Modifications of the Notch Function by Abruptex Mutations in Drosophila Melanogaster

The function of the Notch gene is required in cell interactions defining alternative cell fates in several developmental processes. The Notch gene encodes a transmembrane protein with 36 epidermal growth factor (EGF)-like repeats in its extracellular domain. This protein functions as a receptor that interacts with other transmembrane proteins, such as Serrate and Delta, which also have EGF repeats in their extracellular domain. The Abruptex mutations of the Notch locus are associated with amino acid substitutions in the EGF repeats 24-29 of the Notch protein. We have studied, in genetic combinations, the modifications of Notch function caused by Abruptex mutations. These mutations lead to phenotypes which are opposite to those caused by Notch deletions. The Abruptex phenotypes are modified by the presence of mutations in other loci, in particular in the genes Serrate and Delta as well as Hairless, and groucho. The results suggest that all Abruptex mutations cause stronger than normal Notch activation by the Delta protein. Some Abruptex alleles also display an insufficiency of N function. Abruptex alleles which produce stronger enhancement of Notch activation also display stronger Notch insufficiency. This insufficiency could be due to reduced ability of Abruptex proteins to interact with Notch ligands and/or to form functional Notch dimers.     

Ligand-binding and signaling properties of the Ax[M1] form of Notch

The Abruptex class of Notch alleles has attracted interest because they exhibit some properties that are best explained in terms of increased activity and others that are best explained in terms of reduced activity in vivo. Here, we report a comparison of the properties of Abruptex[M1] and wild-type Notch as ligand binding receptors. Abruptex[M1] showed less activity than wild-type Notch in its ability to bind Delta and Serrate and was expressed at reduced levels on the cell surface. When differences in expression level were taken into account, Abruptex[M1] was comparable to Notch in its sensitivity to ligand-induced activation of reporter gene expression. Abruptex[M1] was also comparable to Notch in its requirement for modification by Fringe and in being sensitive to cis-dowregulation by co-expressed ligands. By the available criteria Abruptex[M1] exhibits less activity than Notch. To explain the ectopic activity of Abruptex[M1] in vivo we suggest that it may be necessary to invoke an altered response to an as yet unidentified ligand or cofactor.     

Interaction of hairless, delta, enhancer of split and notch genes of Drosophila melanogaster as expressed in adult morphology

The interaction of three neurogenic loci viz. Delta, Enhancer of split and Notch, and a related gene, Hairless, of Drosophila melanogaster was investigated at the adult morphology level by measuring the effects of the mutations of the three other genes on the expression of the recessive lethal antimorphic Abruptex mutations of the Notch locus. The Abruptex mutations were also coupled in cis or trans with facet-glossy or split mutations of the Notch locus. In some of the experiments, the genotype of the fly was homozygous for either facet-glossy or split mutation or their wild type alleles but heterozygous for the Abruptex. Facet-glossy is located in a large intron of the locus, whereas split is located in the same exon as Abruptex. In all compounds studied, Delta suppressed the expression of Abruptex while Hairless and Enhancer of split enhanced it. The interactions of the four genes studied were allele specific, suggesting an interaction at the protein level. The comparison of the results presented in this study on the interaction of the neurogenic genes with other results on the same subject suggests that the interactions are similar in embryonic and imaginal development.     

The abruptex mutations of notch disrupt the establishment of proneural clusters in Drosophila

The receptor encoded by the Notch gene plays a central role in preventing cells from making decisions about their fates until appropriate signals are present. This function of Notch requires the product of the Suppressor of Hairless gene. Loss of either Notch or Suppressor of Hairless function results in cells making premature and incorrect cell fate decisions, whilst increases in Notch signalling prevent cells from making these decisions. Here we find that the proneural clusters are not established correctly in certain Abruptex mutations of Notch and this failure to establish proneural clusters correctly is not due to increased Notch signalling during lateral inhibition. In addition we show that the overexpression of certain dominant negative Notch molecules can disrupt the initiation of proneural cluster development in a manner similar to the Abruptex mutants.     

Allelic Negative Complementation at the Abruptex Locus of DROSOPHILA MELANOGASTER

The mutations of the Abruptex locus in Drosophila melanogaster fall into three categories. There are recessive lethal alleles and viable alleles. The latter can be divided into suppressors and nonsuppressors of Notch mutations. The recessive lethals are lethal in heterozygous combination with Notch. As a rule the recessive lethals are lethal also in heterozygous combination with the viable alleles. Heterozygous combinations of certain viable alleles are also lethal. In such heterozygotes, one heteroallele is a suppressor of Notch and the other is a nonsuppressor. Other heterozygous combinations of viable alleles are viable and have an Abruptex phenotype. The insertion of the wild allele of the Abruptex locus as an extra dose (carried by a duplication) into the chromosomal complement of the fly fully restores the viability of the otherwise lethal heterozygotes if two viable alleles are involved. The extra wild allele also restores the viability of heterozygotes in which a lethal and a suppressor allele are present. If, however, a lethal and a nonsuppressor are involved, the wild allele only partly restores the viability, and the effect of the wild allele is weakest if two lethal alleles are involved. It seems likely that of the viable alleles the suppressors of Notch are hypermorphic and the nonsuppressors are hypomorphic. The lethal alleles share properties of both types, and are possibly antimorphic mutations. It is suggested that the locus is responsible for a single function which, however, consists of two components. The hypermorphic mutations are defects of the one component and the hypomorphic mutations of the other. In heterozygotes their cumulative action leads to decreased viability. The lethal alleles are supposed to be defects of the function as a whole. The function controlled by the locus might be a regulative function.     

Dcas is required for importin-alpha3 nuclear export and mechano-sensory organ cell fate specification in Drosophila

We have studied the in vivo function and tissue specificity of Dcas, the Drosophila ortholog of CAS, the importin beta-like export receptor for importin alpha. While dcas mRNA is specifically expressed in the embryonic central nervous system, Dcas protein is maternally supplied to all embryonic cells and its nuclear/cytoplasmic distribution varies in different tissues and times in development. Unexpectedly, hypomorphic alleles of dcas show specific transformations in mechano-sensory organ cell identity, characteristic of mutations that increase Notch signaling. Dcas is essential for efficient importin-alpha3 nuclear export in mechano-sensory cells and the surrounding epidermal cells and is indirectly required for the import of one component of the Notch pathway, but not others tested. We interpret the specificity of the dcas phenotype as indicating that one or more Notch signaling components are particularly sensitive to a disruption in nuclear protein import. We propose that mutations in house keeping genes often cause specific developmental phenotypes, such as those observed in many human genetic disorders.     

Numb suppresses the negative complementation at the Notch locus of Drosophila melanogaster, suggesting a putative mechanism for negative complementation

The mutant form of the intracellular asymmetrically localized Numb membrane-bound protein of Drosophila melanogaster suppresses the negative complementation of certain Abruptex (Ax) mutations of the Notch (N) locus encoding a transmembrane receptor protein in which the Ax mutations are mutations in the epidermal growth factor (EGF)-like repeats of the extracellular domain of the receptor. One model for how Ax mutants affect N function is that they are refractory to an antagonistic signal generated by an excess of N ligands. Genetically numb (nb) is an antagonist of N. In the absence of nb, cells follow the same fate as they would in the presence of a gain-of-function N allele, such as Ax. Numb has been shown to interact with the cytoplasmic domain of Notch. It is therefore suggested that numb counteracts the effect of Abruptex on Notch ligand binding, i.e. that Numb is an antagonist to the activation of the Notch signal generated by Notch ligands. Numb might accomplish this by interfering with the proteolytic cleavage of the Notch intracellular domain at the cell membrane. Thus, it seems possible that the mechanism of negative complementation of certain Ax mutants is the failure of this cleavage. Other possible mechanisms for negative complementation are also discussed.     

The balance between GMD and OFUT1 regulates Notch signaling pathway activity by modulating Notch stability

The Notch signaling pathway plays an important role in development and physiology. In Drosophila, Notch is activated by its Delta or Serrate ligands, depending in part on the sugar modifications present in its extracellular domain. O-fucosyltransferase-1 (OFUT1) performs the first glycosylation step in this process, O-fucosylating various EGF repeats at the Notch extracellular domain. Besides its O-fucosyltransferase activity, OFUT1 also behaves as a chaperone during Notch synthesis and is able to down regulate Notch by enhancing its endocytosis and degradation. We have reevaluated the roles that O-fucosylation and the synthesis of GDP-fucose play in the regulation of Notch protein stability. Using mutants and the UAS/Gal4 system, we modified in developing tissues the amount of GDP-mannose-deshydratase (GMD), the first enzyme in the synthesis of GDP-fucose. Our results show that GMD activity, and likely the levels of GDP-fucose and O-fucosylation, are essential to stabilize the Notch protein. Notch degradation observed under low GMD expression is absolutely dependent on OFUT1 and this is also observed in Notch Abruptex mutants, which have mutations in some potential O-fucosylated EGF domains. We propose that the GDP-fucose/OFUT1 balance determines the ability of OFUT1 to endocytose and degrade Notch in a manner that is independent of the residues affected by Abruptex mutations in Notch EGF domains.     

Functionally redundant peg sensilla on the scorpion pecten

All scorpions have two mid-ventral organs called pectines. Each pecten has thousands of pore-tipped sensilla sensitive to a variety of volatile organic and water-based stimulants. However, it was previously unknown whether individual sensilla were functionally identical or different. The information enhancement hypothesis predicts that all sensilla have similar chemosensitivities such that each is a unit of a parallel processing system. The information segmentation hypothesis states that sensilla differ in their chemosensitivities, a functional arrangement akin to the glomeruli-specific chemical detection system in the moth or human olfactory sense. In this study, we tested these hypotheses by extracellularly tip-recording sensillar responses to three aqueous tastants: 0.01 M KCl, 0.1 M citric acid, and 40% ethanol by volume. We isolated stimulation to one sensillum at a time and compared the chemoresponses. Sensilla appeared to respond similarly to the same stimulant (i.e., sensillar tip-recordings revealed activity of the same cell types), although sometimes a few sensilla responded with higher spike rates than the others. We conclude that our data primarily support the information enhancement hypothesis but for future tests of sensillar function we suggest a new hybrid model, which proposes that a few specialized sensilla exist among a mostly uniform field of identical sensilla.     

Echinoid mutants exhibit neurogenic phenotypes and show synergistic interactions with the Notch signaling pathway

During neurogenesis in Drosophila, groups of ectodermal cells are endowed with the capacity to become neuronal precursors. The Notch signaling pathway is required to limit the neuronal potential to a single cell within each group. Loss of genes of the Notch signaling pathway results in a neurogenic phenotype: hyperplasia of the nervous system accompanied by a parallel loss of epidermis. Echinoid (Ed), a cell membrane associated Immunoglobulin C2-type protein, has previously been shown to be a negative regulator of the EGFR pathway during eye and wing vein development. Using in situ hybridization and antibody staining of whole-mount embryos, we show that Ed has a dynamic expression pattern during embryogenesis. Embryonic lethal alleles of ed reveal a role of Ed in restricting neurogenic potential during embryonic neurogenesis, and result in a phenotype similar to that of loss-of-function mutations of Notch signaling pathway genes. In this process Ed interacts closely with the Notch signaling pathway. Loss of ed suppresses the loss of neuronal elements caused by ectopic activation of the Notch signaling pathway. Using a temperature-sensitive allele of ed we show, furthermore, that Ed is required to suppress sensory bristles and for proper wing vein specification during adult development. In these processes also, ed acts in close concert with genes of the Notch signaling pathway. Thus the extra wing vein phenotype of ed is enhanced upon reduction of Delta (Dl) or Enhancer of split [E(spl)] proteins. Overexpression of the membrane-tethered extracellular region of Ed results in a dominant-negative phenotype. This phenotype is suppressed by overexpression of E(spl)m7 and enhanced by overexpression of Dl. Our work establishes a role of Ed during embryonic nervous system development, as well as adult sensory bristle specification and shows that Ed interacts synergistically with the Notch signaling pathway.     

The cercal sensilla of the blowfly Lucilia cuprina. II. Structure of the enveloping cells and the basal regions of the sensory dendrites

The gustatory, olfactory, touch and stress receptors on the cerci of Lucilia cuprina Wied. (Diptera: Calliphoridae) have either two or three enveloping cells. The gustatory and olfactory sensilla have three enveloping cells: a tormogen, trichogen and thecogen cell. The tormogen and trichogen cells contribute to a sub-cuticular sensillar lumen which divides into two lobes basally. The thecogen cell forms a lumen around the dendrites. Distally the dendrites lie in the contents of the thecogen lumen within the dendritic sheath. Proximally the dendrites embed in the thecogen cell which has an expanded, microlamellate lumen basally. The sensillar lumen of the mechanosensory (trichoid mechanoreceptors and campaniform) sensilla is formed by a single enveloping cell: the presumptive tormogen cell. In trichoid mechanoreceptors the thecogen lumen is restricted to the region of the transitional region of the dendrite whereas the thecogen lumen of campaniform sensilla extends proximally although it is not as well-developed as that of the chemoreceptive sensilla. The dendrites of all sensillum types on the cerci have a granular body in the transitional region: a situation which has not been previously reported in chemoreceptive sensilla although common in the mechanoreceptors of Calliphoridae and Sarcophagidae.     

Functional anatomy of sensory structures on the antennae of Psylliodes chrysocephala L. (Coleoptera: Chrysomelidae)

The ultrastructure and distribution of sensilla on the antennae of the cabbage stem flea beetle, Psylliodes chrysocephala, were investigated using scanning and transmission electron microscopy techniques. Eight different sensillar types were distinguished. These were; hair plate sensilla, sensilla chaetica, three types of sensilla trichodea, sensilla basiconica, grooved peg sensilla and styloconic sensilla. The sensilla chaetica are known to be gustatory receptors. Ultrastructure indicates that the hair plate sensilla and sensilla trichodea type one are probably mechanoreceptors, whilst the sensilla styloconica are probably thermo-hygro receptors. These thermo-hygroreceptors are unusual in that they are innervated by two sensory cells (one hygroreceptor and one thermoreceptor) rather than the more usual triad. The remaining four sensillar types all have a porous hair shaft, indicating an olfactory role. One of these (the grooved peg sensillum) may also have a thermoreceptive function. No sexual dimorphism was found in the structure, number or distribution of the antennal sensilla.     

Electrophysiological evidence of synaptic interactions within chemosensory sensilla of scorpion pectines

The pectines of scorpions are ventral bilateral appendages supporting 10⁴–10⁵ chemosensory sensilla called pegs. Each peg contains 10–18 sensory neurons, some of which show ultrastructural evidence of axo-axonic synapses with other sensory neurons in the same sensillum. In extracellular recordings from single-peg sensilla, individual sensory units can be distinguished by impulse waveform and firing frequency. Cross-correlation analysis of impulse activity showed that at least two of these units, types `A1' and `A2', are inhibited during the 100-ms period immediately following activity of a third unit, type `B'. This interaction between sensory units in a single sensillum also occurs in surgically isolated pectines, indicating that it does not involve efferent feedback from the central nervous system. Other sensillar neurons appear to have excitatory interactions. Thus, in scorpion pectine, chemosensory information undergoes some form of processing within individual sensilla prior to its relay to the CNS, making this an unusually accessible preparation for study of first-order chemosensory processing events. Accepted: 12 April 1997     

Unusual pheromone receptor neuron responses in heliothine moth antennae derived from inter-species imaginal disc transplantation

Single-cell electrophysiological recordings were obtained from olfactory receptor neurons housed in sensilla trichodea along the adult antennae arising from transplantation of the antennal imaginal discs between larval male Helicoverpa zea and Heliothis virescens. The olfactory receptor neurons from the majority of type C sensilla sampled on transplanted antennae displayed response characteristics consistent with those of the species that donated the antennae. However, some of the sensilla type C sampled in either transplant type contained olfactory receptor neurons that responded in a manner typical of the recipient species or other neurons that have not previously been found in the type C sensilla of either species. The single-cell data help to explain behavioral results showing that some transplant males do fly upwind to both species' pheromone blends, an outcome not expected based on known antennal sensory phenotypes. Our results suggest that host tissue can influence antennal olfactory receptor neuron development, and further that because of a common phylogenetic ancestry the donor tissue has the genetic capability to produce a variety of sensillar and receptor types.     

A novel Takeout-like protein expressed in the taste and olfactory organs of the blowfly, Phormia regina

In insects, the functional molecules responsible for the taste system are still obscure. The gene for a 28.5 kDa protein purified from taste sensilla of the blowfly Phormia regina belongs to a gene family that includes takeout of Drosophila melanogaster. Molecular phylogenetic analysis revealed that the Phormia Takeout-like protein is most similar to the protein encoded by a member of the Drosophila takeout gene family, CG14661, whose expression and function have not been identified yet. Western blot analyses revealed that Phormia Takeout-like protein was exclusively expressed in antennae and labellum of the adult blowfly in both sexes. Immunohistochemical experiments demonstrated that Takeout-like protein was localized around the lamella structure of the auxiliary cells and in the sensillar lymph of the labellar taste sensillum. In antennae, Takeout-like protein was distributed at the base of the olfactory sensilla as well. No significant differences in Takeout-like protein expression were found between the sexes. Our results suggest that Phormia Takeout-like protein is involved in some early events concerned with chemoreception in both the taste and olfactory systems.     

Negative Complementation at the Notch Locus of DROSOPHILA MELANOGASTER

Four Abruptex alleles (Ax^E1, Ax^E₂, Ax^₉B₂, and Ax¹⁶¹⁷²) have been mapped within the Notch locus. Based on their visible phenotypes and their interactions with one another and with N mutations, the Ax alleles can be divided into two groups. Heterozygous combinations of members of the same group are intermediate in phenotype compared to the respective homozygotes, whereas heterozygotes of Ax alleles from different groups exhibit negative heterosis, being much less viable and more extremely mutant than either homozygote. It is suggested that the Notch locus is a multi-functional regulator ("integrator") gene, whose product possesses both "repressor" and "activator" functions for the processes it regulates.     

Anatomical pathways connecting lip sensory structures and central nervous system in hirudinid leeches visualized by carbocyanine dyes and laser scanning confocal microscopy

Chemoreception inHirudo medicinalis is thought to be mediated by ciliated cells grouped in sensory structures, the sensilla, arranged in bands on the animal's dorsal lip (Elliott, 1986; Zipseret al., 1994). Furthermore, chemical and/or thermal stimulation of the dorsal lip in reduced preparations evokes changes in the electrical activity of the cephalic nerves that connect the head with the central nervous system. However, the complete trajectory by which the sensory afferents teach the cerebral ganglia has not been demonstrated anatomically. In this study, we traced these pathways following retrograde and/or anterograde transport of carbocyanine dyes (DiI, DiA and DiD) in the cephalic nerves ofHirudo medicinalis and a closely related species,Macrobdella decora. While information regardingMacrobdella's chemoreception is scarce, the two species show some differences with regard to their chemical preferences. Dyes were applied to the sensillar structures along the dorsal lip, or to the cut ends of individual cephalic nerves in fixed preparations that included the lip and attached nerves with or without the head ganglia. After a two week incubation, specimens were mounted and imaged using a confocal microscope.The results show that the axons of the sensory neurons in the sensilla project through the four pairs of cephalic nerves. The sensillar projections are however more numerous in the dorsal nerves than they are in the ventral ones. In addition, the organization of the sensillar bands, the morphology of the pathways and the sensory structures themselves appear to be identical forHirudo andMacrobdella and therefore the behavioral differences in response to appetitive stimuli cannot be readily explained by differences in morphology.     

Analysis of the Negative Complementation of Abruptex Alleles in Gynandromorphs of DROSOPHILA MELANOGASTER

Certain Abruptex alleles in the Notch pseudoallelic series of Drosophila melanogaster show strong negative complementation. Heterozygous combinations of some viable alleles are lethal. As a lethal system this is unique. Analysis of this type of allelic interaction in gynandromorphs suggests that the lethal focus has a fate-map site in the anterior part of the fly, probably close to the central part of the thorax. In addition to the lethal effect, negative interaction of the alleles can also be seen in the morphogenesis of wings and chaetae of thorax and head. At this morphological level, the negative interaction of the alleles appears to be autonomous.     

The development of normal and ectopic sensilla in the wings of hairy and Hairy wing mutants of Drosophila.

We have utilized enhancer trap markers to follow the development of ectopic sensillar precursors in the wings of Drosophila induced by the mutations hairy and Hairy wing. Normal sensilla are still present in these mutations, and can be distinguished from ectopic sensilla based upon both the position and the timing of their development. This correlates well with the development of ectopic achaete expression in these mutations: such staining is detected only after the appearance of normal staining. Thus, neither mutation appears to alter the specification of proneural clusters in the wing, as identified with anti-achaete, or the specification of sensillar precursors within these clusters. Rather, both act to induce the formation of a temporally and spatially distinct phase of sensillar development.