Tiggrin

DEVELOPMENTAL BIOLOGY

Embryonic

A single 7.0 kb Tiggrin transcript is first detected at 6-8 hours of development, increases to a peak level at 12-14 hours and then declines to a low level by the end of embryogenesis. Subsequently the level of Tiggrin mRNA increases to a second peak during the late 2nd to early 3rd larval instar period. In contrast, Tiggrin protein first appears at 8-10 hours and its level then continues to increase throughout embryogenesis, even after Tiggrin mRNA levels have declined (Fogerty, 1994).

Tiggrin protein is found throughout the larval stages with a peak level at 3rd instar. Soluble Tiggrin was detected by Western blot analysis in the hemolymph collected from 3rd instar larvae. At pupation the Tiggrin level declines markedly and the levels of extractable Tiggrin are very low in pupae and adult flies. The apparent discrepancy between changing RNA levels and rate of protein increase is not confined to Tiggrin. The cells that express Tiggrin also make laminin and collagen IV. The rates of formation of these proteins are also not tightly coupled to the corresponding RNA levels, which vary independently of each other over this developmental period. It is concluded that, at least for hemocytes and fat body cells, it is inappropriate to make the common, simplifying assumption of equating developmental RNA patterns with quantitatively matching expression of the corresponding proteins. To determine the spatial expression of Tiggrin during embryogenesis, cDNA probes were hybridized to whole-mount embryos. Transcripts are first detected in hemocytes during germ band retraction. Two hours later many hybridizing hemocytes are distributed throughout the embryo and transcripts are also detected in the fat body. Correspondingly, Tiggrin protein is seen in hemocytes and later in the fat body, indicating that these two tissues are the principal sources of embryonic Tiggrin. Tiggrin accumulates generally in basement membranes, e.g. in those associated with the gut musculature. Early deposits of Tiggrin are found at segmental furrows, at sites where muscle apodemes form, and later it is concentrated at these muscle-to-epidermis attachment sites. A definite staining of the ventral nerve cord commissures is evident with affinity purified antibodies against Tiggrin or the fusion protein. In the larval somatic musculature, Tiggrin is most prominent at the muscle-epidermal attachment sites. It is also present in the ECM surrounding the somatic muscles and in the basement membrane underlying the epidermis. At the Z-bands of adult striated jump muscles, Tiggrin occurs at the muscle surface, where alphaPS2betaPS integrin is found (Fogerty, 1994).

Individual hemocytes each synthesize several of the identified ECM proteins: collagen IV, laminin, papilin and peroxidasin. To assess whether Tiggrin is synthesized by these same hemocytes, whole-mount embryos were immunostained with mouse anti-Tiggrin and rabbit anti-collagen IV antibodies. Of the hemocytes in 10 embryos a total of 614 cells were clearly distinguished; 93-100% of these cells in any embryo have reacted with both antibodies (Fogerty, 1994).

Effects of Mutation or Deletion

Tiggrin deletion (tix x) is lethal to 99% of mutant flies. Surprisingly, homozygous mutant flies appear relatively normal, although escapers do display misshapen (elongated) abdomens. The abdominal phenotype is also seen in tig A1 /tig x and tig O2 /tig x (both semilethal/deletion) mutant adults, though it may not be as severe. Wing defects are found in 7-8% of the tig x homozygous escapers. These defects include notched wings, deformed anterior margins, smaller and rounder wings, and wavy posterior regions. Flies that have just eclosed often show abnormal separation of the dorsal and ventral wing blades in the posterior region of the wing. Wing defects are also observed in tig A1 homozygous and tig A1 /tig x animals. No embryonic lethality is observed. A potential embryonic requirement is not being rescued by a maternal Tiggrin contribution, since a cross of tig x homozygous male and female escapers shows no embryonic lethality and a small percentage of the resulting larvae develop to the adult stage. To determine if Tiggrin is a pupal lethal mutation, homozygous pupae were examined for their ability to eclose to adults. 88% of pupae fail to eclose in this experiment. Most of Tiggrinís lethality can therefore be attributed to the pupal phase (Bunch, 1998).

At pupariation, contraction of bodywall muscles shortens the body. tig x and tig A1 homozygous pupae are 16% longer than wild-type and heterozygous pupae. Pupation, as defined by the appearance of a gas bubble in the abdomen, occurs in tig x and tig A1 mutant animals; however, dispersion of the bubble and head eversion fails to occur in about half of the pupae. This process also requires proper bodywall muscle function. Homozygous tig x and tig A1 larvae show other behavioral abnormalities that are likely to result from muscle defects. Muscle contraction waves that pass from the posterior to anterior of larvae are responsible for locomotion. The duration of these waves were measured in wandering 3rd instar larvae and newly hatched 1st instar larvae. The contraction waves are much slower in the Tiggrin mutants, taking three times as long to pass from posterior tip to anterior tip, as compared with wild-type or Tiggrin heterozyotes in 3rd instar larvae, and twice as long in newly hatched 1st instar larvae. Though a defect in muscle function is a reasonable cause for the slowing of the contraction waves, muscle force and defects in neural function could also contribute to this phenotype. As the muscle contraction wave defect is also observed in 1st instar larvae that have just hatched, it is unlikely to be due to a general weakened condition of the larvae caused by reduced feeding (Bunch, 1998).

Direct examination of muscles in dissected larvae shows severe defects in tig x and tig A1 homozygotes. Large gaps are observed between the dorsal oblique muscles 9 and 10 and between the ventral oblique muscles 15 and 29. At sites where muscles 3, 4, 5, 8 and 16 come together in wild type larvae, muscles 5 and 8 are usually missing. The muscles in Tiggrin mutant animals often appear thinner than in wild type, and other muscles also are missing in these preparations. This is especially true of the large ventral longitudinal muscles 6 and 7. In contrast to the oblique and longitudinal muscles, examination of the transverse muscles 21-24 in tiggrin mutants has not revealed any defects (Bunch, 1998).

In wild-type and tig x /+ animals, strong Tiggrin accumulations are found at the segmentally spaced insertion sites of the major longitudinal muscles 4, 6, 7, 12 and 13, and the wide dorsal oblique muscles 9 and 10. These are the same sites that are observed to be defective in Tiggrin larvae. Notably, only very weak staining for Tiggrin is observed at the attachments of the transverse muscles 21-24 and the ventral attachments of the ventral oblique muscles 15-17. tig x /tig x animals show a complete absence of staining for Tiggrin. tig x /tig + embryos show strong fluorescence at muscle insertions, while tig x /tig x embryos lack all fluorescence. Nevertheless, no abnormal muscle arrangement or structure is detected either in whole mounts or in muscle fillet preparations of tig x /tig x embryos. Thus, the lack of Tiggrin does not seem to interfere with the localization pattern of the somatic embryonic musculature. This suggests that the loss of muscles seen in larvae may be due to muscles in the mutants detaching and/or degenerating during larval life (Bunch, 1998).

Integrins are concentrated within growth cones, but their contribution to axon extension and pathfinding is unclear. Genetic lesion of individual integrins does not stop growth cone extension or motility, but does increase axon defasciculation and axon tract displacement. In this study, a dosage-dependent phenotypic interaction is documented between genes for the integrins, their ligands, and the midline growth cone repellent, Slit, but not for the midline attractant, Netrin. Longitudinal tract axons in Drosophila embryos doubly heterozygous for slit and an integrin gene, encoding alphaPS1, alphaPS2, alphaPS3, or ßPS1, take ectopic trajectories across the midline of the CNS. Drosophila doubly heterozygous for slit and the genes encoding the integrin ligands Laminin A and Tiggrin reveal similar errors in midline axon guidance. It is proposed that the strength of adhesive signaling from integrins influences the threshold of response by growth cones to repellent axon guidance cues (Stevens, 2002).

Tiggrin is a secreted glycoprotein that contains an RGD motif and is considered to be a ligand of the PS2 integrin. Embryos homozygous for a loss of function allele of Tiggrin have a subtle Fas II phenotype reminiscent of integrin mutants. CNS axon tracts are wavy, and no midline axon guidance errors are seen. Labeling of the most lateral axon tract is interrupted between segments. Like the integrin genes, tig also has a semidominant interaction with slit. Fas II labeling of fascicles between segments is reduced. Midline guidance errors are seen in one in three segments (Stevens, 2002).

Drosophila Laminin is a trimer of three proteins: Laminin A, B1, and B2. Laminin is known to be a ligand of PS1 integrin and possibly other integrins as well. Mutants have not been isolated for the B1 and B2 chains; however, a loss of function allele for lanA encoding the A chain has been characterized. The Fas II phenotype of the lanA mutant is nearly wild type, revealing midline guidance errors in 4% of segments. When doubly heterozygous with sli, in sli/+;lanA/+ embryos, the frequency of midline crossovers is >30% (Stevens, 2002).

Does a change in lanA function also affect integrin function in CNS axon tract formation? lanA interaction with scb was examined because Laminin is not known to be a ligand of alphaPS3/4 (encoded by scb), and scb has a strong semidominant interaction with slit. Both lanA and scb reveal midline guidance errors when homozygous. However, in the scb/+;lanA/+ double heterozygote, midline guidance errors are not seen. Nevertheless, this genotype shares aspects of the integrin CNS phenotype: defasciculation and interruptions in Fas II labeling of the most lateral fascicle. This suggests function of both genes in a common or parallel pathway. If the interaction of scb and lanA is independent of the interaction of either gene with sli, then the phenotype of the triple heterozygote scb/sli;lanA/+ would reflect the addition of the scb/sli, scb/+;lanA/+, and sli/+;lanA/+ phenotypes. The degree of defasciculation and midline guidance errors in all axon tracts of the triple heterozygote appears to be additive. However, a narrowing of the CNS and the medial displacement of all axon tracts are also seen in the triple heterozygote. This phenotype is typical of mutants in genes required for midline guidance and is not a component of the integrin mutant phenotype. The synergistic interaction of these three genes suggests dosage-dependent function for each gene in common or parallel pathways (Stevens, 2002).

Thus, a reduced level of expression of the genes for four integrins (alphaPS1, alphaPS2, alphaPS3/4, and ßPS1) or two integrin ligands (Tiggrin and Laminin) increases the probability that CNS axons make pathfinding errors when slit expression is reduced. Expression of the integrins Tiggrin and Laminin A has been demonstrated in the CNS. Integrin expression is not localized and may be expressed in both glia and neurons. Overexpression of alphaPS3 or Laminin A in motoneurons affects axon guidance. Loss of function of the integrins disrupts axon fasciculation and longitudinal axon fascicle placement in the embryonic nerve cord but does not clearly affect axon guidance. These observations have been extended in this study, with different alleles of the integrins, demonstrating a similar function for alphaPS3/4, and revealing axon fascicle phenotypes for loss of function of integrin ligands Tiggrin and Laminin A. The mutant phenotypes share common elements: mild phenotypes show wavy axon tracts and reduced Fas II labeling between segments, whereas severe phenotypes include defasciculation and fascicle displacement, including midline axon guidance errors. The integrins have different extracellular ligands. Therefore, the integrins contribute similarly to axon tract integrity, independent of the ligand that they bind (Stevens, 2002).

Integrin phenotypes in the CNS do not demonstrate a direct role for integrins in growth cone guidance. In contrast, perturbation of midline growth cone repellent signals results in a medial narrowing of the CNS and ectopic midline crossing of longitudinally projecting axons, rather than defasciculation and displacement of axon tracts. One feature of integrin and tiggrin phenotypes shared with robo and dock mutant phenotypes is a thinning or loss of Fas II labeling in the most lateral axon fascicle. This fascicle expresses Fas II late in embryogenesis. This phenotype may reflect impaired or delayed development of independent fascicles in the nerve cord, as implicated by studies of robo function (Stevens, 2002).

The semidominant interaction of all integrins, Tiggrin, and Laminin A with slit is more prevalent than might be expected if the integrins play a specialized role in Slit signaling. scab also has a dramatic semidominant interaction with dock (Nck), which functions in diverse axon guidance events. scb/dock double heterozygotes have disrupted longitudinal, commissural, and peripheral axon tracts. A similar genetic test suggests that ßPS integrin modulates RhoA activity and axon stability in the mushroom body. These diverse phenotypes reflect an adhesive function of the integrins that reduces the responsiveness of growth cones to guidance signals. Independent evidence suggests that this occurs in the growth cone, but a role for integrin in the glia that emit guidance signals cannot be discounted (Stevens, 2002).

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