tube

DEVELOPMENTAL BIOLOGY

Embryonic

Tube mRNA is expressed maximally early in embryogenesis and again late in larval development, corresponding to required periods of tube activity as defined by distinct maternal and zygotic loss-of-function phenotypes in tube mutants (Letsou, 1991).

Effects of Mutation or Deletion

Loss of maternal function of the tube gene disrupts a signaling pathway required for Drosophila pattern formation, causing cells throughout the embryo to adopt the fate normally reserved for those at the dorsal surface. tube mutations also have a zygotic effect on pupal morphology. This phenotype is shared by mutations in Toll and pelle, two genes with apparent intracellular roles in determining dorsoventral polarity (Letsou, 1991).

tube and pelle are two of the maternally transcribed genes required for dorsal-ventral patterning of the Drosophila embryo. Females homozygous for strong alleles of tube or pelle produce embryos that lack all ventral and lateral embryonic pattern elements. Double mutant females carrying dominant ventralizing alleles of Toll and dorsalizing alleles of tube or pelle produce dorsalized embryos, suggesting that tube and pelle act downstream of the membrane protein Toll in the signaling pathway that defines the embryonic dorsal-ventral pattern. Both tube and pelle are also important zygotically for survival: at least 30% of the zygotes lacking either tube or pelle die before adult stages, while 90-95% of tube- pelle- double mutant zygotes die (Hecht, 1993).

Two components are known to mediate the signal transduction between Toll and Dorsal-Cactus: Pelle, a serine/threonine protein kinase, and Tube, a protein with an unknown biochemical activity. Gain-of-function alleles of pelle and tube show that pelle functions downstream of tube. In addition, Pelle and Tube interact directly with one another. Tube is probably a direct activator of the protein kinase Pelle (Grosshans, 1994).

tube mutations have a zygotic effect. Pupae homozygous for strongly dorsalizing tube alleles are noticeable shorter than wild-type pupae (Letsou, 1991).

The Toll signaling pathway functions in several Drosophila processes, including dorsal-ventral pattern formation and the immune response. This pathway is required in the epidermis for proper muscle development. Because Toll mutations affect the development of all 30 muscle fibers in each hemisegment, and not just the several that express Toll, or those closest to the CNS, it semed likely that the epidermal expression is most relevant to muscle development. In the epidermis, Toll expression is highest in the epidermal muscle attachment (EMA) cells, aligned along the segment border; these cells are known to play an important role in muscle patterning. The zygotic Toll protein is necessary for normal muscle development; in the absence of zygotic Toll, close to 50% of hemisegments have muscle patterning defects consisting of missing, duplicated and misinserted muscle fibers (Halfon, 1998).

The requirements for easter, spatzle, tube, and pelle, all of which function in the Toll-mediated dorsal-ventral patterning pathway have now been analyzed. spatzle, tube, and pelle, but not easter, are necessary for muscle development. Mutations in these genes give a phenotype identical to that seen in Toll mutants, suggesting that elements of the same pathway used for Toll signaling in dorsal-ventral development are used during muscle development. By expressing the Toll cDNA under the control of distinct Toll enhancer elements in Toll mutant flies, the spatial requirements for Toll expression were examined during muscle development. Expression of Toll in a subset of epidermal cells that includes the epidermal muscle attachment cells, but not Toll expression in the musculature, is necessary for proper muscle development. A 6.5-kb enhancer element drives expression solely in mesodermally derived tissues. A 1.4-kb enhancer drives expression in the epidermis, CNS midline, gut, salivary glands, Malpighian tubules, pharynx and esophagus, but not in mesodermal tissues. These two enhancers were used to drive expression of Toll in transgenic flies. The 1.4-kb enhancer express Toll in the epidermis (in a narrow strip of cells that includes the EMA cells) as well as in a cluster of cells in the lateral, mid-bodywall region of each segment. This lateral region contains the cells where the lateral transverse muscle fibers have their insertions. Flies with 1.4-kb enhancer driven Toll expression show complete rescue of the muscle error phenotype. These results suggest that signals received by the epidermis early during muscle development are an important part of the muscle patterning process (Halfon, 1998).

Although loss of single minded, a regulator of the Toll pathway in the central midline, causes positioning and insertion errors in a group of the most ventral muscles, these defects are qualitatively different from those observed in Toll mutants. The errors due to sim mutation are thus not likely due to loss of Toll expression; Toll expression in the midline appears to be uninvolved in muscle patterning. It is known that signaling from the muscle fibers induces the expression of beta1-tubulin in the EMA cells and regulates the maintenance of expression of other attachment site-specific genes such as delilah, groovin, and stripe. The nature of the Toll muscle phenotype (most of which consists of duplicated and deleted muscle fibers) suggests that Toll may be acting early in the development process, during the time of founder specification or early muscle fiber growth. The remaining errors (those in muscle insertion) may be either early or late in origin: they may be secondary to mis-specification of muscle identity (early), or alternatively, might indicate a further requirement for Toll during the insertion process (late) (Halfon, 1998).

There are a number of different controls on the expression of the antifungal polypeptide gene Drosomycin in adults: the receptor Toll, intracellular components of the dorsoventral signaling pathway (Tube, Pelle, and Cactus), and the extracellular Toll ligand, Spätzle, but not the NF-kappaB related transcription factor Dorsal. Mutations in the Toll signaling pathway dramatically reduce survival after fungal infection. In Tl-deficient adults, the cecropin A and, to a lesser extent, attacin, drosomycin and defensin genes are only minimally inducible, in contrast with the diptericin and drosocin genes, which remain fully inducible in this context. The drosomycin gene induction is not affected in mutants deficient in gastrulation defective, snake and easter, all upstream of spätzle in the dorsoventral pathway. The involvement of Spätzle in the drosomycin induction pathway is unexpected, since, in contrast with cat, pll, tub, and Tl, the spz mutant shows no striking zygotic phenotype. The partner of Cact in the drosomycin induction pathway has not yet been identified, but it is probably a member of the Rel family, possibly Dorsal-related immunity factor (Lemaitre, 1996).

There are two distinct regulatory pathways controlling the expression of antimicrobial genes, the dorsoventral pathway and the immune deficiency (imd) gene. In contrast to the results with drosomycin, antibacterial genes, cecropin A1, diptericin, drosocin, attacin, and defensin do not give strong constitutive expression in dorsoventral pathway mutants. However, the level of constitutive expression of anti-bacterial genes in dorsoventral pathway mutants is higher than the basal level, and induction of Cecropin A genes is 4-fold lower in dorsoventral pathway mutants. The transcription of cact, dorsal, dif, pll, tub, Tl and spz genes, but not tub, are clearly up-regulated in response to immune challenge. Even though the same components of the dorsoventral pathway that are involved in antifungal response are also involved in antibacterial response, there is an additional requirement for the as yet uncloned imd gene product (Lemaitre, 1996).

Tube and larval hemocyte concentration and the egg encapsulation response in Drosophila

Drosophila larvae defend themselves against parasitoid wasps by completely surrounding the egg with layers of specialized hemocytes called lamellocytes. Similar capsules of lamellocytes, called melanotic capsules, are also formed around 'self' tissues in larvae carrying gain-of-function mutations in Toll and hopscotch. Constitutive differentiation of lamellocytes in larvae carrying these mutations is accompanied by high concentrations of plasmatocytes, the major hemocyte class in uninfected control larvae. The relative contributions of hemocyte concentration vs. lamellocyte differentiation to wasp egg encapsulation are not known. To address this question, Leptopilina boulardi was used to infect more than a dozen strains of host larvae harboring a wide range of hemocyte densities. A significant correlation exists between hemocyte concentration and encapsulation capacity among wild-type larvae and larvae heterozygous for mutations in the Hopscotch-Stat92E and Toll-Dorsal pathways. Larvae carrying loss-of-function mutations in Hopscotch, Stat92E, or dorsal group genes exhibit significant reduction in encapsulation capacity. Larvae carrying loss-of-function mutations in dorsal group genes (including Toll and tube) have reduced hemocyte concentrations, whereas larvae deficient in Hopscotch-Stat92E signaling do not. Surprisingly, unlike hopscotch mutants, Toll and tube mutants are not compromised in their ability to generate lamellocytes. These results suggest that circulating hemocyte concentration and lamellocyte differentiation constitute two distinct physiological requirements of wasp egg encapsulation and Toll and Hopscotch proteins serve distinct roles in this process (Sorrentino, 2004).

These results suggest that the suppression of encapsulation capacity by loss of function of hop, Tl, or tub is likely to be due to distinct requirements of these genes. The suppression of lymph gland response to parasitization in the hop^M4 background is consistent with the observed reduction in hop^M4/Y encapsulation capacity and suggests that the Hopscotch protein is necessary for a parasite-induced signal for lamellocyte differentiation. This signal for lamellocyte differentiation is most likely mediated by the transcription factor Stat92E: Loss of function of one copy of Stat92E suppresses the penetrance of the hop^Tum-l-induced melanotic tumor phenotype and Stat92E is constitutively activated in Drosophila cell cultures that overexpress Hop^Tum-l. These results are consistent with the proposed Stat92E-dependent lamellocyte signal: stat92E larvae are immune compromised and are unable to mount an efficient egg encapsulation response despite exhibiting control circulating hemocyte concentration levels. Additionally, mean circulating lamellocyte percentage in hop^Tum-l/Y; stat92E^HJ/stat92E^HJ larvae that are tumor-free is ~1%, which is indistinguishable from the control value (Sorrentino, 2004).

In contrast to Hop and Stat92E, Toll and Tube appear not to play a role in lamellocyte differentiation; rather, loss-of-function mutations in Toll or tube probably suppress encapsulation via other mechanisms. Since Toll and tube larvae have very few circulating hemocytes, reduction in encapsulation in Tl and tub mutants might be due to defects in wasp egg recognition or a reduction in hemocyte proliferation that normally follows parasitization. The effect of these mutations on crystal cells is unclear. While the possibility that these mutations reduce encapsulation capacity by reducing the crystal cell population cannot be ruled out, this is unlikely, since Black cells mutant larvae without functional crystal cells are immune competent and can still successfully encapsulate wasp eggs. The fact that lymph glands of loss-of-function Tl and tub mutant larvae can support lamellocyte differentiation suggests that the low circulating hemocyte concentration in Tl and tub larvae in itself does not hinder lamellocyte differentiation or the ability of the lymph gland to disperse after the wasp egg is introduced into the hemocoel. Given that gain-of-function Tl alleles induce lamellocyte differentiation, the lack of effect of Tl^- on lamellocyte differentiation is somewhat unexpected, and it is possible that lamellocyte differentiation is in some way secondarily activated in the Tl^10b background. Thus, the wasp egg encapsulation assay is a useful tool for evaluating the genetic requirements for lamellocyte differentiation (Sorrentino, 2004).

In conclusion, this study shows that while there is substantial variation in hemocyte concentration in control larvae, this variation is consistent with a log-normal distribution. Such a distribution could be a result of the inherently logarithmic process of cell division. Using this quantitative method of circulating hemocyte concentration data analysis, it was found that previously reported circulating hemocyte concentration values for mutant larvae that exhibit reduced or increased hemocyte densities are also log-normally distributed and that approximately half of each of these mutant distributions lie beyond the limits of the control distribution, allowing ranges of circulating hemocyte concentration values to be defined as being low, control, and high. In addition, encapsulation capacity in control and DV mutant larvae correlates with circulating hemocyte concentration. Evidence for biological significance of this correlation also comes from observations that D. melanogaster larvae selected for higher resistance against A. tabida have twice as many circulating hemocytes as compared to control larvae. These observations support the notion that circulating hemocytes, possibly plasmatocytes, contribute to the efficiency of the egg encapsulation response. However, high circulating hemocyte concentration alone is insufficient to trigger encapsulation; lamellocytes must be present. For example, massive 20- to 300-fold increases in circulating hemocyte concentration involving plasmatocytes and crystal cells, but not lamellocytes, are insufficient to trigger constitutive encapsulation of self tissue in the larva. The combined use of genetic and immune approaches used in this study demonstrates that different developmental signals independently contribute to the maintenance of the steady-state hemocyte concentration in circulation and the ability to differentiate lamellocytes. Together, these physiological parameters enable larval hosts to efficiently defend themselves against wasp infections (Sorrentino, 2004).

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date revised: 20 March 2007

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