Stage-specific regulation of actin genes in Drosophila wing cells

Extreme and rapid changes in the synthesis of messenger RNAs and proteins accompany differentiation in wing tissues of Drosophila. Of the six actin genes, at least three are expressed in wing cells, some during the most extreme changes in cell shape. However, different messages of the set appear, decay, and reappear on a regulated temporal program. These results show that actin expression is stage-specific in a single cell type.     

Experimental studies on a mutant gene (cl) in the Mexican axoloti which affects cell membrane formation in embryos from cl-cl females

The gene cl exerts a maternal effect in the Mexican axolotl resulting in an abnormal cleavage pattern. The early cleavage furrows appear partially depigmented and never continue completely around the egg. Subsequent divisions display a similar pattern which results in the vegetal hemisphere remaining uncleaved; but some portions of the animal hemisphere continue to cleave normally. Gastrulation is very rarely initiated.Several cytological abnormalities including polyploidy, broken chromosomes and fusion of nuclei are observed in mutant embryos. These abnormalities are likely secondary effects resulting from the absence of cell boundaries in the uncleaved portions of the embryo and account for its limited development. Cytochalasin B treatment of normal fertilized eggs produces phenocopies of the most severely affected mutant embryos. This suggests that the cl gene may directly affect the synthesis and/or distribution of a cell surface component which enables daughter cell membranes to be assembled and to adhere to one another.Cells from mutant blastulae were able to differentiate pigmented epidermis and neural tube when grafted to normal recipient blastulae or neurulae. This suggests that the gene is lethal only to cells derived from the vegetal cytoplasm or cortex, but not lethal to cells inheriting animal cytoplasm from $\text{cl}\text{cl}$ females.     

Patterns of protein synthesis in the imaginal discs of Drosophila melanogaster: a comparison between different discs and stages

High-resolution two dimensional gel electrophoresis has been used to study the patterns of protein synthesis in imaginal discs of Drosophila melanogaster. In this paper we first compare the patterns of protein synthesis in wing, haltere, leg 1, leg 2, leg 3 and eye antenna imaginal discs of late third instar larvae. We have detected only quantitative changes: differences in 17 proteins among the different imaginal discs. In addition, we have analysed the variations in pattern of proteins in the wing disc of the last larval stage and early pupae as well as in wing discs cultured in vivo for 6 days. Variations in these patterns affect more than 20% of the proteins and involve both qualitative and quantitative changes. Some of the changes may correspond to protein phosphorylation. Correlations of these changes between discs and through development are also discussed. Correspondence to: F. Santaren     

Characterization of a silicon transporter gene family in Cylindrotheca fusiformis: sequences, expression analysis, and identification of homologs in other diatoms

The transport of silicon is an integral part of the synthesis of the silicified cell wall of diatoms, yet knowledge of the number, features, and regulation of silicon transporters is lacking. We report the isolation and sequence determination of five silicon transporter (SIT) genes from Cylindrotheca fusiformis, and examine their expression patterns during cell wall synthesis. The encoded SIT amino acid sequences are highly conserved in their putative transmembrane domains. Nine conserved cysteines in this domain may account for the sensitivity of silicon uptake to sulfhydryl blocking agents. A less conserved C-terminal domain is predicted to form coiled-coil structures, suggesting that the SITs interact with other proteins. We show that SIT gene expression is induced just prior to, and during, cell wall synthesis. The genes are expressed at very different levels, and SIT1 is expressed in a different pattern from SIT 2–5. Hybridization experiments show that multiple SIT gene copies are present in all diatom species tested. From the data we infer that individual transporters play specific roles in silicon uptake, and propose that the cell regulates uptake by controlling the amount or location of each. The identification of all SIT genes in C. fusiformis will enhance our understanding of the mechanism and control of silicon transport in diatoms.     

Genetic constraints on wing pattern variation in Lycaeides butterflies: A case study on mapping complex,multifaceted traits in structured populations

Patterns of phenotypic variation within and among species can be shaped and constrained by trait genetic architecture. This is particularly true for complex traits, such as butterfly wing patterns, that consist of multiple elements. Understanding the genetics of complex trait variation across species boundaries is difficult, as it necessitates mapping in structured populations and can involve many loci with small or variable phenotypic effects. Here, we investigate the genetic architecture of complex wing pattern variation in Lycaeides butterflies as a case study of mapping multivariate traits in wild populations that include multiple nominal species or groups. We identify conserved modules of integrated wing pattern elements within populations and species. We show that trait covariances within modules have a genetic basis and thus represent genetic constraints that can channel evolution. Consistent with this, we find evidence that evolutionary changes in wing patterns among populations and species occur in the directions of genetic covariances within these groups. Thus, we show that genetic constraints affect patterns of biological diversity (wing pattern) in Lycaeides, and we provide an analytical template for similar work in other systems.     

The development of wingless,a homeotic mutation of Drosophila

The mutation wingless produces a homeotic transformation in which the distal structures (appendages) of both the wing and haltere discs are replaced by a duplication of the proximal structures (thorax). However, not all of the mutant discs show mutant phenotype; some of them differentiate normal appendages. Gynandromorph and clonal analyses suggest that the phenotype does not result from massive cell death followed by regeneration and/or duplication. We conjecture that the mutant phenotype is caused by a specific failure in the process of compartmentalization. In contrast to other homeotic mutants, wingless is not cell autonomous; that is, mutant clones show wildtype phenotype when produced in wildtype wings.     

Ecological Trade-offs between Migration and Reproduction Are Mediated by the Nutrition-Sensitive Insulin-Signaling Pathway

Crowding and changes in food availability are two critical environmental conditions that impact an animal''s trajectory toward either migration or reproduction. Many insects facing this challenge have evolved wing polyphenisms. When conditions favor reproduction, wing polyphenic species produce adults that either have no wings or short, non-functional wings. Facultative wing growth reflects a physiological and evolutionary trade-off between migration and reproduction, triggered by environmental conditions. How environmental cues are transduced to produce these alternative forms, and their associated ecological shift from migration to reproduction, remains an important unsolved problem in evolutionary ecology. The brown planthopper, a wing polymorphic insect exhibiting strong trade-offs in investment between migration and reproduction, is one of the most serious rice pests in Asia. In this study, we investigated the function of four genes in the insulin-signaling pathway known to couple nutrition with growth, PI3 Kinase (PI3K), PDK1, Akt (Protein Kinase B), and the forkhead gene FOXO. Using a combination of RNA interference and pharmacological inhibitor treatment, we show that all four genes contribute to tissue level regulation of wing polymorphic development in this insect. As predicted, silencing of the NlPI3K, NlAkt and NlPDK1 through dsRNA and with the pharmacological inhibitor Perifosine resulted in short-winged brown planthoppers, whereas knockdown of NlFOXO resulted in long-winged planthoppers. Morphometric analyses confirm that phenotypes from our manipulations mimic what would be found in nature, i.e., major parameters such as bristle number, wing area and body weight are not significantly different from non-experimental animals. Taken together, these data implicate the insulin-signaling pathway in the transduction of environmental factors into condition-dependent patterns of wing growth in insects.     

The cell cycle and its relation to growth during pattern regulation in wing discs of Drosophila

In this paper we have examined the timing of growth-rate changes, of alterations in the cell cycle, and of the reestablishment of epithelial continuity during the early stages of regulative growth of imaginal wing discs cultured in vivo. Tissue organization was disrupted prior to culture, by dissociation, and centrifugally reaggregated pellets of cells were cultured in adult female flies. Significant increases in cell numbers were detectable during the first day of culture. Flow cytometric analysis of the DNA content of cells in reaggregates during culture indicated that during the first half-day of culture, a significant transient increase in the proportions of S-phase and post-S-phase cells occurred. Scanning electron microscopic examination of dissociated cells during culture confirmed that tissue reorganization began during the first day and was nearly complete by the end of the second day. During the second day of culture, when the growth rate was maximal, the proportions of pre-(G₁) and post-S-phase and mitotic (G₂M) cells resembled those of intact premetamorphic wing discs. In contrast to disrupted tissue, intact or minimally wounded wing discs showed practically no change in cell number during culture. However, with both disrupted and intact tissue, the proportion of G₁ cells increased significantly during the culture period so that by the fourth day of culture G₁ cells predominated. Under our conditions all implants grew extensively, but regeneration by cultured reaggregates formed from pure presumptive notum and pure presumptive distal wing fragments was observed in those implants which grew the most.Our results suggest that both DNA synthesis and transient increased proportions of 4C cells may occur as an early response to injury and that these may precede the onset of cell divisions even under conditions where growth is initiated early. Our observations also suggest that the onset of cell divisions, though it occurs well before the end of the first day of culture, may nonetheless be preceded by cellular contacts and intercellular communication.     

Cellular degeneration in the production of some mutant phenotypes in Drosophila melanogaster

Summary A number of mutants of Drosophila melanogaster are characterized by the absence of structures present in the wild type. Imaginal discs from the wing mutants vestigial, apterous-Xasta, Beadex and cut and from the eye mutants Bar, eyeless and lozenge were examined by light and electron microscopy. In all these mutants, with the exception of lozenge, clear evidence of degeneration was found. The onset and duration of degeneration and the number and distribution of dying cells were specific characteristics of each mutant. In most cases the degenerate areas of the disc could be correlated with the missing parts of the adult wing or eye. In contrast, in wild type wing and eye discs and in wing discs from the mutant miniature, which has a wing reduced in size but fully formed, extensive cell death was not observed.The ultrastructural features of the degenerating areas weresimilar in all the mutants studied. Conspicuous aspects of the cytolytic process included condensation and fragmentation of the dying cells followed by phagocytosis of the cell fragments by neighboring disc cells.The results indicate that localized cell death during development is a widespread occurrence among Drosophila mutants which exhibit structural deficiències.     

The Rap GTPase activator Drosophila PDZ-GEF regulates cell shape in epithelial migration and morphogenesis

Epithelial morphogenesis is characterized by an exquisite control of cell shape and position. Progression through dorsal closure in Drosophila gastrulation depends on the ability of Rap1 GTPase to signal through the adherens junctional multidomain protein Canoe. Here, we provide genetic evidence that epithelial Rap activation and Canoe effector usage are conferred by the Drosophila PDZ-GEF (dPDZ-GEF) exchange factor. We demonstrate that dPDZ-GEF/Rap/Canoe signaling modulates cell shape and apicolateral cell constriction in embryonic and wing disc epithelia. In dPDZ-GEF mutant embryos with strong dorsal closure defects, cells in the lateral ectoderm fail to properly elongate. Postembryonic dPDZ-GEF mutant cells generated in mosaic tissue display a striking extension of lateral cell perimeters in the proximity of junctional complexes, suggesting a loss of normal cell contractility. Furthermore, our data indicate that dPDZ-GEF signaling is linked to myosin II function. Both dPDZ-GEF and cno show strong genetic interactions with the myosin II-encoding gene, and myosin II distribution is severely perturbed in epithelia of both mutants. These findings provide the first insight into the molecular machinery targeted by Rap signaling to modulate epithelial plasticity. We propose that dPDZ-GEF-dependent signaling functions as a rheostat linking Rap activity to the regulation of cell shape in epithelial morphogenesis at different developmental stages.     

Neural projection patterns from homeotic tissue of Drosophila studied in bithorax mutants and mosaics.

The central projection patterns of sensory cells from the wing and haltere of Drosophila, as revealed by filling their axons with cobalt, consist of dorsal components arising from small campaniform sensilla and ventral components arising from large campaniform sensilla and from bristles. All of the bristles of the wing are innervated, some singly and some multiply. All three classes of sensilla are strongly represented on the wing, but the haltere carries primarily small campaniform sensilla and has a correspondingly minute ventral projection. In bithorax mutants in which the haltere is transformed into wing, ventral components are added to the projection pattern, while the dorsal components appear as if haltere tissue were still present. Thus, the three classes of receptors not only produce different projection patterns when they develop in their native mesothoracic segment, but also behave differently in the homeotic situation. Consequently, different developmental programs are inferred for each class. When somatic recombination clones of bithorax tissue are generated in phenotypically wild-type flies, they also produce ventral projections. However, these projections of mutant fibers into wild-type ganglia differ in certain details from the projections of mutant fibers into mutant ganglia. Thus, homeotic changes are inferred to occur in the CNS of mutant flies, but these are not required for the execution of those developmental instructions carried in the genome of large campaniform and bristle sensory cells which specify that their axons should grow ventrad in the CNS.     

Detailed analysis of the prothoracic tissues transforming into wings in the Cephalothorax mutants of the Tribolium beetle

Despite the immense importance of the wing in the evolution and successful radiation of the insect lineages, the origin of this critical structure remains a hotly-debated mystery. Two possible tissues have been identified as an evolutionary origin of wings; the lateral expansion of the dorsal body wall (tergal edge) and structures related to an ancestral proximal leg segment (pleural tissues). Through studying wing-related tissues in the red flour beetle, Tribolium castaneum, we have previously presented evidence in support of a dual origin of insect wings, a third hypothesis proposing that wings evolved from a combination of both tergal and pleural tissues. One key finding came from the investigation of a Cephalothorax (Cx) mutant, in which the ectopic wing characteristic to this mutant was found to be formed from both tergal and pleural contributions. However, the degree of contribution of the two tissues to the wing remains elusive. Here, we took advantage of multiple Cx alleles available in Tribolium, and produced a variety of degrees and types of ectopic wing tissues in their prothoracic segments. Through detailed phenotypic scoring of the Cx phenotypes based on nine categories of mutant traits, along with comprehensive morphological analysis of the ectopic wing tissues, we found that (i) ectopic wing tissues can be formed at various locations in the prothorax, even internally, (ii) the lateral external ectopic wing tissues have tergal origin, while the internal and posterior external ectopic wing tissues appear to be of pleural origin, and (iii) the ectopic wing tissues of both tergal and pleural origin are capable of transforming into wing surface tissues. Collectively, these outcomes suggest that the evolutionary contribution of each tissue to a complete wing may be more complex than the simple binary view that is typically invoked by a dual origin model (i.e. the wing blade from the tergal contribution + musculature and articulation from the pleural contribution).     

Regulation and Coordination of Purine and Pyrimidine Biosyntheses in Yeast: I. Regulation of Purine Biosynthesis and Its Relation to Transient Changes in Intracellular Nucleotide Levels

The control of purine biosynthesis in a yeast mutant deficient for uracil, adenine, and histidine has been studied in vivo. The adenine mutation causes accumulation of aminoimidazole ribotide in the cells. The control curve relating steady-state purine nucleotide level in the cell to rate of synthesis in the de novo purine synthetic pathway has been determined. Control in the cell depends on a feedback mechanism involving end-product inhibition. The transient responses of the purine nucleotide pool to changes in adenine input have been studied. Under certain conditions the pool overshoots when shifting from one steady-state to another. Transient changes in nucleotide levels are followed by inverse changes in the rate of attempted de novo purine synthesis. A study of the transient responses of specific intracellular nucleotides suggests that inosinic acid controls the rate of attempted purine synthesis. The transient response of nucleic acid synthesis rate to changes in nucleotide levels was studied and the implications for regulation of nucleic acid synthesis discussed.     

Mutations in the polycomb group gene polyhomeotic lead to epithelial instability in both the ovary and wing imaginal disc in Drosophila

Background

Most human cancers originate from epithelial tissues and cell polarity and adhesion defects can lead to metastasis. The Polycomb-Group of chromatin factors were first characterized in Drosophila as repressors of homeotic genes during development, while studies in mammals indicate a conserved role in body plan organization, as well as an implication in other processes such as stem cell maintenance, cell proliferation, and tumorigenesis. We have analyzed the function of the Drosophila Polycomb-Group gene polyhomeotic in epithelial cells of two different organs, the ovary and the wing imaginal disc.

Results

Clonal analysis of loss and gain of function of polyhomeotic resulted in segregation between mutant and wild-type cells in both the follicular and wing imaginal disc epithelia, without excessive cell proliferation. Both basal and apical expulsion of mutant cells was observed, the former characterized by specific reorganization of cell adhesion and polarity proteins, the latter by complete cytoplasmic diffusion of these proteins. Among several candidate target genes tested, only the homeotic gene Abdominal-B was a target of PH in both ovarian and wing disc cells. Although overexpression of Abdominal-B was sufficient to cause cell segregation in the wing disc, epistatic analysis indicated that the presence of Abdominal-B is not necessary for expulsion of polyhomeotic mutant epithelial cells suggesting that additional POLYHOMEOTIC targets are implicated in this phenomenon.

Conclusion

Our results indicate that polyhomeotic mutations have a direct effect on epithelial integrity that can be uncoupled from overproliferation. We show that cells in an epithelium expressing different levels of POLYHOMEOTIC sort out indicating differential adhesive properties between the cell populations. Interestingly, we found distinct modalities between apical and basal expulsion of ph mutant cells and further studies of this phenomenon should allow parallels to be made with the modified adhesive and polarity properties of different types of epithelial tumors.     

Some required events in conidial germination of Neurospora crassa.

A temperature-sensitive mutant of Neurospora crassa, with reduced levels of protein synthesis at 37°C, was used to identify some essential events in conidial germination. Conidia of mutant strain psi-1 were incubated for 2 hr at 37°C and then shifted to 20°C. Germination was inhibited at 37°C, but commenced after 1.5 hr at 20°C. Increases in aspartate transcarbamylase activity, cell wall synthesis, and nuclear number preceded germination. However, increases in glutamate dehydrogenase activity, amino acid uptake, and DNA synthesis were inhibited prior to germination. Although all of these events were correlated with germination in control cultures of the mutant at 20°C and of its parent strain at 20 and 37°C, some events were apparently not essential for germination. The requirement for aspartate transcarbamylase activity was demonstrated independently by the failure of strain pyr-3d (lacking the activity) to germinate in the absence of uridine. The dispensability of glutamate dehydrogenase activity and DNA synthesis for the germination of some conidia was verified by the germination of strain am-1 (lacking glutamate dehydrogenase activity) in the absence of glutamate and by the germination of the parent strain in the presence of hydroxyurea (an inhibitor of DNA synthesis). These findings identify some landmarks in germination which may be useful in further studies of the regulation of a developmental program. They also provide preliminary evidence that the resting conidia may contain nuclei arrested at different stages of their division cycle.