InteractiveFly: GeneBrief

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

Gene name - Tiggrin

Synonyms -

Cytological map position - 26D1--26D2

Function - extracellular matrix protein

Keywords - mesoderm, muscle attachment

Symbol - Tig

FlyBase ID: FBgn0011722

Genetic map position - 2-[20]

Classification - novel multidomain protein

Cellular location - extracellular

NCBI link: Entrez Gene
Tig orthologs: Biolitmine
Recent literature
Green, N., Odell, N., Zych, M., Clark, C., Wang, Z. H., Biersmith, B., Bajzek, C., Cook, K. R., Dushay, M. S. and Geisbrecht, E. (2016). A common suite of coagulation proteins function in Drosophila muscle attachment. Genetics [Epub ahead of print]. PubMed ID: 27585844
The organization and stability of higher order structures that form in the extracellular matrix (ECM) to mediate the attachment of muscles are poorly understood. The surprising discovery was made that a subset of clotting factor proteins are also essential for muscle attachment in the model organism Drosophila melanogaster. One such coagulation protein, Fondue (Fon), was identified as a novel muscle mutant in a pupal lethal genetic screen. Fon accumulates at muscle attachment sites and removal of this protein results in decreased locomotor behavior and detached larval muscles. A sensitized genetic background assay reveals that fon functions with the known muscle attachment genes thrombospondin (Tsp) and tiggrin (Tig). Interestingly, Tig is also a component of the hemolymph clot. It was further demonstrated that an additional clotting protein, Larval serum protein 1γ (Lsp1γ), is also required for muscle attachment stability and accumulates where muscles attach to tendons. While the local biomechanical and organizational properties of the ECM vary greatly depending on the tissue microenvironment, it is proposed that shared extracellular protein-protein interactions influence the strength and elasticity of ECM proteins in both coagulation and muscle attachment.
Zhang, C. U. and Cadigan, K. M. (2017). The matrix protein Tiggrin regulates plasmatocyte maturation in Drosophila larva. Development [Epub ahead of print]. PubMed ID: 28526755
The lymph gland (LG) is a major source of hematopoiesis during Drosophila development. In this tissue, prohemocytes differentiate into multiple lineages including macrophage-like plasmatocytes, which comprise the vast majority of mature hemocytes. Previous studies have uncovered genetic pathways that regulate prohemocyte maintenance and some cell fate choices between hemocyte lineages. However, less is known about how the plasmatocyte pool of the LG is established and matures. This study reports that Tiggrin, a matrix protein expressed in the LG, is a specific regulator of plasmatocyte maturation. Tiggrin mutants exhibit precocious maturation of plasmatocytes, while Tiggrin overexpression blocks this process, resulting in a buildup of intermediate progenitors (IPs) expressing prohemocyte and hemocyte markers. These IPs likely represent a transitory state in prohemocyte to plasmatocyte differentiation. It was also found that overexpression of Wee1 kinase, which slows G2/M progression, results in a phenotype similar to Tiggrin overexpression while String/Cdc25 expression phenocopies Tiggrin mutants. Further analysis revealed that Wee1 inhibits plasmatocyte maturation through up-regulation of Tiggrin transcription. These results elucidate connections between the extracellular matrix and cell cycle regulators in the regulation of hematopoiesis.

Tiggrin is a component of the extracellular matrix (ECM), found at sites for muscle insertion or attachment known as apodemes (Fogarty, 1994). The concentration of Tiggrin at these segmentally repeated sites gives embryos stained with anti-Tiggrin antibodies the striped appearance associated with Tigger, a tiger-like character introduced in the 1938 children's classic by A.A. Milne, The House at Pooh Corner.

A few words on other components of the ECM are in order, before reviewing Tiggrin. Other ECM proteins that like Tiggrin have been identified at Drosophila muscle insertions are collagen IV, papilin (a glycoprotein containing a TSP1 [properidin] domain). and Glutactin (a glycoprotein containing carboxyesterase homology). Transcripts of Tenascin A (see Drosophila Tenascin-major), a member of the tenascin family, are also found in cells located at apodemes (Baumgartner, 1993). Dachsous, a cadherin, and Laminin A have both been associated with apodemes. Mutants of crocodile, coding for a forkhead domain transcription factor, lack certain apodemes. Action of stripe, coding for a EGR type zinc finger transcription factor, has been found to be both necessary and sufficient to initiate the developmental program of epidermal muscle attachment. Ectopic expression of Stripe in various epidermal cells transforms these into muscle-attachment cells expressing an array of epidermal muscle attachment cell-specific markers. These markers include Goovin, Delilah, and beta1 tubulin (Becker, 1997).

The extracellular matrix itself is an interconnected network of glycoproteins, proteoglycans and glycosaminoglycans secreted and assembled by cells. At the apodeme sites the PS2 integrins function to maintain muscle attachments and Tiggrin has been shown to be is a ligand of the PS2 integrins. This suggests that Tiggrin in the ECM binds to the PS2 integrins and mediates PS2-ECM interactions. Along striated muscles Tiggrin and the PS2 integrins colocalize at Z-bands (Fogerty, 1994). PS1 (Multiple edematous wings) and PS2 (Inflated) integrins contain a common betaPS subunit (Myospheroid) that is associated with either an alphaPS1 or an alphaPS2 subunit. Thus, PS1 and PS2 integrins are respectively alphaPS1betaPS and alphaPS2betaPS heterodimers (Fogarty, 1994).

Three observations suggest that Tiggrin interacts with the PS2 integrins:

If all functions of the PS2 integrins are mediated by their interactions with Tiggrin then Tiggrin mutants would be expected to display the same range of phenotypes that are found in mutations that remove the PS2 integrins. This would include complete detachment of most muscles from their attachment sites, and central nervous system, wing and gut defects. More likely, Tiggrin may be only one of several ligands used by the PS2 integrins, and Tiggrin may have additional functions that are independent of its interactions with the PS2 integrins. Tiggrin mutant phenotypes are shown to be complex, affecting some but not all of the tissues that are defective in PS2 integrin mutants. In these tissues, Tiggrin mutants display novel phenotypes not described for PS2 integrin mutants (Bunch, 1998).

At segment borders where ends of multiple muscles attach to epidermal tendon cells, and where accumulations of Tiggrin protein are found in wild-type animals, the gaps between muscles increase from 7 mm in wild-type third instar larvae to 30 mm in Tiggrin mutant larvae. One model that could explain these gaps is that the PS2 integrins are involved in two adhesion sites when neighboring muscles make attachments to the same or neighboring epidermal tendon cells. The first site is the well-documented muscle-epidermal attachment. This attachment may or may not utilize Tiggrin. The second site involves muscle-muscle attachment. Muscle-muscle attachments have not been described in detail; however, experiments that genetically remove the epidermal tendon cells result in muscles that detach from the epidermis but remain attached to each other (Martin-Bermudo, 1996). A recently isolated mutant, rhea, also displays muscles that detach from the epidermis but remain attached to each other (Prout, 1997). This strongly suggests that muscle-muscle attachments exist in normal animals. The results reported here suggest that Tiggrin is required to maintain and/or establish these specialized muscle-muscle junctions (Bunch, 1998).

In contrast to the longitudinal and oblique bodywall muscles, transverse bodywall muscles appear to attach only to the epidermis, do not show strong Tiggrin staining and do not show defects in the Tiggrin mutants. This indicates that these two muscle attachment sites are different and is consistent with the model that Tiggrin is involved mainly with the muscle-muscle junctions and not so critical at the muscle-epidermal junctions. Prokop (1998a) has observed that these two muscle junctions are ultrastructurally quite distinct. The transverse muscles display a close apposition (30-40 nm) between muscle and epidermis; in contrast to this, at segment borders where longitudinal and oblique muscles converge at sites on the epidermis, large accumulations of tendon matrix, including Tiggrin, separate cells by several mm. In this model, the absence of Tiggrin results in muscles remaining attached to the epidermal cells but detaching from each other, resulting in the separated, but well-ordered, muscle termini. The PS2 integrins are involved in both attachments because inflated mutants (in which the alphaPS2 subunits are removed) result in completely detached and rounded up muscles. This model suggests that the PS2 integrins may use different ligands for the muscle-epidermal attachment. Tenascin major is an example of a potential ECM component that may carry out this function. It is found at attachment sites, has an RGD sequence and interacts with the PS2 integrins in cell spreading assays (Baumgartner, 1994). Other extracellular molecules known to function or locate to these attachment areas include Laminin, Slit, Masquerade, m-Spondin, Collagen IV and Groovin. By interacting directly or indirectly with the PS2 integrins and other cell surface receptors, these proteins may further support the muscle-epidermal attachment in the absence of Tiggrin (Bunch, 1998).

For Tiggrin to mediate a direct link between cells via the PS2 integrins would require two PS2 integrin-binding sites, but Tiggrin has only one RGD sequence. Biochemical data are consistent with Tiggrin forming extended rod-like homodimers or homotrimers that are approximately 180 nm in length (Fogerty, 1994). If two Tiggrin molecules dimerize in an anti-parallel fashion, this would place the RGD integrin-binding domain on each end of the rod and could serve as a direct link between PS2 integrins on adjacent cells. However, the 180 nm length of such a dimer or trimer is not consistent with the distance of several mm between neighboring muscles visualized by actin staining or at the EM level (Prokop, 1998a). Furthermore, Tiggrin is anchored to the ECM by interactions with other matrix components, as evidenced by the correct localization of Tiggrin in myospheroid mutants, which lack PS2 integrins (Fogerty, 1994). Therefore Tiggrin may provide a link to the ECM rather than a direct linkage between the PS2 integrins on neighboring cells (Bunch, 1998).

Integrins regulate responsiveness to Slit repellent signals

Integrins are concentrated within growth cones, but their contribution to axon extension and pathfinding is unclear. Genetic lesionof 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).


Protein Interactions

Primary Drosophila embryo cells from postgastrulation embryos differentiate when cultured on a substratum of Drosophila laminin or human vitronectin. Tiggrin, and a fusion protein of Tiggrin (residues 1,891-2,161) that contains the RGD binding site have been tested as substrata. Excellent differentiation of several different cell types occurs; e.g. abundant multinucleate myotubes and neurites form and both hemocytes and clusters of epidermal cells are present after 18 hours of culture at 22 degrees C. On control coverslips coated with bovine serum albumin very limited differentiation is observed (Fogerty, 1994).

The finding of an RGD cell attachment motif in Tiggrin, which conspicuously colocates with both alphaPS integrins at muscle apodemes and at Z-bands, emphasized the need to test Tiggrin as an integrin ligand. To do this, the ability of Tiggrin-coated substratum to mediate the spreading of Drosophila S2 cells that have been transformed with genes for alphaPS2 and betaPS integrin chains was assayed. A pair of alternative RNA splice variants codes for two forms of the alphaPS2 integrin chain that differ by 25 amino acids near the putative ligand binding site. Correspondingly, two transformed S2 cell lines were created: HSPS2(C), which expresses the alphaPS2 chain that contains the additional 25 amino acids, and HSPS2(m8), a line in which these same 25 amino acids (coded for by exon 8) are missing. Both cell lines also express the betaPS chain and make comparable amounts of integrins. Tiggrin serves as a substratum for alphaPS2betaPS integrin-mediated cell spreading. HSPS2(C) cells show no spreading when applied to tissue culture plastic that has been blocked with milk solids to prevent nonspecific interactions. When the plastic is first coated with Tiggrin, these cells show extensive cell spreading. Similar but less extensive spreading is seen for HSPS2(m8) cells. The untransformed S2 cells show no spreading on Tiggrin, demonstrating that the observed spreading is mediated by the integrins expressed from the transgenes. Spreading of both cell lines is abolished when a monoclonal antibody that blocks integrin-mediated aggregation of Drosophila cells is added to the spreading medium. In contrast, the control CF.6G11 antibody, which binds to betaPS integrin chains on cells but does not block their function, has no effect on cell spreading. Tiggrin that was eluted from an SDS-PAGE band also promotes cell adhesion and spreading, providing further evidence that the active cell adhesive component is Tiggrin and not some chromatographically unresolved contaminant. The fusion protein that contains 270 amino acids from the C-terminal region of Tiggrin (residues 1,891-2,161), including the RGD sequence, but that lacks both of the putative LRE cell attachment sequences and the central repeat domain, also promotes adhesion and spreading of >65% of HSPS2(m8) cells and >85% of HSPS2(C) cells. The amounts of intact purified Tiggrin or Tiggrin fusion protein required to promote spreading are similar. A control fusion protein, derived from the antisense strand of the cDNA that codes for the RGD-containing fusion protein, has no effect on cell attachment and spreading. Cell spreading was quantitated on surfaces coated with 1 µg/ml or 5 µg/ml tiggrin solutions. Tiggrin mediates the spreading of a high percentage of cells expressing either alphaPS2betaPS integrin. However, on plastic coated with 1 µg/ml Tiggrin the HSPS2(C) cells spread better than the HSPS2(m8) cells (Fogerty, 1994).

To test the functional significance of Tiggrin's RGD sequence in these cell spreading experiments, the effects of increasing concentrations of an added, soluble RGD peptide (GRGDSP) was tested. This peptide inhibits spreading of both HSPS2(C) and HSPS2(m8) cells on tiggrin. Inhibition of HSPS2(m8) cells is achieved with much lower levels of soluble peptide than is seen for the HSPS2(C) cells. This is probably due to the lower initial levels of spreading seen for the HSPS2(m8) cells in the absence of any peptide. The control RGE peptide (GRGESP) has relatively little effect on cell spreading (Fogerty, 1994).

Drosophila cells expressing PS2 integrins spread on Tiggrin purified from Drosophila cells or Tiggrin fusion proteins expressed in bacteria. This spreading is inhibitable by RGD peptides, which suggests that the RGD sequence in Tiggrin is critical for the integrin-tiggrin interaction (Fogerty, 1994). This hypothesis was further tested in cultured cells and the whole fly. Using in vitro mutagenesis the sequence encoding RGD in Tiggrin was changed to encode LGA. Fusion proteins containing the final 333 amino acids of Tiggrin (of a total of 2186 amino acids) were produced in bacteria. These proteins are identical with the exception of having RGD in one case and LGA in the other (TIG-RGD and TIG-LGA respectively). A standard cell spreading assay tested the ability of each fusion protein to promote PS2 integrin-mediated cell spreading. There are four potential forms of the PS2 integrin that are generated by alternative splicing of mRNA encoding the alphaPS2 and betaPS subunits All four forms were tested for their ability to interact with TIG-RGD and TIG-LGA. The TIG-RGD readily promotes cell spreading (Fogerty, 1994).

To test the importance of the RGD sequence in the whole organism, an RGD-to-LGA mutant tiggrin transgene (UASTig LGA ) was constructed and this was introduced into flies. This mutant transgene fails to rescue tiggrin mutant flies to the levels of the wild-type transgene. It shows little rescue of the muscle defects observed in dissected larvae, as muscles are often missing and gaps are observed between muscles 9 and 10. However, measurement of these gaps indicates that they are intermediate in distance between wild-type, or UASTig + rescued, larvae. UASTig LGA-rescued tig x /tig x larvae have muscle contraction waves that are twice as fast as tig x mutants; however, this is still much slower than wild-type or tig x flies rescued by UASTig +. Thus, Tiggrin lacking an RGD sequence shows partial activity in these two assays. Lethality can be rescued by the UASTig LGA up to a maximum of 30%. tig x /tig A1 and tig x /tig O2 mutants are also rescued much more efficiently by wild-type tiggrin trangenes as compared with tig LGA transgenes (Bunch, 1998).

In the Drosophila embryo, the correct association of muscle cells with their specific ectodermally derived tendon cells, also known as epidermal muscle attachment or EMA cells, isachieved through reciprocal interactions between these two distinct cell types. Vein, a neuregulin-like factor secreted by the approaching myotube, activates the EGF-receptor signaling pathway within the tendon cells to initiate tendon cell differentiation. kakapo is expressed in the tendons and is essential for muscle-dependent tendon cell differentiation. Kakapo is a large intracellular protein and contains structural domains also found in cytoskeletal-related vertebrate proteins (including plakin, dystrophin, and Gas2 family members). kakapo mutant embryos exhibit abnormal muscle-dependent tendon cell differentiation. The expression of delilah, stripe, and beta1 tubulin is induced in the epidermal attachment cells as a result of the EGF-receptor pathway activation by the neuregulin-like growth factor, Vein. Vein is secreted by mesodermal cells underlying the EMA cells. Vein protein localization is restricted to the muscle-tendon junctional site in wild-type embryos. However, in kak mutant embryos, Vein protein is not localized and appears rather diffuse. This altered pattern of Vein may explain the multiple number of cells expressing delilah and stripe: since Vein is not strictly localized at a given muscle-tendon junction site, it apparently weakly activates the EGF-receptor pathway in neighboring cells as well. It is presumed that the only cells that can respond to the ectopic Vein protein are the competent population of EMA cells, defined by the early expression of stripe. These cells express stripe during early developmental stages in a muscle-independent manner and normally lose their stripe expression by stage 16 of embryonic development. When these competent EMA cells receive the muscle-derived Vein signal, the expression of stripe and delilah is reactivated. It appears that only this population of cells is capable of responding to Vein, since the pattern of the ectopic Stripe- or Delilah-expressing cells in the kak mutant embryos resembles that of the early population of Stripe-expressing cells. The reduced levels of beta1 tubulin mRNA in the mutant tendon cells may also result from the abnormal pattern of Vein localization, since lower levels of Vein may not be sufficient to induce maximal beta1 tubulin expression. It therefore appears that the primary defect in kak mutant embryos stems from the lack of Vein accumulation at the muscle-tendon junctional site (Strumpf, 1998).

How could this intracellular protein affect the localization of Vein at the extracellular matrix surrounding the EMA cell? At least two possibilities, which are not mutually exclusive, are considered. The first is the association of Kak with the unique cytoskeletal network of the EMA cell, which is critical for the cell's polarized organization. Tendon cell polarity may be essential for maintaining the characteristic junctional complexes formed between the basal surfaces of the EMA cell and the muscle cells. The space between these junctional complexes contains many extracellular matrix proteins, some of which may possess a Vein binding function. Impaired tendon cell polarity may lead to the loss of the putative Vein-binding component(s). Alternatively, Kak may be associated with a transmembrane protein(s) responsible for Vein localization either by direct binding or by association with additional extracellular matrix components that may directly bind Vein. Immunoprecipitation experiments with anti-Kak antibody indicated that Kakapo forms protein complexes containing the extracellular protein Tiggrin. These results favor the latter possibility that Kak is directly associated with protein complexes that may be important for Vein binding. The reduced amount of electron-dense material observed at the muscle-tendon junction site in the kak mutant embryos described in Prokop, et al. (1998b) is in agreement with both mechanisms mentioned above (Strumpf, 1998).

Drosophila integrins have been expressed on the surfaces of cultured cells and tested for adhesion and spreading on various matrix molecules. PS1 integrin (Multiple edematious wings) is a laminin receptor, PS1 and PS2 (inflated) integrins promote cell spreading on two different Drosophila extracellular matrix molecules, laminin and Tiggrin: PS1 on laminin, and PS2 on Tiggrin. The differing ligand specificities of these two integrins, combined with data on the in vivo expression patterns of the integrins and their ligands, lead to a model for the structure of integrin-dependent attachments in the pupal wings and embryonic muscles of Drosophila. Specifically it is thought that PS2 integrins are restricted to cells of the presumptive ventral layer of the wing and PS1 is restricted to the presumptive dorsal cells. Clones of wing tissue mutant for the PS2 integrin result in blisters on the ventral, but not the dorsal wing surface. It is not known, however, how the opposing cell layers are linked extracellularly. It is proposed that both laminin and Tiggrin are involved in these linkages. Either Tiggrin and laminin bind to one another or bind via other, as yet unidentified ECM molecules (Gotwals, 1994).

Splice variants of the Drosophila PS2 integrins differentially interact with RGD-containing fragments of the extracellular proteins tiggrin, ten-m, and D-laminin 2

Two new potential ligands of the Drosophila PS2 integrins have been characterized by functional interaction in cell culture. These potential ligands are a new Drosophila laminin alpha2 chain encoded by the wing blister locus and Ten-m, an extracellular protein known to be involved in embryonic pattern formation. As with previously identified PS2 ligands, both contain RGD sequences, and RGD-containing fragments of these two proteins (DLAM-RGD and TENM-RGD) can support PS2 integrin-mediated cell spreading. In all cases, this spreading is inhibited specifically by short RGD-containing peptides. As previously found for the PS2 ligand tiggrin (and the tiggrin fragment TIG-RGD), TENM-RGD induces maximal spreading of cells expressing integrin containing the alphaPS2C splice variant. This is in contrast to DLAM-RGD, which is the first Drosophila polypeptide shown to interact preferentially with cells expressing the alphaPS2 m8 splice variant. The betaPS integrin subunit also varies in the presumed ligand binding region as a result of alternative splicing. For TIG-RGD and TENM-RGD, the beta splice variant has little effect, but for DLAM-RGD, maximal cell spreading is supported only by the betaPS4A form of the protein. Thus, the diversity in PS2 integrins due to splicing variations, in combination with diversity of matrix ligands, can greatly enhance the functional complexity of PS2-ligand interactions in the developing animal. The data also suggest that the splice variants may alter regions of the subunits that are directly involved in ligand interactions, and this is discussed with respect to models of integrin structure (Graner, 1998full text of article).



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).


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).


Search PubMed for articles about Drosophila Tiggrin

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

Milne, A. A. (1928). The House at Pooh Corner, New York: E. P. Dutton & Co., Inc.

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

Prokop, A., et al. (1998b). The kakapo mutation affects terminal arborization and central dendritic sprouting of Drosophila motorneurons. J. Cell Biol. 143(5): 1283-94. PubMed ID: 9832556

Prout, M., Damania, Z., Soong, J., Fristrom, D. and Fristrom, J. W. (1997). Autosomal mutations affecting adhesion between wing surfaces in Drosophila melanogaster. Genetics 146: 275-285

Stevens, A. and Jacobs, J. R. (2002). Integrins regulate responsiveness to Slit repellent signals. J. Neurosci. 22(11): 4448-4455. 12040052

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

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