, we describe the identification and characterisation of fly homologs for the mammalian vascular endothelial growth factor/platelet derived growth factor (VEGF/PDGF) and the VEGF receptor. The Drosophila factor (DmVEGF-1) gene has two splice variants and is expressed during all stages, the signal distribution during embryogenesis being ubiquitous. The receptor (DmVEGFR) gene has several splice variants; the variations affecting only the extracellular domain. The most prominent form is expressed in cells of the embryonic haematopoietic cell lineage, starting in the mesodermal area of the head around stage 10 of embryogenesis. Expression persists in hemocytes as embryonic development proceeds and the cells migrate posteriorly. In a fly strain carrying a deletion uncovering the DmVEGFR gene, hemocytes are still present, but their migration is hampered and the hemocytes remain mainly in the anterior end close to their origin. These data suggest that the VEGF/PDGF signalling system may regulate the migration of the Drosophila embryonic haemocyte precursor cells.

Electron micrograph map of the Drosophila melanogaster polytene chromosome 3R divisions 91 through 100.

The banding pattern of the distal half of the polytene salivary gland 3R chromosome of Drosophila melanogaster was studied by means of the thin section electron microscopy. Bands were identified according to the revised light microscopic map of Bridges. Bridges' map contains 332 single bands and 137 double bands within the region 91 through 100. This makes a total of 606 bands when the doublets are counted as two bands each, but 469 bands when the doublets are counted as one band. In the electron micrographs we found a total of 443 bands within this region. 109 Bridges' singlets were easily detected in almost all thin sections, while 144 mainly faint bands could be seen only in some micrographs. 79 Bridges' single bands and one doublet (94D7-8) could not be found. 42 Bridges' doublets were made up of two separate bands each, 87 Bridges' doublets looked single, and three pairs of Bridges' doublets formed dark complexes in the thin sections. The telomere region with the most distal band 100F4-5 was gray. A total of 15 new bands, which are not drawn on Bridges' map, were detected. Most of the new bands were in the divisions 96 and 99.

Polytene chromosomes show normal gene activity but some mRNAs are abnormally accumulated in the pseudonurse cell nuclei of Drosophila melanogaster otu mutants.

Certain mutant alleles of the ovarian tumor (otu) locus give rise to polytene chromosomes in the pseudonurse cells (PNCs). We have previously shown that the banding pattern of these germ line-derived chromosomes is similar to that in the larval salivary gland chromosomes. In this study, we have examined the gene activity of these chromosomes. General gene expression from these chromosomes was studied by uridine autoradiography. The expression of specific genes was monitored by in situ hybridisation to mRNA and also by combining enhancer trap lines with otu mutants. We found that most of the genes studied were expressed in the PNCs as they were in the wild-type nurse cells. Four out of the 12 mRNAs studied accumulated in the nuclei instead of migrating to the cytoplasm. The intensity of accumulation directly correlated with the extent of polytenisation in the PNC nuclei. We suggest that the otu mRNA remains partly attached to the polytene chromosome template after transcription and discuss the effects of this phenomenon on polytenisation of the PNC chromosomes.

Two otu transcripts are selectively localised in Drosophila oogenesis by a mechanism that requires a function of the otu protein.

The ovarian tumour gene (otu) is required for several processes during Drosophila oogenesis. The locus encodes two protein isoforms that have been proposed to act during different stages of oogenesis. Here we show that the corresponding otu mRNAs display a dynamic pattern of expression during oogenesis. The 4.1 kb mRNA encoding the 104 kDa isoform is expressed throughout adult oogenesis, but is mainly restricted to nurse cells. The 3.2 kb mRNA encoding the 98 kDa protein isoform is selectively localised in the oocyte up to stage 9. Both mRNAs are expressed abundantly in nurse cells at stages 10-11. We propose that the oocyte-specific function of otu is realised by the 98 kDa isoform. We show that the export of the 3.2 kb mRNA from the nurse cell nuclei requires a functional otu protein. The otu protein is also required for the correct distribution of the pumilio and oskar mRNAs, while the Bic-D, K10 and staufen mRNAs are localised in wild type fashion in otu mutants. Furthermore, we have observed a region of homology between the carboxy-terminal part of the otu protein and the mammalian microtubule associated proteins. The more severe the mutation in this region of homology, the more disturbed mRNA distribution is observed in otu mutants.

Polytene chromosomes from ovarian pseudonurse cells of the Drosophila melanogaster otu mutant. II. Photographic map of the X chromosome.

The banding pattern of the polytene chromosomes of the ovarian pseudonurse cells (PNC) of the Drosophila melanogaster otu mutant were compared with larval salivary gland (SG) polytene chromosomes. The X chromosome was studied and no significant differences were found in the banding pattern between these functionally very different tissues. Most of the differences result from differential puffing activity. In situ hybridisation with five different DNA probes located along the X chromosome was used to cross-check the results obtained by morphological mapping. The constrictions present in the SG chromosomes were found to be absent in the germ line derived PNC chromosomes. There are prominent puffs in the PNC chromosomes at certain locations where genes known to be transcriptionally active in the germ line reside. This suggests that at least some of the genes active in the wild-type nurse cells may also be active in the PNC cells.

Electron micrograph map of the Drosophila melanogaster polytene chromosome 3R divisions 81 through 90.

The banding pattern of the proximal half of the polytene salivary gland 3R chromosome of Drosophila melanogaster was studied with thin section electron microscopy. Bands were identified according to Bridges' revised light microscopic map, which contains 330 single and 121 double bands within the regions 81 through 90. We found a total of 447 bands in this region. 97 Bridges' single bands were easily detected in almost all thin sections, while 177 faint bands could be seen only in some micrographs. 56 Bridges' single bands could not be found. 32 Bridges' doublets were made up of two separate bands each in thin sections. The other 89 Bridges' doublets seemed to be either single bands or remained obscure. A total of 20 small new bands, which were not drawn on Bridges' map, were detected.

Electron microscopic analysis of the banding pattern in the salivary gland chromosomes of Drosophila melanogaster. Divisions 11 through 20 of X.

The banding pattern of the proximal half of the polytene salivary gland X chromosome of Drosophila melanogaster was studied using thin section electron microscopy. The bands were identified according to Bridges' revised light microscopic map. On Bridges' map, the divisions 11 to 20 contain 112 single bands and as many as 181 double bands. A majority of Bridges' single bands were identified in the thin sections. A total of 23 Bridges' single bands (and 4 bands of division 20) could not be found; in particular, bands were missing from the difficult regions 11DE, 15A, 15C and 16A. Electron microscopy showed the existence of 18 additional faint bands, 4 at the region 18D and 7 at 19EF. Bridges' faintest single bands and the new bands were best seen in formaldehyde fixed material. About 1/4 of Bridges' double bands were found to be made up of two separate bands each. The remaining Bridges' doublets include all kinds of bands: broad, narrow, dark, faint, puffed. Many of them look single in thin sections.

Analyses of the Drosophila quit, ovarian tumor and shut down mutants in oocyte differentiation using in situ hybridisation.

We have studied the role of three loci, quit, ovarian tumor and shut down during oocyte differentiation in Drosophila by using in situ hybridisation and double mutant analyses. Mutations in qui and otu disturb the cystocyte divisions and the oocyte determination, while mutations in shu affect the cystocyte integrity, nevertheless allowing differentiation of normal-looking egg chambers with an oocyte. In all mutants the transport of molecules towards the posterior end of the egg chamber takes place as revealed by the accumulation of Bic-D or K10 transcripts. We show that the transport is ineffective in the qui and otu mutants apparently due to the lack of a properly differentiated oocyte. In the shu mutant the transport collapses and the oocyte is lost, leading to egg chambers with 15 nurse cells. We also show that one function of qui+ is to enhance otu+ mRNA expression, suggesting that these genes control the cystocyte maturation via the same pathway.

Effect of otu mutations on male fertility and spermatogenesis in Drosophila melanogaster.

The 17-ethyl-methyl-sulphonate (EMS) induced female sterile alleles of the ovarian tumour (otu) locus show a wide spectrum of phenotypes and affect various processes of Drosophila oogenesis. These phenotypes have been previously studied in detail, but the exact molecular function of the otu locus in the different processes of oogenesis is only poorly known. To date, no effect of otu mutations have been reported in the males. However, separate species of otu mRNAs are expressed in the testes and the thorax of the adult male, but their role is not known. In this study we analysed the effects of EMS-induced otu mutations on male fertility. We observed that the proportion of totally sterile males is significantly higher in most of the tested otu strains as compared to the wild type. There was a strong correlation between male sterility and severity of impairment in the female phenotype. Spermatogenesis of these semi-sterile strains was analysed by phase contrast microscopy, Hoechst 33258 and Feulgen stain, and by in situ hybridisation with testis-specific probes. No changes which could account for the induction of sterility were recorded and normal amounts of motile sperm were observed in all strains. Sterility turned out to be a consequence of a failure in mating behaviour. The wild type females refused to react to the courtship attempts of the mutant males. We propose two alternative explanations for this. Either the otu locus may play some important role in male somatic tissue, or some germ line function is necessary for correct mating behaviour.

Electron micrograph maps of divisions 51 through 60 of thin sectioned polytene 2R chromosome of Drosophila melanogaster.

The banding pattern of the distal half, i.e., the divisions 51 through 60, of the salivary gland 2R chromosome in Drosophila melanogaster was studied using thin section electron microscopy. This chromosome region contains 280 single and 157 double bands on the Bridges' revised light microscopic map. Most of the single bands were identified in thin sections; 34 bands were regarded as missing or they could not be reliably shown. In addition, about 20 new, mainly very faint single bands were found. 21 Bridges' double bands were found to be made up of two separate bands each in thin sections.

Polytene chromosomes from ovarian pseudonurse cells of the Drosophila melanogaster otu mutant. I. Photographic map of chromosome 3.

Certain mutant alleles of the otu locus in Drosophila melanogaster produce abnormal nurse cells in the ovaries. These cells are called pseudonurse cells (PNC), since they generate polytene chromosomes instead of endopolyploid ones and do not normally have an oocyte to nurse. The banding pattern of polytene chromosome 3 from the salivary glands (SG) and from PNCs of homozygous otu1 females was compared and a detailed photomap of PNC chromosomes with different degrees of polyteny is presented. The banding pattern was found to be strikingly similiar in the two tissues. The puffing pattern of the PNC chromosomes is discussed. No constrictions or breaks were found in the PNC chromosomes which seems to indicate that these sites, which are known to be underreplicated in the SG chromosomes, are equally replicated along with the rest of the chromosomes in the PNC nuclei.

Electron microscopic photomap of divisions 64 through 70 of the salivary gland 3L chromosome in Drosophila melanogaster.

The study is a part of an electron microscopic mapping project on the salivary gland chromosomes of D. melanogaster. Chromosomes were fixed with acetic methanol and, alternatively, with formaldehyde-Ringer solution, squashed and thin sectioned for electron microscopy. The banding pattern was compared with the drawn light microscopic map of Bridges, which shows 209 single and 93 double bands within the divisions 64 through 70. Thirteen Bridges' singlets could not be consistently documented in the electron micrographs. About 50 new, mainly very faint bands were found, most of them following formaldehyde fixation. Again, about 40 bands marked double by Bridges were interpreted to be two separate single bands. This paper completes the electron microscopic mapping of the 3L chromosome.