Tiggrin


DEVELOPMENTAL BIOLOGY

Embryonic

Tiggrin, a novel Drosophila extracellular matrix protein that functions as a ligand for Drosophila alphaPS2betaPS integrins

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

The PS2 integrin ligand tiggrin is required for proper muscle function in Drosophila

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).


REFERENCES

Baumgartner, S. and Chiquet-Ehrismann, R. (1993). Ten[a], a Drosophila gene related to tenascin, shows selective transcript localization. Mech. Dev. 40: 165--176

Baumgartner, S., Martin, D., Hagios, C. and Chiquet-Ehrismann, R. (1994). ten m, a Drosophila gene related to tenascin, is a new pair-rule gene EMBO J. 13: 3728-3740

Becker, S., et al. (1997). Reciprocal signaling between Drosophila epidermal muscle attachment cells and their corresponding muscles. Development 124(13): 2615-2622. PubMed ID: 9217003

Bunch, T. A., et al. (1998). The PS2 integrin ligand tiggrin is required for proper muscle function in Drosophila. Development 125: 1679-1689. PubMed ID: 9521906

Fogerty, F. J., et al. (1994). Tiggrin, a novel Drosophila extracellular matrix protein that functions as a ligand for Drosophila alphaPS2betaPS integrins. Development 120(7):1747-58. PubMed ID: 7924982

Gotwals, P. J., et al. (1994). Drosophila PS1 integrin is a laminin receptor and differs in ligand specificity from PS2. Proc. Natl. Acad. Sci. 91: 11447-51

Graner, M. W., Bunch, T. A., Baumgartner, S., Kerschen, A. and Brower, D. L. (1998). Splice variants of the Drosophila PS2 integrins differentially interact with RGD-containing fragments of the extracellular proteins tiggrin, ten-m, and D-laminin 2. J. Biol. Chem. 273: 18235-18241. Medline abstract: 9660786

MartÌn-Bermudo, M. and Brown, N. (1996). Intracellular signals direct integrin localization to sites of function in embryonic muscles. J. Cell Biol. 134: 217-226

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Prokop, A., et al. (1998a). Absence of PS integrins or Laminin A affects extracellular adhesion, but not intracellular assembly, of hemiadherens and neuromuscular junctions in Drosophila embryos. Dev. Biol. 196(1): 58-76. PubMed ID: 9527881

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Strumpf, D. and Volk, T. (1998). Kakapo, a novel cytoskeletal-associated protein is essential for the restricted localization of the neuregulin-like factor, vein, at the tion site. J. Cell Biol. 143(5): 1259-70. PubMed ID: 9832554


Tiggrin: Biological Overview | Regulation | Developmental Biology | Effects of Mutation

date revised: 20 August 2002

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