The Wingless pathway plays an essential role during synapse development. Recent studies at Drosophila glutamatergic synapses suggest that Wingless is secreted by motor neuron terminals and binds to postsynaptic Drosophila Frizzled-2 (DFz2) receptors. DFz2 is, in turn, endocytosed and transported to the muscle perinuclear area, where it is cleaved, and the C-terminal fragment is imported into the nucleus, presumably to regulate transcription during synapse growth. Alterations in this pathway interfere with the formation of new synaptic boutons and lead to aberrant synaptic structures. This study shows that the 7 PDZ protein dGRIP is necessary for the trafficking of DFz2 to the nucleus. dGRIP is localized to Golgi and trafficking vesicles, and dgrip mutants mimic the synaptic phenotypes observed in wg and dfz2 mutants. DFz2 and dGRIP colocalize in trafficking vesicles, and a severe decrease in dGRIP levels prevents the transport of endocytosed DFz2 receptors to the nucleus. Moreover, coimmunoprecipitation experiments in transfected cells and yeast two-hybrid assays suggest that the C terminus of DFz2 interacts directly with the PDZ domains 4 and 5. These results provide a mechanism by which DFz2 is transported from the postsynaptic membrane to the postsynaptic nucleus during synapse formation and implicate dGRIP as an essential molecule in the transport of this signal (Ataman, 2006: full text of article).
At the NMJ, the Wg pathway is initiated by the secretion of Wg from the presynaptic cells and its binding to DFz2 receptors present at the postsynaptic muscle cells. Upon Wg binding to DFz2, the receptor is internalized and transported to the perinuclear area, where it is cleaved, and the C-terminal DFz2 fragment (DFz2-C) is imported into the nucleus. Although the evidence suggested that endocytosis at the postsynaptic membrane and transport via microtubules are required for DFz2 trafficking from synapses to the muscle nucleus, the exact molecular mechanisms of trafficking were unknown. In this study, evidence is provided that the transport of DFz2 to the nucleus depends on interactions between a PDZ-binding motif at the C terminus of DFz2 and PDZ domains of dGRIP. In postsynaptic muscles, dGRIP is present in Golgi bodies and in a subset of vesicles that is highly concentrated at the postsynaptic area. These vesicles move along microtubules and colocalize with internalized DFz2. DFz2 and dGRIP can directly interact when expressed in heterologous systems. Manipulations that lead to severe reduction of dGRIP mimic all of the synaptic phenotypes resulting from mutations in wg or dfz2. Furthermore, in these dgrip mutants, internalized DFz2 accumulates at the postsynaptic region and is not transported to the nucleus. It is suggested that dGRIP is required at synapses to mediate the trafficking of DFz2 to the nucleus to properly regulate the expansion of the NMJ during muscle growth (Ataman, 2006).
Studies suggest that GRIP is involved in the clustering and trafficking of AMPA receptors at mammalian synapses. GRIP has also been found to interact with Ephrin ligands and Eph receptors, neuronal RAS guanine nucleotide exchange factor (GRASP1), members of the Liprin-α/syd2 family of proteins, the KIF5 microtubule motor kinesin, and extracellular matrix protein FRAS1. This large number of partners identified is perhaps not surprising, given that GRIP contains at least seven modular protein-interaction domains (Ataman, 2006).
Studies have shown that, similar to dGRIP, rat GRIP is also localized to both presynaptic axons and postsynaptic dendritic structures and is enriched in vesicular profiles that closely associate with microtubules. Furthermore, a recent study shows that knockdown of GRIP-1 using siRNA in primary hippocampal neurons interfered with the formation and growth of dendrites in developing neurons and the maintenance of dendrites in mature neurons. In this study, it was similarly found that interfering with GRIP function hampered synaptic bouton formation and growth in larval glutamatergic synapses. Additionally, elimination of postsynaptic dGRIP led to loss of the postsynaptic apparatus and presynaptic active zones and, presumably, to either the retraction or deficient stabilization of new synaptic boutons. These results imply that GRIP family proteins have a conserved role in both the formation and the stabilization of synapses in the nervous system (Ataman, 2006).
In Drosophila, dGRIP is involved in the guidance of embryonic muscle precursors to establish the proper body-wall muscle pattern, whereas this study shows that dGRIP is required for synapse differentiation. These results are not surprising, given the recurrent theme that many molecules necessary for early pattern formation in the embryo, such as members of the TGF-β pathway, the Wg pathway, and the tumor suppressor proteins DLG and Scribble (Scrib) are used again during synapse development (Ataman, 2006).
Evidence that dGRIP and DFz2 might interact arises from the observation that manipulations that lead to alterations in both proteins give rise to remarkably similar phenotypes, including decreased NMJ expansion and the presence of ghost boutons, which lack all postsynaptic proteins studied and the subsynaptic reticulum, and are devoid of active zones but filled with synaptic vesicles. These ghost boutons may represent boutons that initiated their differentiation presynaptically but never fully matured by forming corresponding pre- and post-synaptic specializations. Alternatively, the ghost boutons may represent boutons that are initially formed (including differentiation of both pre- and post-synaptic specializations) but subsequently retracted. However, studies have suggested that retraction of mature boutons at the Drosophila NMJ is accompanied by the presence of 'synaptic footprints', in which postsynaptic proteins are still present, despite the absence of a presynaptic bouton. In mutants in the current study, the opposite phenotype was observed, where synapses have some presynaptic proteins but are devoid of a postsynaptic apparatus (Ataman, 2006).
Interestingly, synaptic footprints are observed when the retrograde signaling mediated by TGFβ is disrupted. In contrast, ghost boutons are observed when the Wg pathway is abnormal. The Wg pathway at the NMJ has been shown to function in an anterograde manner, but the possibility that it also functions in a retrograde manner has not been studied. These findings suggest that, at the Drosophila NMJ, synapse retraction can be induced both pre- and post-synaptically, as in the vertebrate NMJ. It would be interesting to determine whether anterograde Wg and retrograde TGF-β signaling pathways crosstalk and coordinate synapse stability during development. Taken together, these studies suggest that one function of dGRIP at the NMJ is in trafficking DFz2 to the nucleus, which, in turn, regulates synaptic growth (Ataman, 2006).
Glutamate receptor interacting protein (GRIP) homologues, initially characterized in synaptic glutamate receptor trafficking, consist of seven PDZ domains (PDZDs), whose conserved arrangement is of unknown significance. The Drosophila GRIP homologue (DGrip) is needed for proper guidance of embryonic somatic muscles towards epidermal attachment sites, with both excessive and reduced DGrip activity producing specific phenotypes in separate muscle groups. These phenotypes were utilized to analyze the molecular architecture underlying DGrip signaling function in vivo. Surprisingly, removing PDZDs 1-3 (DGripΔ1-3) or deleting ligand binding in PDZDs 1 or 2 convert DGrip to excessive in vivo activity mediated by ligand binding to PDZD 7. Yeast two-hybrid screening identifies the cell adhesion protein the Echinoid (Ed) type II PDZD-interaction motif as binding PDZDs 1, 2 and 7 of DGrip. ed loss-of-function alleles exhibit muscle defects, enhance defects caused by reduced DGrip activity and suppress the dominant DGripΔ1-3 effect during embryonic muscle formation. It is proposed that Ed and DGrip form a signaling complex, where competition between N-terminal and the C-terminal PDZDs of DGrip for Ed binding controls signaling function (Swan, 2006).
This study used genetics to develop a mechanistic model concerning a well-defined function mediated by Drosophila Grip-embryonic muscle guidance. Functional requirements were not transmitted by single domains, but were found to be distributed over the whole length of this 7 PDZD protein in an unexpectedly complex manner. Binding ligands via PDZDs 1 and 2 repressed the activity of the protein, binding to PDZD 3 was involved in de-repression, and PDZ-ligand binding via PDZD 7-mediated DGrip function after its de-repression. Despite the fact that there was no critical dependence on PDZDs 4-5 or interdomains for function, it cannot be excluded that interactions over these domains play a subthreshold role. In fact, the DGripΔ1-3x7 construct showed some residual functionality in terms of muscle rescue. Thus, the whole protein might be used as an 'intelligent frame' designed to execute fine controls such as thresholding functions or coincidence detections. In fact, all attempts to provide DGrip activity or to repress DGrip activities with only partial fragments (DGripΔ4-7, DGripΔ1-5) failed, leading to the belief that DGrip is responsible for the organization of a macromolecular complex, of which the transmembrane protein Ed is part (Swan, 2006).
This analysis suggests that a critical number of PDZDs are utilized for DGrip function, with both negative and positive interactions occurring. Such dependence between PDZDs may be due to structural chaperoning. Alternatively, a fixed orientation might be required for high-affinity binding to its targets as found for tandem PDZDs 1 and 2 in PSD-95, with a complex of two PDZDs having higher binding affinity than either PDZD alone. Moreover, allosteric changes upon PDZD-ligand binding could change binding affinities of neighboring domains or via bridging interactions where one molecule contacts multiple sites on a PDZ protein to effect conformational change. Such mechanisms might be the substrate for integrating ligand binding and functional output over a large 'multivalent' PDZD protein (Swan, 2006).
Point mutations of PDZD 1 and PDZD 2 recapitulated the DGripΔ1-3 phenotype in the lateral transverse muscles (LTM) group of muscles, indicating that the repressive function of the PDZDs 1-3 region is not 'structural' (i.e. by covering other PDZDs on the protein). Instead, it is suggested that ligand interactions are communicated over the whole protein to steer equilibrium between two different functional modes of DGrip signaling (Swan, 2006).
Ed was identified as a novel DGrip interactor. Ed is cell adhesion protein with 7 Ig and 2 FNIII domains, described to have both adherence and signaling roles in Drosophila tissues. It is highly conserved among invertebrates and its closest vertebrate homologues are Nectins, which exhibit 3 Ig domains and end in the PDZ-binding motif E/A-X-Y-V. Functionally, both protein families are similar: although not functionally redundant with Ed, Nectins are present at mammalian adherens junctions (AJs) along with l-afadin and, like Ed, regulate Cadherin-based adherence at AJs. Several lines of evidence link Ed to DGrip:Ed interacted with DGrip in a yeast two-hybrid screen, dependent on the C-terminal EIIV motif, mediated via PDZDs 1, 2 or 7. Myc-tagged DGrip specifically interacts with a peptide representing the last 10 amino acids of the Ed protein, including the EIIV PDZ-binding motif. ed zygotic mutants have defects in the morphogenesis of embryonic muscles qualitatively similar to DGripΔ1-3 overexpression. The dgripex36 muscle phenotype in embryos is enhanced by heterozygosity for ed1x5. Here, LTMs (unaffected in pure dgripex36) are affected as well. dgripex36 mutant muscles (both VLMs and LTMs) are sensitive to Ed overexpression. These synthetic defects suggest that DGrip, while itself not essential for LTM morphogenesis, regulates Ed in this group of muscles. Homozygosity for hypomorphic edslH8 chromosome strongly reduced the severity of the phenotype evoked by pan-muscular expression of DGripΔ1-3, indicating that Ed acts downstream of activated DGrip (Swan, 2006).
Notably, the pattern of Ed-PDZD binding correlates with the DGrip-dependent LTM phenotype. Expression of DGrip missing PDZDs 1, 2 and 3 together, or ligand binding in PDZD 1 and PDZD 2 only, showed a strong dominant active phenotype. Mutation of PDZD 2 caused a dominant phenotype in LTMs. In a yeast-two hybrid test, Ed interacted strongly with PDZD 2 with and PDZD 7, and more weakly with PDZD 1 (Swan, 2006).
In imaginal discs, Ed binds two different PDZD proteins via its EIIV motif: Canoe, an F-actin interacting protein and PAR-3/Bazooka. This interaction is mutually exclusive, thereby influencing cell adhesion and the remodeling of subcortical actin at AJs. This study proposes a similar mechanism, in that both functional states of Ed are established via binding to the same protein (DGrip) at different sites. In this model, DGrip may assist in maintaining equilibrium between active and inactive signaling states of Ed, which in its inactive state binds to PDZDs 1 and 2, and in its active form to PDZD 7 of DGrip. This interaction appears tissue specific in nature, since DGrip mutants do not display the full spectrum of defects of ed mutants (such as neurogenic phenotypes) and since there are as yet unknown members of the DGrip-Ed complex, such as that member that binds to the 'de-repressing' PDZD 3 (Swan, 2006).
Both Ed loss of function and overexpression can produce similar phenotypes in muscles, which are strongly enhanced by the absence of DGrip. Ed is described as a homophilic cell adhesion molecule, and is maternally expressed in the epidermis, over which nascent muscles 'crawl' during the muscle guidance process to reach their target apodeme. ed clones in wing discs show cell sorting behavior, causing aggregation and adhesion of only those cells expressing the same complement of cell adhesion molecules. Thus, both reduction and excess of Ed on the 'muscle side' of transient muscle-epidermal adhesions could lead to significant changes in the cell adhesion properties of the developing muscle. The experiments shown in this study for DGripΔ1-3 overexpression in muscle 5 and for VLMs in dgrip mutants imply that a tight balance of DGrip activity might particularly be needed to keep navigating muscle projections motile and to avoid their premature stabilization at ectopic epidermis contacts during the 'steering' process-ultimately instructed by Slit/Robo or other guidance systems. It is likely that Ed and DGrip form complexes enriched at muscle projection membranes to locally control adhesiveness. Ectopic adhesions among muscles cells with aberrant DGrip activity are in fact indicative of changes in muscle adhesiveness (Swan, 2006).
Natural variants of mGRIP missing PDZDs 1-3 have been localized to mammalian synapses, and it has recently been found that the type 5 metalloproteinase MT5-MMP is recruited by GRIP1/2 to growth-cone filopodia and to both mature and developing synapses, where it proteolyses N-cadherins. GRIP2 was also observed to be a member of a Δ-catenin containing complex. Drosophila Echinoid is known to regulate DE-Cadherin in homeotypic cell-cell junctions. Given these promising indications, it will prove interesting to see whether in the context Grip proteins became famous for synapse assembly -- similar molecular strategies are used by the GRIP protein as those described here in the context of muscle morphogenesis (Swan, 2006).
To examine the expression pattern of Grip, an in situ hybridization against Grip was performed on Drosophila embryos. Until germ band extension, no Grip expression was detected. From stage 10 onward, staining in the posterior half of the segments is observed, indicating expression in developing somatic muscles. In fact, in stage 16 embryos, a strong Grip mRNA expression is observed specifically within muscles, whereas at this stage neither epidermis nor central nervous system seems to express Grip mRNA (Swan, 2004).
Grip participates in the reception of an attractive signal that emanates from the epidermal attachment sites to direct the motility of developing muscles. To correlate the embryonic expression with the mutant phenotype, embryos were stained for Grip protein expression by using anti-Grip antibody. In immunostainings of both Gripex36/Y and Gripex122/Y embryos, no signal was observed, proving the specificity of this Grip antibody in embryo stainings. Stage-16 embryos, in which muscles are fully attached, were examined. Here, Grip protein is localized to both the anterior and posterior edges of the VLMs, where the muscles are in contact with their attachment sites. Weaker expression of Grip is also detected in the contact regions of more dorsal muscles attaching to the segment border, whereas no Grip expression could be detected at the contact sites of LTMs and other directly attaching muscles. This agrees with the mRNA distribution of Grip, which also shows strong staining in the VLM region. A GFP-tagged variant of Grip expressed using 24B-gal4 rescued the Grip phenotype and is found to accumulate at muscle edges of VLMs as well. The temporal profile of Grip expression in embryonic muscles was also examined. Consistent with the distribution of the Grip transcript, Grip protein is absent from early embryos and is first detected in developing mesoderm from stage 13 on. Obviously already in early stage 14, Grip starts accumulating at both anterior and posterior muscle ends. The protein then progressively concentrates to become very sharply localized at muscle ends in stage 16. To examine Grip expression early in an identified VLM, 5053-gal4 was used to stain muscle 12 (muscle myosin is not yet expressed in these early stages). Even before proper attachment of muscle 12 at the segment border is established, staining at both the posterior and anterior end of muscle 12 is observed. As expected, Grip is sharply localized in stage 16. It should be noted that Grip staining is certainly not restricted to the labeled VLM (Swan, 2004).
Grip staining in embryonic muscle appears as discrete punctae, suggesting that the protein accumulates in distinct intracellular compartments. To learn about its subcellular distribution, Grip was expressed in COS-7 cells. Here, Grip was also found expressed in discrete punctae, which in terms of size and distribution appeared very similar to Grip punctae of embryonic muscles. Colocalization experiments using established markers for intracellular compartments showed a substantial overlap with markers labeling the endocytic compartment. In contrast, no overlap with markers of endoplasmic reticulum, Golgi, cell membrane, or other organelles as lysosomes or mitochondria was observed (Swan, 2004).
The presented data show that Grip is expressed in those muscles, which are affected by the absence of the gene product. The site of Grip localization is in agreement with the argument that the protein participates in the process of muscle guidance, possibly executing its function in an endosomal compartment. Consistent with Grip having a transient function needed for embryonic muscle patterning, Grip expression at muscle ends vanishes in postembryonic development (Swan, 2004).
The Grip locus maps to position 5C10 on chromosome X. To generate mutations, use was made of the P-element insertion P(GT1)BG01736 located 2 kb downstream of the Grip stop codon. Upon remobilization of the P-element, the small deletion Gripex36 was recovered, in which the whole transcription unit but no other annotated gene is deleted. Alternatively, P(KG)02862, which is inserted just upstream of the Grip transcription start, was used to recover Gripex122, in which the first exon, including the predicted start codon of the Grip locus, is deleted. Individuals of the genotype Gripex36/Y, Gripex122/Y, and Gripex36/Gripex122 are semilethal. Precise excisions of the parental P-lines instead were fully viable and did not present any of the phenotypes observed in Gripex36 and Gripex122. Embryos hemizygous for both Gripex36 and Gripex122 were negative for Grip mRNA in the in situ hybridization. To examine protein expression in Grip mutant embryos, a polyclonal antibody against PDZ domains 6 and 7 of the protein was affinity-purified. The Grip encoding cDNAs predict a protein of 112 kD. Consistently, Western blot analysis of wild-type Drosophila embryo extracts (stage 10-17), probed with antibody, detected a single band of ~110 to 120 kD apparent size, that comigrated with recombinant Grip expressed in insect cells. In contrast, embryo extracts derived from a Gripex122 homozygous strain were negative for Grip protein on Western blot. It is therefore concluded that both Gripex36 and Gripex122 represent protein null alleles of the Grip locus. Consistently, both alleles resulted in identical phenotypes. Moreover, phenotypes were identical in Gripex122/Y irrespective of whether animals were obtained from Gripex122 homozygous or heterozygous mothers. Examining this together with the in situ results, it is therefore concluded that no maternal activity of Grip is present and that both alleles thus establish true Grip null situations in embryos (Swan, 2004).
To examine the embryonic development of mesoderm in the absence of Grip activity, the somatic muscle pattern of mutant embryos was visualized by myosin stainings. Control embryos showed the typical pattern of somatic muscles. However, an abnormal patterning of the VLMs was easily detected in embryos of the genotype Gripex36/Y, Gripex122/Y, and Gripex36/Gripex122. The defective VLMs of the mutants appeared rounded instead of stretched between the attachment sites at the segment borders. Mesodermal expression of Grip using the 24B-gal4 or twist-gal4 driver rescued the VLM patterning defect of Gripex36/Y to the wild-type muscle pattern. The epidermal gal4 driver lines, engrailed-gal4 and stripe-gal4, with the latter specifically expressing in tendon cells, were tested. Driving Grip expression with these lines did not rescue muscle defects of Gripex36/Y. These results indicate that Grip is required in the developing muscle but not in the epidermis for proper muscle guidance (Swan, 2004).
Muscle patterning defects in the absence of Grip were further characterized at the cellular level by using confocal microscopy. In Grip mutants, the VLMs differed markedly from the elongated cylindrical appearance of wild-type VLMs. The mutant VLMs appeared atypically compact and rounded. Such strongly affected VLMs position themselves randomly, more at either the anterior or posterior segment end. In weaker cases, while still attached to both segment borders, the mutant VLMs appeared irregularly shaped and did not align in register at the segment borders, a defect not observed for wild-type VLMs. Greater that 95% of all VLMs 6/7 were affected in Grip mutant embryos, with 40% of the cells of this VLM type showing a full 'rounding up' of the muscle cells. VLMs 12/13 were affected to 80% with ~10% fully rounded up. Defects within other muscles apart from the VLMs were less obvious in Grip mutants. With a frequency of ~5%, another type of indirectly attaching muscle, muscle 8, was strongly affected. Some defects in segment border attachment were also recognized in other muscles: muscle 4, the ventral oblique muscles 14 and 30, and the dorsal muscles 1 and 2 showed milder defects in ~10% to 20% of the cells counted. Importantly however, directly attaching muscles such as the LTMs are absolutely unaffected in Grip mutants. In summary, it is concluded that Grip represents an essential component needed to establish the correct patterning within VLMs and to a lesser extent in other indirectly associating muscles during Drosophila embryogenesis (Swan, 2004).
Defective muscle adhesion, such as that found after interfering with integrin function, often results in muscle detachment. This effect is caused in response to contractile force in the affected muscle, a condition usually fatal in late embryogenesis. However, Gripex36/Y, Gripex122/Y, and Gripex36/Gripex122 individuals develop into larvae, which maintain the defective VLM pattern observed in embryos. Within these animals, the embryonically affected muscles have obviously grown and elongated throughout larval development. No sign of muscle degeneration is recognizable. Affected VLMs had produced ectopic intrasegmental attachment sites instead of the normal intersegmental attachments in wild type. Microscopic inspection of Grip mutant larvae shows that these attachments form at the inner layer of muscles and not to the epidermis, showing that in the absence of Grip function, muscle-muscle junctions are formed. Such muscle-muscle junctions, instead of normal tripartite muscle-muscle-epidermis junctions, have also been reported for other mutants such as kakapo. Both in embryos and larvae, even the most strongly affected Grip mutant VLMs form multiple extensions, implying that Grip-deficient muscles still seek attachments (Swan, 2004).
That Grip-deficient larvae are capable of sustained locomotion and show the absence of detached muscles strongly suggests that muscle attachments are functional in this mutant. Based on phalloidin stainings and electron microscopy, the defective VLMs had normal organization of the contractile apparatus as well. Moreover, escaping Gripex36/Y, Gripex122/Y, or Gripex36/Gripex122 adults showed a shrunken abdomen and defective head posture, phenotypes likely due to adult muscle patterning defects. However, no other defects -- in particular, no signs of a general impairment of cell adhesion -- were observed in these animals (Swan, 2004).
Drosophila muscles are highly differentiated concerning their attachment site and shape. Fate changes among VLM founder cells could therefore be responsible for the observed muscle phenotype. To examine this possibility, the VLM pattern was monitored in Grip mutant embryos by staining with the VLM-specific differentiation marker Vestigial. Vestigial was expressed even in the most strongly affected muscles of Gripex36/Y, indicating that the muscles develop according to their proper fate. Furthermore, use was made of 5053-gal4, which specifically drives expression in VLM 12 from stage 12 on. Expression of ß-Galactosidase (ß-Gal) driven by 5053-gal4 develops normally in Grip mutant embryos. Collectively, these data show that VLMs still develop proper fate in the absence of Grip activity. In Grip mutants, muscle 12 is frequently attached to ectopic positions, as shown by ßPS-integrin staining, a marker of muscle attachment sites. Thus, VLMs lacking Grip activity are able to attach to ectopic intrasegmental attachment sites by contacting other muscles (Swan, 2004).
To evaluate myoblast fusion, the number of nuclei was determined in Grip mutant muscle 12 of stage-17 embryos after Hoechst staining. The number of nuclei was only slightly lower in Gripex36/Y than in wild type (8.7 ± 1.7 versus 10.0 ± 2.0, respectively; P < 0.08). Moreover, because correct muscle attachment is observed in the absence of myoblast fusion, a defect in myoblast fusion can be excluded as the primary cause of the muscle defects observed (Swan, 2004).
Finally, possible differentiation defects were also examined within apodemes. In mutants for the transcription factor stripe, such defects provoke defective attachment of somatic muscles somewhat similar to those observed in Grip mutants. However, neither Grip mRNA nor protein was detectable in the epidermis throughout the period of attachment formation. Given that Grip is specifically expressed within the affected muscles and that the defect can be rescued by purely mesodermal expression, a direct role of Grip in apodeme differentiation appears very unlikely. To positively exclude a role of epidermal cells with respect to the observed phenotype, staining was carried out for the apodeme differentiation markers Delilah and Stripe in Grip mutant embryos of stage 17. Both the number and position of apodemes are unchanged in Grip mutants compared with wild type. Consistent with the formation of pure muscle-muscle junctions by displaced VLMs, no sign of additional apodemes attached to VLMs was observed in Grip mutants (Swan, 2004).
In summary, these results establish that muscle cell differentiation, myoblast fusion, and epidermal attachment sites are not affected by the lack of Grip activity, leaving the option that the mutant muscles are misguided and fail to properly reach their target sites (Swan, 2004).
To explore the possibility that Grip may function in the guidance of developing VLMs toward their individual epidermal attachment sites, the development of individual VLMs was followed in Grip mutants. For this task VLM 12 was labeled by expressing UAS-lacZ with the aid of the 5053-gal4 driver (Swan, 2004).
The developing muscle 12 precursor was first observed at stage 12 in both control and Grip mutant embryos. Throughout further development, muscles then expand by the integration of naïve myoblasts. In control embryos of late stage 13, the growing muscle is still found in the posterior portion of the hemisegments, extending a single cellular extension invariably toward anterior. Already at stage 13, VLMs are apparently defective in the absence of Grip. Specifically in Grip mutants, cellular extensions were observed that were pointing in 'wrong directions'. Moreover, muscle 12 precursor cells in Grip mutant embryos often appeared bipolar, forming extensions in both anterior and posterior direction. In wild type, extensions extend further in stage 14 until they finally contact the epidermal attachment sites at the anterior border of the segment at late stage 14/early stage 15. In Grip mutants of this stage, cell extensions often appear collapsed, or if developed, they miss their proper target sites at the segment borders. This observation is consistent with the fact that muscles finally are often unable to form proper contact with their normal attachment sites. These results thus explain the appearance of the misattached rounded muscles that are observed in the Grip mutants from stage 16 onward. As mentioned before, in Grip mutant embryos the patterning of VLM 12 is somewhat less severely affected than that of VLMs 6/7. There was no muscle 6/7-specific driver available to explore the possibility of very likely even more penetrant guidance defects of this muscle pair in the Grip mutant. It is concluded that the cellular behavior of Grip mutant muscles becomes aberrant significantly before the developmental time point at which segment border attachment normally is established. In Grip mutants, directionality of the cellular extensions normally mediating guidance seems essentially randomized. This is concluded, since cells extending in anterior or posterior direction as well as 'bipolar' cells are observed in similar quantities. Consistently, Grip mutant VLMs are found attached to either the anterior or posterior segment border only. VLMs in Grip mutants often stretch over segment borders, indicating that the muscles have missed their attachment sites at the segment borders. These results are consistent with the interpretation that prior to any attachment, during stage 13, Grip mutant muscles fail to respond to attractive cues by directing the outgrowth of cellular extensions. These results thus suggest a direct role of Grip to mediate the response to an essential attractive signal within the developing muscles. This attractive signal seems to emanate from the segment border in order to direct and/or stabilize cellular extensions of developing muscles (Swan, 2004).
This study shows that Grip is essential to mediate a motility response in the VLMs toward the anterior segment border. This is based on the finding that in the absence of the Grip, the cellular extensions of developing VLMs no longer form properly. In contrast, the motility of LTMs is undisturbed in the absence of Grip, consistent with the observation that these muscles do not seem to normally express Grip. It was asked whether an ectopic activity of Grip would influence muscle motility. To achieve Grip expression in all muscle cells, the gene was expressed in response to either 24B-gal4 or twist-gal4, both driving expression in all myogenic cells of Drosophila embryos, together with two copies of UAS-Grip. Grip overexpression in muscles was confirmed by immunofluorescence stainings using the anti-Grip antibody. In embryos overexpressing the Grip gene by using either 24B-gal4 or twist-gal4 , muscle morphology was only slightly affected in VLMs and other indirectly attaching muscles. Similarly, direct overexpression of Grip in VLM 12 using 5053-gal4 was without phenotypic consequence. In contrast, LTMs were very sensitive with respect to Grip overexpression. Such LTMs adopted an irregular morphology, bent to attach at the segment border), or produced thin cellular extensions, which connected them to the segment borders. In controls, such cellular extensions formed by LTMs or bending of whole LTMs toward segment borders were never observed; neither was formation of ectopic cellular extensions from directly attaching muscles overexpressing Grip. Thus, ectopic expression of Grip can efficiently provoke LTMs to attach to other muscles at the segment border. Muscles that display both direct and indirect attachment modes, such as the ventral oblique and acute muscles, display misrouted processes at the directly attaching end of the muscle, whereas the indirectly attaching ends of these muscles are essentially unaffected as for VLMs. Conversely, in the Grip mutant the indirectly attaching ends of these muscles is affected. No sign of ectopic tendon cell differentiation was observed in embryos overexpressing Grip in developing muscles, indicating that misattached muscles were misguided toward preexisting segment border tendon cells (Swan, 2004).
It is concluded that Grip is a key player in organizing different patterns of muscle attachment between different groups of muscles. The factor is both necessary and sufficient to promote the formation of muscle cell extensions, which has been implicated in sensing and reacting toward a guidance signal expressed at the segment border (Swan, 2004).
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date revised: 20 January 2007
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