spalt-induced specification of distinct dorsal and ventral domains is required for Drosophila tracheal patterning

Morphogenesis of the Drosophila tracheal system relies on different signalling pathways that have distinct roles in specifying both the migration of the tracheal cells and the particular morphological features of the primary branches. The current view is that the tracheal cells are initially specified as an equivalent group of cells whose diversification depends on signals from the surrounding cells. In this work, we show that the tracheal primordia are already specified as distinct dorsal and ventral cell populations. This subdivision depends on the activity of the spalt (sal) gene and occurs prior to the activity of the signalling pathways that dictate the development of the primary branches. Finally, we show that the specification of these two distinct cell populations, which are not defined by cell lineage, are critical for proper tracheal patterning. These results indicate that tracheal patterning depends not only on signalling from surrounding cells but also in the different response of the tracheal cells depending on their allocation to the dorsal or ventral domains.     

The hernandez and fernandez genes of Drosophila specify eye and antenna

The formation of different structures in Drosophila depends on the combined activities of selector genes and signaling pathways. For instance, the antenna requires the selector gene homothorax, which distinguishes between the leg and the antenna and can specify distal antenna if expressed ectopically. Similarly, the eye is formed by a group of "eye-specifying" genes, among them eyeless, which can direct eye development ectopically. We report here the characterization of the hernandez and fernandez genes, expressed in the antennal and eye primordia of the eye-antenna imaginal disc. The predicted proteins encoded by these two genes have 27% common amino acids and include a Pipsqueak domain. Reduced expression of either hernandez or fernandez mildly affects antenna and eye development, while the inactivation of both genes partially transforms distal antenna into leg. Ectopic expression of either of the two genes results in two different phenotypes: it can form distal antenna, activating genes like homothorax, spineless, and spalt, and it can promote eye development and activates eyeless. Reciprocally, eyeless can induce hernandez and fernandez expression, and homothorax and spineless can activate both hernandez and fernandez when ectopically expressed. The formation of eye by these genes seems to require Notch signaling, since the induction of ectopic eyes and the activation of eyeless by the hernandez gene are suppressed when the Notch function is compromised. Our results show that the hernandez and fernandez genes are required for antennal and eye development and are also able to specify eye or antenna ectopically.     

Dorso-ventral asymmetric functions of teashirt in Drosophila eye development depend on spatial cues provided by early DV patterning genes

The teashirt (tsh) gene has dorso-ventral (DV) asymmetric functions in Drosophila eye development: promoting eye development in dorsal and suppressing eye development in ventral by Wingless mediated Homothorax (HTH) induction [Development 129 (2002) 4271]. We looked for DV spatial cues required by tsh for its asymmetric functions. The dorsal Iroquois-Complex (Iro-C) genes and Delta (Dl) are required and sufficient for the tsh dorsal functions. The ventral Serrate (Ser), but not fringe (fng) or Lobe (L), is required and sufficient for the tsh ventral function. We propose that DV asymmetric function of tsh represents a novel tier of DV pattern regulation, which takes place after the spatial expression patterns of early DV patterning genes are established in the eye.     

Genetic and biochemical analysis of brown eye mutation in Drosophila nasuta nasuta and Drosophila nasuta albomicans

By analyzing the progeny of crosses involving brown eye mutants and the wild types in two members of Drosophila nasuta subgroup namely D. n. nasuta and D. n. albomicans we could show that the mutant gene is recessive, located in the chromosome 2 and the alleles of this gene are present at different loci. A study of fitness in the eye color mutants in comparison with the wild types revealed that D. n. nasuta mutant has higher viability at both 25 ± 1°C and ambient temperatures; while D. n. albomicans mutant has faster rate of development only at 25 ± 1°C. Quantitative analysis of eye pigments in the mutants revealed that there is biosynthesis of both pteridines and xanthommatins unlike in bw/bw of D. melanogaster, where only xanthommatins are synthesized. In both the species, the pteridine quantities in mutants are similar; whereas xanthommatin quantity in is 10 times higher than that of . Further, the F₁ progeny of intraspecific crosses (wild type X mutant) are found to have high amounts of pteridine, even when compared with parental wild type. This revised version was published online in July 2006 with corrections to the Cover Date.     

Development of neural lineages derived from the sine oculis positive eye field of Drosophila

The Anlage of the Drosophila visual system, called eye field, comprises a domain in the dorso-medial neurectoderm of the embryonic head and is defined by the expression of the early eye gene sine oculis (so). Beside the eye and optic lobe, the eye field gives rise to several neuroblasts that contribute their lineages to the central brain. Since so expression is only very short lived, the later development of these neuroblasts has so far been elusive. Using the P-element replacement technique [Genetics, 151 (1999) 1093] we generated a so-Gal4 line driving the reporter gene LacZ that perdures in the eye field derived cells throughout embryogenesis and into the larval period. This allowed us to reconstruct the morphogenetic movements of the eye field derived lineages, as well as the projection pattern of their neurons. The eye field produces a dorsal (Pc1/2) and a ventral (Pp3) group of three to four neuroblasts each. In addition, the target neurons of the larval eye, the optic lobe pioneers (OLPs) are derived from the eye field. The embryonically born (primary) neurons of the Pp3 lineages spread out at the inner surface of the optic lobe. Together with the OLPs, their axons project to the dorsal neuropile of the protocerebrum. Pp3 neuroblasts reassume expression of so-Gal4 in the larval period and produce secondary neurons whose axonal projection coincides with the pattern formed by the primary Pp3 neurons. Several other small clusters of neurons that originate from outside the eye field, but have axonal connections to the dorsal protocerebrum, also express so and are labeled by so-Gal4 driven LacZ. We discuss the dynamic pattern of the so-positive lineages as a tool to reconstruct the morphogenesis of the larval brain.     

Genetic interactions between the Polycomb locus and the Antennapedia and Bithorax complexes of Drosophila

Summary We have studied the embryonic and adult phenotypes of genetic combinations between Polycomb (Pc), Regulator of bithorax (Rg-bx) and the genes of the Bithorax complex (BX-C) and the Antennapedia complex (ANT-C). The products of Pc and Rg-bx genes act antagonistically, their mutant combinations leading to the ectopic expression of genes of the BX-C and ANT-C. The genetic analysis of the Pc locus suggests it is a complex gene. Pc⁺ products behave as members of a regulatory set that negatively control the expression of BX-C and ANT-C genes. Genetic combinations between different doses of Pc, Rg-bx and the genes of the BX-C and ANT-C have phenotypes which may be interpreted as resulting from ectopic derepression of posterior selector genes repressing selector genes of anterior segments. The transformation phenotypes of certain genetic combinations differ in embryos and adults. A model of regulation of the BX-C and the ANT-C genes during the imaginal cell proliferation is presented, in which the specification state is maintained by self-activation of a given selector gene and down modulation of other selector genes in the same cell.     

CYFIP dependent actin remodeling controls specific aspects of Drosophila eye morphogenesis

Cell rearrangements shape organs and organisms using molecular pathways and cellular processes that are still poorly understood. Here we investigate the role of the Actin cytoskeleton in the formation of the Drosophila compound eye, which requires extensive remodeling and coordination between different cell types. We show that CYFIP/Sra-1, a member of the WAVE/SCAR complex and regulator of Actin remodeling, controls specific aspects of eye architecture: rhabdomere extension, rhabdomere terminal web organization, adherens junctions, retina depth and basement membrane integrity. We demonstrate that some phenotypes manifest independently, due to defects in different cell types. Mutations in WAVE/SCAR and in ARP2/3 complex subunits but not in WASP, another major regulator of Actin nucleation, phenocopy CYFIP defects. Thus, the CYFIP-SCAR-ARP2/3 pathway orchestrates specific tissue remodeling processes.     

The pleiohomeotic gene is required for maintaining expression of genes functioning in ventral appendage formation in Drosophila melanogaster

Polycomb group (PcG) proteins are negative regulators that maintain the expression of homeotic genes and affect cell proliferation. Pleiohomeotic (Pho) is a unique PcG member with a DNA-binding zinc finger motif and was proposed to recruit other PcG proteins to form a complex. The pho null mutants exhibited several mutant phenotypes such as the transformation of antennae to mesothoracic legs. We examined the effects of pho on the identification of ventral appendages and proximo-distal axis formation during postembryogenesis. In the antennal disc of the pho mutant, Antennapedia (Antp), which is a selector gene in determining leg identity, was ectopically expressed. The homothorax (hth), dachshund (dac) and Distal-less (Dll) genes involved in proximo-distal axis formation were also abnormally expressed in both the antennal and leg discs of the pho mutant. The engrailed (en) gene, which affects the formation of the anterior-posterior axis, was also misexpressed in the anterior compartment of antennal and leg discs. These mutant phenotypes were enhanced in the mutant background of Posterior sex combs (Psc) and pleiohomeotic-like (phol), which are another PcG genes. These results suggest that pho functions in maintaining expression of genes involved in the formation of ventral appendages and the proximo-distal axis.     

Phylogenetic relationships in the spoon tarsus subgroup of Hawaiian Drosophila: conflict and concordance between gene trees

The Hawaiian Drosophilidae contains approximately 1000 species, placed in species groups and subgroups based largely on secondary sexual modifications to wings, forelegs and mouthparts. Members of the spoon tarsus subgroup possess a cup-shaped structure on the foretarsi of males. Eight of the twelve species in this subgroup are found only on the Big Island of Hawaii, suggesting that they have diverged within the past 600,000 years. This rapid diversification has made determining the relationships within this group difficult to infer. We use 13 genes, including nine rapidly evolving nuclear loci, to estimate relationships within the spoon tarsus species, as well as to test the monophyly of this subgroup. A variety of analytical approaches are used, including individual and concatenated analyses, Bayesian estimation of species trees and Bayesian untangling of concordance knots. We find widespread agreement between phylogenetic estimates derived from different methods, although some incongruence is present. Notably, our analyses suggest that the spoon tarsus subgroup, as currently defined, is not monophyletic.     

Second-site modifiers of the split mutation of Notch define genes involved in neurogenesis in Drosophila melanogaster

Summary We have searched for dominant modifiers, i.e., enhancers and suppressors, of the compound eye phenotype of split, a recessive viable allele of Notch. Among the spl modifiers found, we have detected mutations in loci whose functions were previously known to cooperate with Notch in embryonic neurogenesis, such as daughterless, master mind, Delta and Hairless. In addition, other spl modifier mutations have been found in loci that were not previously known to interact with Notch, such as scabrous, glass, roughened eye, and several other genes that have not yet been assigned to known loci. The phenotypes associated with mutations in some of these latter loci suggest the participation of the corresponding genes in embryonic neurogenesis. We show that in some cases the observed interactions are due to genetic haplo-insufficent expression of the genes, whereas allele-specific interactions with spl are observed in master mind and Delta alleles. From this observation, we propose a direct functional association between the proteins encoded by Notch, Delta and master mind.     

Localized repressors delineate the neurogenic ectoderm in the early Drosophila embryo

The Dorsal gradient produces sequential patterns of gene expression across the dorsoventral axis of early embryos, thereby establishing the presumptive mesoderm, neuroectoderm, and dorsal ectoderm. Spatially localized repressors such as Snail and Vnd exclude the expression of neurogenic genes in the mesoderm and ventral neuroectoderm, respectively. However, no repressors have been identified that establish the dorsal limits of neurogenic gene expression. To investigate this issue, we have conducted an analysis of the ind gene, which is selectively expressed in lateral regions of the presumptive nerve cord. A novel silencer element was identified within the ind enhancer that is essential for eliminating expression in the dorsal ectoderm. Evidence is presented that the associated repressor can function over long distances to silence neighboring enhancers. The ind enhancer also contains a variety of known activator and repressor elements. We propose a model whereby Dorsal and EGF signaling, together with the localized Schnurri repressor, define a broad domain of ind expression throughout the entire presumptive neuroectoderm. The ventral limits of gene expression are defined by the Snail and Vnd repressors, while the dorsal border is established by the newly defined silencer element.     

Nucleotide divergence of the rp49 gene region between Drosophila melanogaster and two species of the Obscura group of Drosophila

Summary A 2.1-kb SStI fragment including the rp49 gene and the 3 end of the -serendipity gene has been cloned and sequenced in Drosophila pseudoobscura. rp49 maps at region 62 on the tip of chromosome II of this species. Both the coding and flanking regions have been aligned and compared with those of D. subobscura. There is no evidence for heterogeneity in the rate of silent substitution between the rp49 coding region and the rate of substitutions in flanking regions, the overall silent divergence per site being 0.19. Noncoding regions also differ between both species by different insertions/deletions, some of which are related to repeated sequences. The rp49 region of D. pseudoobscura shows a strong codon bias similar to those of D. subobscura and D. melanogaster. Comparison of the rates of silent (K _S ) and nonsilent (K _a ) substitutions of the rp49 gene and other genes completely sequenced in D. pseudoobscura and D. melanogaster confirms previous results indicating that rp49 is evolving slowly both at silent and nonsilent sites. According to the data for the rp49 region, D. pseudoobscura and D. subobscura lineages would have diverged some 9 Myr ago, if one assumes a divergence time of 30 Myr for the melanogaster and obscura groups.Offprint requests to: C. Segarra     

Larval cells become imaginal cells under the control of homothorax prior to metamorphosis in the Drosophila tracheal system

In Drosophila melanogaster, one of the most derived species among holometabolous insects, undifferentiated imaginal cells that are set-aside during larval development are thought to proliferate and replace terminally differentiated larval cells to constitute adult structures. Essentially all tissues that undergo extensive proliferation and drastic morphological changes during metamorphosis are thought to derive from these imaginal cells and not from differentiated larval cells. The results of studies on metamorphosis of the Drosophila tracheal system suggested that large larval tracheal cells that are thought to be terminally differentiated may be eliminated via apoptosis and rapidly replaced by small imaginal cells that go on to form the adult tracheal system. However, the origin of the small imaginal tracheal cells has not been clear. Here, we show that large larval cells in tracheal metamere 2 (Tr2) divide and produce small imaginal cells prior to metamorphosis. In the absence of homothorax gene activity, larval cells in Tr2 become non-proliferative and small imaginal cells are not produced, indicating that homothorax is necessary for proliferation of Tr2 larval cells. These unexpected results suggest that larval cells can become imaginal cells and directly contribute to the adult tissue in the Drosophila tracheal system. During metamorphosis of less derived species of holometabolous insects, adult structures are known to be formed via cells constituting larval structures. Thus, the Drosophila tracheal system may utilize ancestral mode of metamorphosis.     

The EGFR ligands Spitz and Keren act cooperatively in the Drosophila eye

The EGFR signalling cascade is responsible for coordinating a wide variety of events during Drosophila eye development. It remains something of a mystery how it is that cells are able to interpret the signal so as to choose the appropriate response from the battery of possibilities: division, differentiation, cell shape change and so on. Since the cascade is essentially linear below the receptor, different cellular responses cannot be regulated by alternative signal transduction pathways. The main diversity lies upstream, in the multiple activating ligands. Spitz, Gurken and Vein have been long studied, but little is known about the physiological functions of the fourth ligand, Keren, although various roles have been predicted based on the differences between mutants in the known ligands and those of the receptor. Here, we have isolated a mutant in the keren gene, and demonstrate that Keren does indeed participate in EGFR signalling in the eye, where it acts redundantly with Spitz to control R8 spacing, cell clustering and survival. Thus, specificity cannot be determined by ligand choice, and must instead be a consequence of cell-intrinsic factors, although we speculate that there may be some quantitative differences in signalling elicited by the two ligands.