Increased levels of the Drosophila Abelson tyrosine kinase in nerves and muscles: subcellular localization and mutant phenotypes imply a role in cell-cell interactions.

Mutations in the Drosophila Abelson tyrosine kinase have pleiotropic effects late in development that lead to pupal lethality or adults with a reduced life span, reduced fecundity and rough eyes. We have examined the expression of the abl protein throughout embryonic and pupal development and analyzed mutant phenotypes in some of the tissues expressing abl. abl protein, present in all cells of the early embryo as the product of maternally contributed mRNA, transiently localizes to the region below the plasma membrane cleavage furrows as cellularization initiates. The function of this expression is not yet known. Zygotic expression of abl is first detected in the post-mitotic cells of the developing muscles and nervous system midway through embryogenesis. In later larval and pupal stages, abl protein levels are also highest in differentiating muscle and neural tissue including the photoreceptor cells of the eye. abl protein is localized subcellularly to the axons of the central nervous system, the embryonic somatic muscle attachment sites and the apical cell junctions of the imaginal disk epithelium. Evidence for abl function was obtained by analysis of mutant phenotypes in the embryonic somatic muscles and the eye imaginal disk. The expression patterns and mutant phenotypes indicate a role for abl in establishing and maintaining cell-cell interactions.     

Roles of wingless in patterning the larval epidermis of Drosophila.

The larval epidermis of Drosophila shows a stereotyped segmentally repeating pattern of cuticular structures. Mutants deficient for the wingless gene product show highly disrupted patterning of the larval cuticle. We have manipulated expression of the wg gene product to assess its role in this patterning process. We present evidence for four distinct phases of wg function in epidermal cells: (1) an early requirement in engrailed-expressing cells to establish and maintain stable expression of en, (2) a discrete period when wg and en gene products act in concert to generate positional values in the anterior portion of the ventral segment and all values of the dorsal and lateral epidermis, (3) a progressive function (dependent on prior interaction with the en-expressing cells) in conferring positional values to cells within the posterior portion of the segment, and (4) a late continuous requirement for maintaining some ventral positional values.     

Secretion and movement of wingless protein in the epidermis of the Drosophila embryo.

The segment polarity gene wingless encodes a cysteine rich protein which is essential for pattern formation in Drosophila. Using polyclonal antibodies against the product of the wingless gene, we demonstrate that this protein is secreted in the embryo and that it is taken up by neighbouring cells. The protein can be found two or three cell diameters away from the cells in which it is synthesized. We discuss the possible mechanisms which are responsible for this spatial distribution and its regulation during embryogenesis.     

The consequences of ubiquitous expression of the wingless gene in the Drosophila embryo.

The segment polarity gene wingless has an essential function in cell-to-cell communication during various stages of Drosophila development. The wingless gene encodes a secreted protein that affects gene expression in surrounding cells but does not spread far from the cells where it is made. In larvae, wingless is necessary to generate naked cuticle in a restricted part of each segment. To test whether the local accumulation of wingless is essential for its function, we made transgenic flies that express wingless under the control of a hsp70 promoter (HS-wg flies). Uniform wingless expression results in a complete naked cuticle, uniform armadillo accumulation and broadening of the engrailed domain. The expression patterns of patched, cubitus interruptus Dominant and Ultrabithorax follow the change in engrailed. The phenotype of heatshocked HS-wg embryos resembles the segment polarity mutant naked, suggesting that embryos that overexpress wingless or lack the naked gene enter similar developmental pathways. The ubiquitous effects of ectopic wingless expression may indicate that most cells in the embryo can receive and interpret the wingless signal. For the development of the wild-type pattern, it is required that wingless is expressed in a subset of these cells.     

Isolation of a human gene with protein sequence similarity to human and murine int-1 and the Drosophila segment polarity mutant wingless.

An expressed gene sequence which was identified by the isolation of a methylation free CpG island from human chromosome 7 has been cloned from a human lung cDNA library. The deduced protein sequence contains 360 amino acids and has several features of a secreted protein; it is cysteine rich with a signal peptide sequence and two potential asn-linked glycosylation sites. The protein sequence shows marked similarity with human and murine int-1 and their Drosophila homolog wingless (Dint-1). This human int-1 related protein, int-1 and Dint-1 have diverse patterns of expression, but the inferred structural similarities suggest that some of the functional characteristics of these proteins may be shared.     

Thenar and hypothenar muscles and their innervation by the ulnar and median nerves in the human hand.

The thenar and hypothenar muscles as well as their supplying nerves were analyzed with an improved dissecting method. Among the four thenar muscles, the m. abductor pollicis brevis (AbPB) has a separate muscle belly, whereas the m. opponens pollicis (OP), the superficial and deep heads of the flexor pollicis brevis (sFPB and dFPB), and the adductor pollicis (AdP) are fused with each other to make a single mass (deep thenar muscle group). These muscles are innervated by branches of the recurrent nerve and the accessory recurrent nerve from the median nerve as well as by terminal branches of the deep branch (ramus profundus) of the ulnar nerve. These three nerves frequently form a loop within the deep thenar muscle group (thenar loop), and a branch to the OP and one to deep parts of the sFPB often make a smaller loop (intrathenar loop), whereas the AbPB receives a separate nerve branch. Among the hypothenar muscle, the m. abductor digiti minimi and the m. flexor digiti minimi brevis are fused with each other, and their supplying nerves frequently form a loop in these muscles (intrahypothenar loop), whereas the m. opponens digiti minimi is separated from the others and receives a separate nerve branch. In the distribution pattern of supplying nerves to the thenar and hypothenar muscles, we find regularities in that they branch off in a regular manner from the ulnar and the median nerve, and that nerve branches to those muscles with fused bellies frequently communicate with each other to make loops.     

Directionality of wingless protein transport influences epidermal patterning in the Drosophila embryo.

Active endocytotic processes are required for the normal distribution of Wingless (Wg) protein across the epidermal cells of each embryonic segment. To assess the functional consequences of this broad Wg distribution, we have devised a means of perturbing endocytosis in spatially restricted domains within the embryo. We have constructed a transgene expressing a dominant negative form of shibire (shi), the fly dynamin homologue. When this transgene is expressed using the GAL4-UAS system, we find that Wg protein distribution within the domain of transgene expression is limited and that Wg-dependent epidermal patterning events surrounding the domain of expression are disrupted in a directional fashion. Our results indicate that Wg transport in an anterior direction generates the normal expanse of naked cuticle within the segment and that movement of Wg in a posterior direction specifies diverse denticle cell fates in the anterior portion of the adjacent segment. Furthermore, we have discovered that interfering with posterior movement of Wg rescues the excessive naked cuticle specification observed in naked (nkd) mutant embryos. We propose that the nkd segment polarity phenotype results from unregulated posterior transport of Wg protein and therefore that wild-type Nkd function may contribute to the control of Wg movement within the epidermal cells of the segment.     

The development of adult abdominal muscles in Drosophila: myoblasts express twist and are associated with nerves.

During metamorphosis, the adult muscles of the Drosophila abdomen develop from pools of myoblasts that are present in the larva. The adult myoblasts express twist in the third larval instar and the early pupa and are closely associated with nerves. Growing adult nerves and the twist-expressing cells migrate out across the developing abdominal epidermis, and as twist expression declines, the myoblasts begin to synthesize beta 3 tubulin. There follows a process involving cell fusion and segregation into cell groups to form multinucleate muscle precursors. These bipolar precursors migrate at both ends to find their correct attachment points. beta 3 tubulin expression continues at least until 51 h APF by which time the adult muscle pattern has been established.     

Actin III and myofilaments in different muscles of wildtype and the mutant raised of Drosophila melanogaster

The mutation raised (rsd, 3-95.4) of Drosophila melanogaster causes flightlessness as a consequence of abnormalities in the fibrillar flight muscles (FFMs). In this muscle type actin III is neither synthesized nor accumulated while adult tubular muscles of rsd flies are indistinguishable from wildtype. This paper demonstrates ultrastructural defects in rds FFMs and extends the biochemical comparison of adult wildtype and rsd muscles to larval muscles and to embryo cells differentiating in culture. The FFMs of mature rsd flies contain thick filaments in irregular bundles, but no thin filaments. Normal Z-discs are virtually absent. Instead, a large number of Z-disc residues are present in stacks attached to short filaments on either side. In newly emerged rsd flies the disorganization is less pronounced. The adult tubular muscles and the supercontracting muscles of third-instar larvae of rsd can ultrastructurally not be distinguished from wildtype. The present biochemical results indicate that not only FFMs of mature and newly emerged adults are affected by the rsd genotype. Synthesis of actin III is not detectable in rsd FFMs which corresponds to the heavy structural defects. In addition to the lack of actin III synthesis in rsd FFMs, three unidentified proteins (52 kDa, 80 kDa, 90 kDa) which are specific for wildtype FFMs are also not synthesized in rsd flies. Among all other muscle types studied, all of which are morphologically unaffected, only adult tubular muscle of rsd genotype showed no biochemical effect. Larval supercontracting muscle as well as embryo cells differentiating in culture failed to synthesize actin III in the case of rsd cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Secretory and inductive properties of Drosophila wingless protein in Xenopus oocytes and embryos.

Like its vertebrate homologues, Xenopus wnt-8 and murine wnt-1, we find that Drosophila wingless (wg) protein causes axis duplication when overexpressed in embryos of Xenopus laevis after mRNA injection. In many cases, the secondary axes contain eyes and cement glands, which reflect the induction of the most dorsoanterior mesodermal type, prechordal mesoderm. We show that the extent of axis duplication is dependent on the embryonic site of expression, with ventral expression leading to a more posterior point of axis bifurcation. The observed duplications are due to de novo generation of new axes as shown by rescue of UV-irradiated embryos. The true dorsal mesoderm-inducing properties of wg protein are indicated by its ability to generate extensive duplications after mRNA injection into D-tier cells of 32-cell embryos. As revealed by lineage mapping, the majority of these D cell progeny populate the endoderm; injections into animal blastomeres at this stage are far less effective in inducing secondary axes. However, when expressed in isolated animal cap explants, wg protein induces only ventral mesoderm, unless basic fibroblast growth factor is added, whereupon induction of muscle and occasionally notochord is seen. We conclude that in intact embryos, wg acts in concert with other factors to cause axis duplication. Immunolocalisation studies in embryos indicate that wg protein remains localised to the blastomeres synthesizing it and has a patchy, often perinuclear distribution within these cells, although some gets to the surface. In oocytes, the pool of wg protein is entirely intracellular and relatively unstable. When the polyanion suramin is added, most of the intracellular material is recovered in the external medium.     

Biochemical characterization of the Drosophila wingless signaling pathway based on RNA interference

Regulation of Armadillo (Arm) protein levels through ubiquitin-mediated degradation plays a central role in the Wingless (Wg) signaling. Although zeste-white3 (Zw3)-mediated Arm phosphorylation has been implicated in its degradation, we have recently shown that casein kinase Ialpha (CKIalpha) also phosphorylates Arm and induces its degradation. However, it remains unclear how CKIalpha and Zw3, as well as other components of the Arm degradation complex, regulate Arm phosphorylation in response to Wg. In particular, whether Wg signaling suppresses CKIalpha- or Zw3-mediated Arm phosphorylation in vivo is unknown. To clarify these issues, we performed a series of RNA interference (RNAi)-based analyses in Drosophila S2R+ cells by using antibodies that specifically recognize Arm phosphorylated at different serine residues. These analyses revealed that Arm phosphorylation at serine-56 and at threonine-52, serine-48, and serine-44, is mediated by CKIalpha and Zw3, respectively, and that Zw3-directed Arm phosphorylation requires CKIalpha-mediated priming phosphorylation. Daxin stimulates Zw3- but not CKIalpha-mediated Arm phosphorylation. Wg suppresses Zw3- but not CKIalpha-mediated Arm phosphorylation, indicating that a vital regulatory step in Wg signaling is Zw3-mediated Arm phosphorylation. In addition, further RNAi-based analyses of the other aspects of the Wg pathway clarified that Wg-induced Dishevelled phosphorylation is due to CKIalpha and that presenilin and protein kinase A play little part in the regulation of Arm protein levels in Drosophila tissue culture cells.     

Interactions of decapentaplegic,wingless, and Distal-less in the Drosophila leg

The genes decapentaplegic, wingless, and Distalless appear to be instrumental in constructing the anatomy of the adult Drosophila leg. In order to investigate how these genes function and whether they act coordinately, we analyzed the leg phenotypes of the single mutants and their inter se double mutant compounds. In decapentaplegic the tarsi frequently exhibit dorsal deficiencies which suggest that the focus of gene action may reside dorsally rather than distally. In wingless the tarsal hinges are typically duplicated along with other dorsal structures, confirming that the hinges arise dorsally. The plane of symmetry in double-ventral duplications caused by decapentaplegic is virtually the same as the plane in double-dorsal duplications caused by wingless. It divides the fate map into two parts, each bisected by the dorsoventral axis. In the double mutant decapentaplegic wingless the most ventral and dorsal tarsal structures are missing, consistent with the notion that both gene products function as morphogens. In wingless Distal-less compounds the legs are severely truncated, indicating an important interaction between these genes. Distal-less and decapentaplegic manifest a relatively mild synergism when combined. Correspondence to: L.I. Held     

A screen for genes regulating the wingless gradient in Drosophila embryos

During the development of the Drosophila embryonic epidermis, the secreted Wingless protein initially spreads symmetrically from its source. At later stages, Wingless becomes asymmetrically distributed in a Hedgehog-dependent manner, to control the patterning of the embryonic epidermis. When Wingless is misexpressed in engrailed cells in hedgehog heterozygous mutant embryos, larvae show a dominant phenotype consisting of patches of naked cuticle in denticle belts. This dose-sensitive phenotype is a direct consequence of a change in Wg protein distribution. We used this phenotype to carry out a screen for identifying genes regulating Wingless distribution or transport in the embryonic epidermis. Using a third chromosome deficiency collection, we found several genomic regions that showed a dominant interaction. After using a secondary screen to test for mutants and smaller deficiencies, we identified three interacting genes: dally, notum, and brahma. We confirmed that dally, as well as its homolog dally-like, and notum affect Wingless distribution in the embryonic epidermis, directly or indirectly. Thus, our assay can be used effectively to screen for genes regulating Wingless distribution or transport.