Patterning defects in the primary axonal scaffolds caused by the mutations of the extradenticle and homothorax genes in the embryonic Drosophila brain

During early brain development in Drosophila a highly stereotyped pattern of axonal scaffolds evolves by precise pioneering and selective fasciculation of neural fibers in the newly formed brain neuromeres. Using an axonal marker, Fasciclin II, we show that the activities of the extradenticle (exd) and homothorax (hth) genes are essential to this axonal patterning in the embryonic brain. Both genes are expressed in the developing brain neurons, including many of the tract founder cluster cells. Consistent with their expression profiles, mutations of exd and hth strongly perturb the primary axonal scaffolds. Furthermore, we show that mutations of exd and hth result in profound patterning defects of the developing brain at the molecular level including stimulation of the orthodenticle gene and suppression of the empty spiracles and cervical homeotic genes. In addition, expression of a Drosophila Pax6 gene, eyeless, is significantly suppressed in the mutants except for the most anterior region. These results reveal that, in addition to their homeotic regulatory functions in trunk development, exd and hth have important roles in patterning the developing brain through coordinately regulating various nuclear regulatory genes, and imply molecular commonalities between the developmental mechanisms of the brain and trunk segments, which were conventionally considered to be largely independent. Received: 4 October 1999 / Accepted: 10 January 2000     

Natural hyperthermia and expression of the heat shock protein Hsp70 affect developmental abnormalities in Drosophila melanogaster

We demonstrate that natural heat stress on wild larval Drosophila melanogaster results in severe developmental defects in >10% of eclosing adults, and that increased copy number of the gene encoding the major inducible heat shock protein of D. melanogaster, Hsp70, is sufficient to reduce the incidence of such abnormalities. Specifically, non-adult D. melanogaster inhabiting necrotic fruit experienced severe, often lethal heat stress in natural settings. Adult flies eclosing from wild larvae that had survived natural heat stress exhibited severe developmental anomalies of wing and abdominal morphology, which should dramatically affect fitness. The frequency of developmental abnormalities varied along two independent natural thermal gradients, exceeding 10% in adults eclosing from larvae developing in warm, sunlit fruit. When exposed to natural heat stress, D. melanogaster larvae with the wild-type number of hsp70 genes (n=10) developed abnormal wings significantly more frequently than a transgenic sister strain with 22 copies of the hsp70 gene. Received: 19 April 1999 / Accepted: 16 July 1999     

The wingless gene is required for embryonic brain development in Drosophila

 We have studied the role of the wingless gene in embryonic brain development of Drosophila. wingless is expressed in a large domain in the anlage of the protocerebrum and also transiently in smaller domains in the anlagen of the deutocerebrum and tritocerebrum. Elimination of the wingless gene in null mutants has dramatic effects on the developing protocerebrum; although initially generated, approximately one half of the protocerebrum is deleted in wingless null mutants by apoptotic cell death at late embryonic stages. Using temperature sensitive mutants, a rescue of the mutant phenotype can be achieved by stage-specific expression of functional wingless protein during embryonic stages 9–10. This time period correlates with that of neuroblast specification but preceeds the generation and subsequent loss of protocerebral neurons. Ectopic wingless over-expression in gain-of-function mutants results in dramatically oversized CNS. We conclude that wingless is required for the development of the anterior protocerebral brain region in Drosophila. We propose that an important role of wingless in this part of the developing brain is the determination of neural cell fate. Received: 7 October 1997 / Accepted: 30 December 1997     

Distinct functions of the Drosophila genes Serrate and Delta revealed by ectopic expression during wing development

 The Drosophila gene Serrate encodes a transmembrane protein with 14 epidermal growth factor-(EGF)-like repeats in its extracellular portion. It has been suggested to act as a signal in the developing wing from the dorsal side to induce the organising centre at the dorsal/ventral compartment boundary, which is required for growth and patterning of the wing. Ectopic expression of Serrate during wing development induces ectopic outgrowth of ventral wing tissue and the formation of an additional wing margin. Here we present data to suggest that both events are mediated by genes that are required for normal wing development, including Notch as receptor. In order for Serrate to elicit these responses the concomitant expression of wingless seems to be required. The lack of wings in flies devoid of Serrate function can be partially restored by Gal4-mediated expression of Serrate, whilst expression of wingless is not sufficient. Ectopic expression of Delta, which encodes a structurally very similar transmembrane protein with EGF-like repeats, provokes wing outgrowth and induction of a new margin under all conditions tested here, both on the dorsal and ventral side. Our data further suggest that Serrate can act as an activating ligand for the Notch receptor only under certain circumstances; it inhibits Notch function under other conditions. Received: 26 april 1996 / Accepted: 24 May 1996     

The genital disc of Drosophila melanogaster

 The genital disc of Drosophila, which gives rise to the genitalia and analia of adult flies, is formed by cells from different embryonic segments. To study the organization of this disc, the expressions of segment polarity and homeotic genes were investigated. The organization of the embryonic genital primordium and the requirement of the engrailed and invected genes in the adult terminalia were also analysed. The results show that the three primordia, the female and male genitalia plus the analia, are composed of an anterior and a posterior compartment. In some aspects, each of the three primordia resemble other discs: the expression of genes such as wingless and decapentaplegic in each anterior compartment is similar to that seen in leg discs, and the absence of engrailed and invected cause duplications of anterior regions, as occurs in wing discs. The absence of lineage restrictions in some regions of the terminalia and the expression of segment polarity genes in the embryonic genital disc suggest that this model of compartmental organization evolves, at least in part, as the disc grows. The expression of homeotic genes suggests a parasegmental organization of the genital disc, although these genes may also change their expression patterns during larval development. Received: 4 February 1997 / Accepted: 22 May 1997     

Molecular thermal telemetry of free-ranging adult Drosophila melanogaster

The expression of two temperature-sensitive reporter genes, hsp70 and an hsp70-LacZ fusion, in free-ranging adult Drosophila melanogaster indicates that natural thermal stress experienced by such small and mobile insects may be either infrequent or not severe. Levels of the heat-shock protein Hsp70, the major inducible Hsp of Drosophila, were similar in most wild Droso- phila captured after warm days to levels previously reported for unstressed flies in the laboratory. In a transgenic strain transformed with an hsp70-LacZ fusion (i.e., the structural gene encoding bacterial β-galactosidase under control of a heat shock promoter), exposure to temperatures ≥32°C in the laboratory typically resulted in β-galactosidase activities exceeding 140 mOD₄₅₀ h^–1μg^–1 soluble protein. Flies caged in sun frequently had β-galactosidase activities in excess of this level, whereas flies caged in shade and flies released and recaptured on cool days did not. Most flies (>80%) released on warm, sunny days had low β-galactosidase activities upon recapture. Although the balance of recaptured flies had elevated β-galactosidase activities on these days, their β-galactosidase activities were <50% of levels for flies caged in direct sunlight or exposed to laboratory heat shock. These data suggest that even on warm days most flies may avoid thermal stress, presumably through microhabitat selection, but that a minority of adult D. melanogaster undergo mild thermal stress in nature. Both temperature-sensitive reporter genes, however, are limited in their ability to infer thermal stress and demonstrate its absence. Received: 14 July 1999 / Accepted: 21 December 1999     

The yan gene is highly conserved in Drosophila and its expression suggests a complex role throughout development

 Competence for cell fate determination and cellular differentiation is under tight control of regulatory genes. Yan, a nuclear target of receptor tyrosine kinase (RTK) signaling, is an E twenty six (ETS) DNA-binding protein that functions as a negative regulator of cell differentiation and proliferation in Drosophila. Most members of RTK signaling pathways are highly conserved through evolution, yet no yan orthologues have been identified to date in vertebrates. To investigate the degree of yan conservation during evolution, we have characterized a yan homologue from a sibling species of D. melanogaster, D. virilis. Our results show that the organization, primary structure and expression pattern of yan are highly conserved. Both genes span over 20 kb and contain four exons with introns at identical positions. The areas with highest amino acid similarity include the Pointed and ETS domain but there are other discrete regions with a high degree of similarity. Phylogenetic analysis reveals that yan’s closest relative is the human tel gene, a negative regulator of differentiation in hematopoetic precursors. In both species, Yan is dynamically expressed beginning as early as stage 4/5 and persisting throughout embryogenesis. In third instar larvae, Yan is expressed in and behind the morphogenetic furrow of the eye imaginal disc as well as in the laminar precursor cells of the brain. Ovarian follicle cells also contain Yan protein. Conservation of the structure and expression patterns of yan genes strongly suggests that regulatory mechanisms for their expression are also conserved in these two species. Received: 3 November 1998 / Accepted: 9 December 1998     

Apoptosis of nurse cells at the late stages of oogenesis of Drosophila melanogaster

 In Drosophila a remarkable feature of oogenesis is the regression of the nurse cells after dumping their cytoplasmic contents into the oocyte. We have studied the nature of this process at the late stages of egg chamber development. In egg chambers DAPI staining shows highly condensed chromatin from stage 12 and TUNEL labelling shows DNA fragmentation up to stage 14. Gel electrophoresis of the end-labelled DNA, extracted from isolated egg chambers at the same stages of development, shows a ladder typical of apoptotic nuclei. This provides evidence that, during Drosophila oogenesis, the nurse cells undergo apoptosis. Apoptotic nuclei have also been detected in dumping-defective egg chambers, indicating that the cytoplasmic depletion of nurse cells is concurrent with but apparently not the cause of the process. Received: 12 December 1997 / Accepted: 6 January 1998     

An amphioxus netrin gene is expressed in midline structures during embryonic and larval development

Members of the netrin gene family have been identified in vertebrates, Drosophila and Caenorhabditis elegans and found to encode secreted molecules involved in axon guidance. Here I use the conserved function of netrins in triploblasts, coupled with the phylogenetic position of amphioxus (the closest living relative of the vertebrates), to investigate the evolution of an axon guidance cue in chordates. A single amphioxus netrin gene was isolated by PCR and cDNA library screening and named AmphiNetrin. The predicted AmphiNetrin protein showed high identity to other netrin family members but differed in that the third of three EGF repeats found in other netrins was absent. Molecular phylogene-tic analysis showed that despite the absent EGF repeat AmphiNetrin is most closely related to the vertebrate netrins. AmphiNetrin expression was identified in embryonic notochord and floor plate, a pattern similar to that of vertebrate netrin-1 expression. AmphiNetrin expression was also identified more widely in the posterior larval brain, and in the anterior extension of the notochord that underlies the anterior of the amphioxus brain. All of these areas of expression are correlated with developing axon trajectories: The floor plate with ventrally projecting somatic motor neurons and Rohde cell projections, the posterior brain with the ventral commissure and primary motor centre and the anterior extension of the notochord with ventrally projecting neurons associated with the median eye. Amphioxus is naturally cyclopaedic and also lacks the ventral brain cells that the induction of which results in the splitting of the vertebrate eye field and, when missing, result in cyclopaedia. These cells normally express netrins required for developing axon tracts in the brain, and the expression of AmphiNetrin in the anterior extension of the notochord underlying the brain may explain how amphioxus is able to maintain ventral guidance cues while lacking these cells. Received: 15 November 1999 / Accepted: 27 January 2000     

Glial cells phagocytose neuronal debris during the metamorphosis of the central nervous system in Drosophila melanogaster

 Using electron microscopy we demonstrate that degenerating neurons and cellular debris resulting from neuronal reorganization are phagocytosed by glial cells in the brain and nerve cord of the fruitfly Drosophila melanogaster during the first few hours following pupariation. At this stage several classes of glial cells appear to be engaged in intense phagocytosis. In the cell body rind, neuronal cell bodies are engulfed and phagocytosed by the same glial cells that enwrap healthy neurons in this region. In the neuropil, cellular debris in tracts and synaptic centres resulting from metamorphic re-differentiation of larval neurons is phagocytosed by neuropil-associated glial cells. Phagocytic glial cells are hypertrophied, produce large amounts of lysosome-like bodies and contain a large number of mitochondria, condensed chromatin bodies, membranes and other remains from neuronal degeneration in phagosomes. Received: 23 January 1996 / Accepted in revised form: 21 May 1996     

Hox C6 expression during development and regeneration of forelimbs in larval Notophthalmus viridescens

 A central theme concerning the epimorphic regenerative potential of urodele amphibian appendages is that limb regeneration in the adult parallels larval limb development. Results of previous research have led to the suggestion that homeobox containing genes are ”re-expressed” during the epimorphic regeneration of forelimbs of adult Notophthalmus viridescens in patterns which retrace larval limb development. However, to date no literature exists concerning expression patterns of any homeobox containing genes during larval development of this species. The lack of such information has been a hindrance in exploring the similarities as well as differences which exist between limb regeneration in adults and limb development in larvae. Here we report the first such results of the localization of Hox C6 (formerly, NvHBox-1) in developing and regenerating forelimbs of N. viridescens larvae as demonstrated by whole-mount in situ hybridization. Inasmuch as the pattern of Hox C6 expression is similar in developing forelimb buds of larvae and epimorphically regenerating forelimb blastemata of both adults and larvae, our results support the paradigm that epimorphic regeneration in adult newts parallels larval forelimb development. However, in contrast with observations which document the presence of Hox C6 in both intact, as well as regenerating hindlimbs and tails of adult newts, our results reveal no such Hox C6 expression during larval development of hindlimbs or the tail. As such, our findings indicate that critical differences in larval hindlimb and tail development versus adult expression patterns of this gene in these two appendages may be due primarily to differences in gene regulation as opposed to gene function. Thus, the apparent ability of urodeles to regulate genes in such a highly co-ordinated fashion so as to replace lost, differentiated, appendicular structures in adult animals may assist, at least in part, in better elucidating the phenomenon of epimorphic regeneration. Received: 6 November 1998 / Accepted: 12 December 1998     

A new family of adenylyl cyclase genes in the male germline of Drosophila melanogaster

We describe the cloning and characterization of a new gene family of adenylyl cyclase related genes in Drosophila. The five adenylyl cyclase-like genes that define this family are clearly distinct from previously known adenylyl cyclases. One member forms a unique locus on chromosome 3 whereas the other four members form a tightly clustered, tandemly repeated array on chromosome 2. The genes on chromosome 2 are transcribed in the male germline in a doublesex dependent manner and are expressed in postmitotic, meiotic, and early differentiating sperm. These genes therefore provide the first evidence for a role for the cAMP signaling pathway in Drosophila spermatogenesis. Expression from this locus is under the control of the always early, cannonball, meiosis arrest, and spermatocyte arrest genes that are required for the G₂/M transition of meiosis I during spermatogenesis, implying a mechanism for the coordination of differentiation and proliferation. Evidence is also provided for positive selection at the locus on chromosome 2 which suggests this gene family is actively evolving and may play a novel role in spermatogenesis. Received: 26 September 1999 / Accepted: 27 October 1999     

The Drosophila melanogaster homologue of the hsp60 gene is encoded by the essential locus l(1)10Ac and is differentially expressed during fly development

 The hsp60 (heat-shock protein 60) gene family of molecular chaperones has been a subject of study in numerous systems due to its important role in the correct folding of non-native proteins in development as well as after heat-shock treatment. Here we present the characterization of the first Drosophila hsp60 homologue. Drosophila HSP60 is most closely related (72% identity across the entire protein sequence) to the mouse mitochondrial HSP60. Western blot experiments indicate that Drosophila HSP60 is enriched in the mitochondrial fraction. The distribution of HSP60 protein is dynamic during fly embryogenesis, suggesting that various cell types might have different HSP60 requirements. The molecular analysis of a P-element-induced mutation that affects the l(1)10Ac locus shows that the transposon is inserted in a 3-kb intron present in the hsp60 gene. By genetic rescue experiments we prove that Drosophila HSP60 is encoded by the essential locus l(1)10Ac opening the possibility for detailed genetic analysis of HSP60 functions in the fly. Received: 24 March 1997 / Accepted: 16 June 1997