org Interactive Fly, Drosophila Laminin A: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References
Gene name - Laminin A

Synonyms -

Cytological map position - 65A10-11

Function - Extracellular matrix, Axon guidance

Developmental family - gut endoderm, muscle, eye, ocelli, wing, heart, CNS

, basement membrane
Symbol - LanA

FlyBase ID:FBgn0002526

Genetic map position - 3-[21]

Classification - Laminin-A

Cellular location - Secreted



NCBI links: Precomputed BLAST | Entrez Gene
Recent literature
Diaz de la Loza, M. C., Diaz-Torres, A., Zurita, F., Rosales-Nieves, A. E., Moeendarbary, E., Franze, K., Martin-Bermudo, M. D. and Gonzalez-Reyes, A. (2017). Laminin levels regulate tissue migration and anterior-posterior polarity during egg morphogenesis in Drosophila. Cell Rep 20(1): 211-223. PubMed ID: 28683315
Summary:
Basement membranes (BMs) are specialized extracellular matrices required for tissue organization and organ formation. The role of laminin and its integrin receptor were studied in the regulation of tissue migration during Drosophila oogenesis. Egg production in Drosophila involves the collective migration of follicle cells (FCs) over the BM to shape the mature egg. Laminin content in the BM was shown to increase with time, whereas integrin amounts in FCs do not vary significantly. Manipulation of integrin and laminin levels reveals that a dynamic balance of integrin-laminin amounts determines the onset and speed of FC migration. Thus, the interplay of ligand-receptor levels regulates tissue migration in vivo. Laminin depletion also affects the ultrastructure and biophysical properties of the BM and results in anterior-posterior misorientation of developing follicles. Laminin emerges as a key player in the regulation of collective cell migration, tissue stiffness, and the organization of anterior-posterior polarity in Drosophila.
Sessions, A. O., Kaushik, G., Parker, S., Raedschelders, K., Bodmer, R., Van Eyk, J. E. and Engler, A. J. (2017). Extracellular matrix downregulation in the Drosophila heart preserves contractile function and improves lifespan. Matrix Biol 62: 15-27. PubMed ID: 27793636
Summary:
Aging is associated with extensive remodeling of the heart, including basement membrane (BM) components that surround cardiomyocytes. Remodeling is thought to impair cardiac mechanotransduction, but the contribution of specific BM components to age-related lateral communication between cardiomyocytes is unclear. Using a genetically tractable, rapidly aging model with sufficient cardiac genetic homology and morphology, e.g. Drosophila melanogaster, this study observed differential regulation of BM collagens between laboratory strains, correlating with changes in muscle physiology leading to cardiac dysfunction. Therefore, attempts were made to understand the extent to which BM proteins modulate contractile function during aging. Cardiac-restricted knockdown of ECM genes Pericardin, Laminin A, and Viking in Drosophila prevented age-associated heart tube restriction and increased contractility, even under viscous load. Most notably, reduction of Laminin A expression correlated with an overall preservation of contractile velocity with age and extension of organismal lifespan. Global heterozygous knockdown confirmed these data, which provides new evidence of a direct link between BM homeostasis, contractility, and maintenance of lifespan.
Sanchez-Sanchez, B. J., Urbano, J. M., Comber, K., Dragu, A., Wood, W., Stramer, B. and Martin-Bermudo, M. D. (2017). Drosophila embryonic hemocytes produce laminins to strengthen migratory response. Cell Rep 21(6): 1461-1470. PubMed ID: 29117553
Summary:
The most prominent developmental function attributed to the extracellular matrix (ECM) is cell migration. While cells in culture can produce ECM to migrate, the role of ECM in regulating developmental cell migration is classically viewed as an exogenous matrix presented to the moving cells. In contrast to this view, this study shows that Drosophila embryonic hemocytes deposit their own laminins in streak-like structures to migrate efficiently throughout the embryo. With the help of transplantation experiments, live microscopy, and image quantification, it was demonstrated that autocrine-produced laminin regulates hemocyte migration by controlling lamellipodia dynamics, stability, and persistence. Proper laminin deposition is regulated by the RabGTPase Rab8, which is highly expressed and required in hemocytes for lamellipodia dynamics and migration. These results thus support a model in which, during embryogenesis, the Rab8-regulated autocrine deposition of laminin reinforces directional and effective migration by stabilizing cellular protrusions and strengthening otherwise transient adhesion states.
BIOLOGICAL OVERVIEW

Laminin is an enormous complex extracellular, composed of three different huge polypeptide chains. The longest is Laminin A, made up of 3712 amino acids. In Drosophila laminin is involved in heart, gut, muscle, wing, and leg morphogenesis. Laminins are known to interact with a variety of proteins including integrins (See Myospheroid) and lectins. In tissue culture, Laminin is both a promoter and substratum for neurite outgrowth. In the fly, only one form of laminin has been characterized, but many proteins show homology to various domains of the laminin polypeptide. For example, Netrins, involved in axon guidance in many species, show extensive homology to common domains in each of the Laminin polypeptides. In vertebrates laminin is not a single molecular complex, but a family of at least seven different complexes using alternative subunits. In the mouse, mutation in one laminin subunit results in a form of muscular dystrophy, while mutation in another leads to defects at the neuromuscular junction (García-Alonso, 1996 and references).

Below, the involvement of laminin in the pathfinding process of ocellar axons will be described, following a short explanation of the ocellar axon pathfinding process.

Adult flys have three simple eyes (ocelli) located near the midline on the dorsal surface of the head. Left and right ocelli are derived from the left and right eye-antennal imaginal discs, while the median ocellus derives equally from both discs once they fuse together after puparium formation. In the adult, the ocellar photoreceptors (about 80 per ocellus) have short axons that synapse on the dendrites of ocellar giant interneurons (about 4 per ocellus) in the neuropil of the ocellar ganglion that lies just below the ocelli. The axons of the ocellar giant interneurons project to the brain from within the ocellar nerve. In the adult this nerve contains about 12 giant interneuron axons. The ocellar nerve, projecting from the brain to the ocelli of the adult, is in reality a follower and not a pioneer. Instead, the ocellar nerve is pioneered by about 200 axons from a transient population of ocellar pioneers that appear around the time of puparium formation. The axons from four separate transient populations of about 50 ocellar pioneer neurons (one from each lateral ocelli, and one each from right and left rudiments of the median ocellus) project from the pupal ocelli to the brain. These four populations form four fascicles or axon bundles that extend towards the brain along a non-cellular substratum. At a later pupal stage, the adult ocellar photoreceptors are born and differentiate concentrically outside the cluster of pioneers. The pioneers then die. The axons of the giant ocellar interneurons extend backward from the brain to the ocelli along the pathway laid out earlier by the pioneer neurons. By the time of adult eclosion, the 200 or so ocellar pioneer axons are gone, and only the approximately 12 giant axons of the ocellar interneurons remain in the ocellar nerve (García-Alonso, 1996).

Laminin A is involved in pathfinding of the pioneer interneurons. In some LanA mutants these axons do not grow as far as in wild type, while in others, axons do not form the characteristic pair of ocellar pioneer axon bundles, but rather form multiple axons fascicles, some of which enter the brain at abnormal positions. In the more extreme case of defects, the ocellar pioneer axons do not form the normal projection that traverses the head capsule from the epidermis to the brain, but rather extend for a short distance in the epidermis, and then stall, forming large fasciculated masses, and occasional whirls of axons. These stalled axons remain attached to the head epidermis. There are also striking defects in compound eye retinal axon pathfinding in LanA mutants, but there is also an abnormal distribution of glial cells. It is therefore not possible to know whether the pathfinding defects of compound eye axons is a primary or secondary defect. ECM containing Laminin A is not required for pathfinding by neighboring mechanosensory (bristle) axons in the head or bristle axons in the wing (García-Alonso, 1996).

Extracellular matrix containing Laminin A cannot be the entire story for the guidance of ocellar axons towards the brain, since guidance in mutants is not always defective. This suggests that in the absence of Laminin A, the pioneer axons can still read directional cues pointing them towards the brain. While laminin-rich extracellular matrix provides the appropriate growth promoting substratum, some other signal must provide directional cue (García-Alonso, 1996).

Activity-dependent retrograde laminin A signaling regulates synapse growth at Drosophila neuromuscular junctions

Retrograde signals induced by synaptic activities are derived from postsynaptic cells to potentiate presynaptic properties, such as cytoskeletal dynamics, gene expression, and synaptic growth. However, it is not known whether activity-dependent retrograde signals can also depotentiate synaptic properties. This study shows that laminin A (LanA) functions as a retrograde signal to suppress synapse growth at Drosophila neuromuscular junctions (NMJs). The presynaptic integrin pathway consists of the integrin subunit βν and focal adhesion kinase 56 (Fak56), both of which are required to suppress crawling activity-dependent NMJ growth. LanA protein is localized in the synaptic cleft and only muscle-derived LanA is functional in modulating NMJ growth. The LanA level at NMJs is inversely correlated with NMJ size and regulated by larval crawling activity, synapse excitability, postsynaptic response, and anterograde Wnt/Wingless signaling, all of which modulate NMJ growth through LanA and βν. These data indicate that synaptic activities down-regulate levels of the retrograde signal LanA to promote NMJ growth (Tsai, 2012).

This study proposes a plasticity mechanism by which the synapse growth (or size) can be modulated by larva crawling and synaptic activities. These activities modulate LanA-integrin signaling that functions to constrain NMJ growth. This trans-synaptic signaling functions in a retrograde manner, which requires postsynaptic muscle-derived LanA and presynaptic integrin. The model suggests various activities modulate NMJ growth by regulating the LanA level and integrin signaling (Tsai, 2012).

Regulation of LanA levels at NMJs is the major mechanism underlying this synaptic structural plasticity. The LanA levels at NMJs are tightly coupled to several synaptic activities that are involved in synaptic structural plasticity at NMJs. Wg signaling in both pre- and postsynaptic compartments are shown to modify synaptic structure at Drosophila NMJs. The channel mutations para and eag Sh alter both synaptic potential and NMJ size. Finally, manipulation of postsynaptic responses by altering the GluRIIA and GluRIIB compositions also fine tunes synapse size and pFAK levels. Activities that promote NMJ growth also down-regulated LanA levels at NMJs. In contrast, NMJ growth suppression was accompanied with LanA accumulation, establishing an inverse correlation between the LanA level and the NMJ size. Importantly, manipulation of the gene dosage of LanA (or βν) could override these synaptic activities in NMJ growth regulation. This study also showed that LanA down-regulation at NMJs preceded synaptic structural remodeling induced by larval crawling, further supporting that LanA is a major mediator of these activities to modulate NMJ growth (Tsai, 2012).

Integrin signaling activities play important roles in synapse development and plasticity. In mammalian central synapses, various integrin subunits are important to transmit postsynaptic signaling in various plasticity models may function redundantly with βν to mediate integrin signaling. This study indicates a distinct presynaptic integrin pathway that is likely composed of βν and αPS3 (encoded by Volado), as suggested by their strong genetic interaction in NMJ growth. In response to postsynapse-secreted LanA signals, activation of the presynaptic integrin is transmitted through Fak56 activation. Interestingly, the signaling activity is rather local, limited by the range of LanA distribution, and shown by muscle 6-specific rescue, although this does not exclude the involvement of signaling to the nuclei of motor neurons. The presynaptic integrin/Fak56 signaling is in turn mediated by two downstream signaling activities. The activation of NF1/cAMP signaling, which suppressed NMJ overgrowth induced by crawling activity or βν mutation. The integrin/Fak56 pathway also suppresses Ras/MAPK signaling Tsai, 2008), as shown by diphospho-ERK (dpERK) accumulation and Fas2 reduction at NMJs in high crawling condition. These pathways have been shown to regulate cell adhesion and cytoskeletal organization, leading to the stabilization of synapses. The activity-dependent depletion of the LanA laminins in the synaptic cleft would allow the remodeling of synapses and further growth of NMJs (Tsai, 2012).

The activity-dependent structural plasticity is specific to the presynaptic integrin pathway. hiw mutants that show large NMJ size still retained the structural plasticity and constant pFAK levels at NMJs. Interestingly, LanA levels were increased in hiw mutants, in contrast to other NMJ overgrown mutants. Two nonmutually exclusive mechanisms can regulate activity-dependent LanA expressions at NMJs. First, within hours of activity induction, the LanA levels can be regulated at NMJs by putative ECM regulators such as matrix metalloproteinases. Second, transcription regulation of LanA can provide long-term changes of LanA levels at NMJs. Activity-triggered presynaptic Wg secretion promotes Wg receptor DFz2 activation on both post- and presynaptic compartments. The LanA level is regulated by the anterograde Wg signaling that is transduced through nuclear entry of the DFz2 intracellular domain and its transcription activity. However, LanA is unlikely to mediate all aspects of Wg signaling activity as overexpression of LanA in postsynapses suppressed ghost bouton formation, a hallmark in disrupting Wg signaling. Postsynaptic BMP/Gbb functions as a retrograde signal to activate presynaptic BMP type II receptor Wit in response to synaptic activity. With the lack of genetic interaction between BMP/Gbb and integrin signaling components, and constant levels of phosphorylated Mothers against dpp (pMad) in different crawling activities, it is proposed that both BMP/Gbb and LanA pathways can function in parallel by retrograde mechanisms to regulate NMJ growth (Tsai, 2012).

A moving source of matrix components is essential for de novo basement membrane formation

The basement membrane (BM) is a thin layer of extracellular matrix (ECM) beneath nearly all epithelial cell types that is critical for cellular and tissue function. It is composed of numerous components conserved among all bilaterians; however, it is unknown how all of these components are generated and subsequently constructed to form a fully mature BM in the living animal. Although BM formation is thought to simply involve a process of self-assembly, this concept suffers from a number of logistical issues when considering its construction in vivo. First, incorporation of BM components, including Col IV, Perl and LanA appears to be hierarchical, yet it is unclear whether their production during embryogenesis must also be regulated in a temporal fashion. Second, many BM proteins are produced not only by the cells residing on the BM but also by surrounding cell types, and it is unclear how large, possibly insoluble protein complexes are delivered into the matrix. This study exploited the ability to live image and genetically dissect de novo BM formation during Drosophila development. This reveals that there is a temporal hierarchy of BM protein production that is essential for proper component incorporation. Furthermore, it was shown that BM components require secretion by migrating macrophages (hemocytes) during their developmental dispersal, which is critical for embryogenesis. Indeed, hemocyte migration is essential to deliver a subset of ECM components evenly throughout the embryo. This reveals that de novo BM construction requires a combination of both production and distribution logistics allowing for the timely delivery of core components (Matsubayashi, 2017).

To analyze de novo basement membrane (BM) formation, developing Drosophila embryos were used. The developmental profile of BM components was analyzed from the Drosophila modENCODE project. This revealed that, while Laminin mRNAs are observed early in development, extracellular matrix (ECM) components associated with a mature BM, such as Collagen IV (Vkg in Drosophila) and Perlecan (Trol in Drosophila), are expressed later, suggesting that there is a temporal hierarchy of BM production during embryogenesis (Matsubayashi, 2017).

Embryonic BM protein production was examined using endogenously tagged BM fly lines. Homozygous viable GFP-protein traps were used in Collagen IV (Col IV) and Perlecan (Perl) as well as a recently generated line containing GFP-tagged Lamininα (LanA). This LanA-GFP is capable of biochemically interacting with other Laminin subunits to form a mature Laminin trimer, and it rescued LanA mutant embryos. Furthermore, when expressed in a Lamininβ (LanB1) mutant background, LanA levels were severely reduced, suggesting that subunit trimerization is indeed essential for Laminin production and secretion. Using these GFP-tagged lines, the dynamics of BM production were analyzed by quantifying GFP intensity over time during development. This revealed that expression of BM components peaked immediately prior to embryonic hatching. Furthermore, components showed precise temporal regulation with LanA expressed first, followed by Col IV, and finally Perl. A second GFP-tagged construct was examined of the sole Drosophila Lamininβ isoform (LanB1), which was previously confirmed to be fully functional, and this also revealed Laminin expression to occur prior to Col IV or Perl (Matsubayashi, 2017).

In Drosophila embryos, hemocytes are known to produce BM. However, it has been unclear what proportion of the embryonic BM is hemocyte dependent. When GFP-tagged BM proteins were expressed in a mutant background in which hemocytes failed to develop, it was revealed that BM components are differentially hemocyte dependent. This showed that 70% of Col IV and 50% of Perl are dependent on hemocytes. In contrast, hemocytes contribute only 30% of embryonic LanA, with most of the hemocyte-derived Laminin induced at later stages of development. As the mesoderm expresses LanA, it was hypothesize that its early expression is likely dependent on this tissue. For Col IV and Perl, the remaining protein was expressed in the fat body at late stages of development, which is known to be the major source of larval BM (Pastor-Pareja, 2011; Matsubayashi, 2017 and references therein).

To investigate the functional importance of the temporal hierarchy of BM component expression, embryos were generated expressing the GFP-tagged LanA, Col IV, and Perl in all possible mutant backgrounds of opposite components. This revealed that, while LanA incorporation or levels were unaffected by the absence of subsequent components, Col IV and Perl formed disorganized extracellular deposits in the absence of Laminin. It was hypothesized that these aggregates are the specific result of Col IV aggregation, as the Perl deposits were absent in a Col IV/Laminin double mutant. Finally, Perl, which is expressed last in the temporal hierarchy, required prior production of Laminin and Col IV for proper expression and incorporation into the BM, which is similar to what was previously reported (Hollfelder, 2014). These results suggest that proper de novo BM formation requires temporal regulation of component production. A similar temporal hierarchy of BM production may be critical for BM formation in other species, as disorganized ECM deposits have also been observed in laminin mutant mice (Smyth, 1999) and C. elegans (Huang, 2003; Matsubayashi, 2017).

Differences were observed in the appearance of Col IV and Laminin in the wild-type background, with Laminin showing a much more diffuse distribution. These differences were investigated by time-lapse microscopy during hemocyte migration along the ventral nerve cord (VNC), which is a known migratory route that is readily amenable to live imaging. Both LanA and LanB1 subunits were observed to form 'halos' of graded expression surrounding migrating hemocytes, with trails of Laminin forming as cells moved within the acellular fluid-filled cavity of the embryo (hemocoel). These halos of Laminin were identical to expression of secreted-GFP, suggesting that Laminin is simply filling the hemocoel. In contrast, while Col IV and Perl decorated the surface of the VNC, there was no observable fluorescence filling the hemocoel. Whether the differences in BM component localization were the result of their differing diffusive characteristics was examined by performing fluorescence recovery after photobleaching (FRAP) analysis. This showed that LanA had a significant mobile fraction unlike Col IV, which failed to show any recovery. To understand why Laminin formed halos surrounding hemocytes along the VNC, the ventral hemocoel was examined by transmission electron microscopy (TEM). This revealed that the ventral hemocoel is highly confined, with the VNC in physical contact with the overlying epithelium. Therefore the halos of Laminin and its trails following hemocyte movement represent hemocytes separating the VNC from the overlying epithelium, allowing Laminin diffusion. These data highlight that different BM components have distinct diffusive properties within the developing embryo (Matsubayashi, 2017).

The apparent absence of soluble Col IV in the hemocoel suggested that Col IV might require a local mechanism of deposition by hemocytes. However, while it was possible to observe some BM material deposited beneath migrating hemocytes by TEM, it was difficult to examine the dynamics of Col IV deposition beneath hemocytes by standard confocal microscopy due to the low level of fluorescence and small size of the deposits. Therefore, lattice light-sheet microscopy, which allows for enhanced spatiotemporal resolution with reduced phototoxicity, was used. Indeed, hemocyte motility within the ventral hemocoel was highly amenable to lattice light-sheet imaging at early stages of hemocyte dispersal with minimal photobleaching (Matsubayashi, 2017).

Imaging by lattice light-sheet microscopy revealed that, at the stage when hemocytes are aligned on the ventral midline, Col IV is primarily localized beneath hemocytes on the surface of the nerve cord and in the segmentally spaced dorsoventral channels of the VNC. Subsequently, when hemocytes left the midline and migrated laterally, they appeared to deposit Col IV in a local fashion leaving puncta of matrix that eventually developed into longer fibrils. Additionally, simultaneous imaging of Col IV and the hemocyte actin cytoskeleton showed that Col IV colocalized with actin fibers within lamellae, suggesting hemocyte secretion of Col IV may require release along actin fibers or that recently released Col IV is rapidly remodelled by hemocytes using their actin network. Indeed, tracking movements in the Col IV matrix at high temporal resolution by particle image velocimetry revealed strong regions of ECM deformation beneath hemocyte lamellae, suggesting hemocyte traction forces are being exerted on the developing BM (Matsubayashi, 2017).

As time-lapse imaging suggested that hemocytes are 'plastering' embryonic surfaces with Col IV, it was hypothesized that hemocyte developmental dispersal may be a critical part of the BM deposition process. Hemocytes develop in the anterior of the embryo, and after stage 10 of embryogenesis they disperse within the hemocoel using a combination of external guidance cues and contact inhibition of locomotion, resulting in an evenly tiled cellular distribution. Therefore, how the timing of BM component production correlated with the dispersal of hemocytes was examined. While LanA was expressed during initial stages as hemocytes migrated from their source in the head of the embryo, Col IV production lagged behind by approximately 5 hr. As the induction of Col IV expression occurred largely after hemocyte dispersal, this suggested that hemocyte spreading within the embryo might be a prerequisite for Col IV delivery (Matsubayashi, 2017).

It was previously proposed that hemocytes were required for BM deposition specifically around the renal tubules during embryogenesis (Bunt, 2010); however, this was only interrogated in mutant embryos that were defective in both hemocyte migration and their survival. To directly examine the role of hemocyte migration in BM component deposition, aberrant hemocyte dispersal was caused by misexpression of Pvf2, a platelet-derived growth factor (PDGF)-like chemotactic cue for hemocytes. Overexpressing Pvf2 during hemocyte dispersal caused hemocytes to aggregate in the embryonic head, which was likely due to a distraction of hemocytes from their normal Pvf source. LanA in wild-type embryos initially spread down the midline of the VNC, and this was unaffected by the inhibition of hemocyte migration. Subsequently, in control embryos, a sheet-like structure containing Laminin extended from the middle of the VNC to lateral positions. These nascent Laminin sheets were stable in time compared to the halos/trails of Laminin following migrating hemocytes, which fluctuated on the order of seconds. Therefore, it was hypothesize that the extension of the Laminin sheets reflects the incorporation and growth of the polymerized matrix from a soluble source of Laminin residing predominantly on the midline. The initiation of Laminin incorporation was unaffected by Pvf2 overexpression. However, in Pvf2-expressing embryos, the Laminin sheets failed to continue extending, leaving large gaps that increased in size by later stages of development. This apparent breakdown of the Laminin matrix was similar to embryos lacking hemocytes. Therefore, Laminin produced by hemocytes may be critical for proper Laminin incorporation or that hemocyte movement, which opens up spaces between tissues, could be aiding the growth of the Laminin matrix by enhancing its diffusion in the hemocoel. In contrast, despite an increase in Col IV upon Pvf2 overexpression, confocal microscopy and lattice light-sheet imaging revealed that there was an uneven coverage of Col IV within the embryo, with most Col IV surrounding hemocytes in the head. A similar local deposition of Col IV around hemocytes was also observed when hemocyte migration was disturbed by the expression of dominant-negative Rac (RacN17) or constitutively active Rac (RacV12). These results further suggest that Laminin deposition requires its diffusion within the embryonic hemocoel while Col IV is locally deposited by hemocytes (Matsubayashi, 2017).

While these data suggested a highly local mechanism of Col IV delivery by migrating hemocytes, a more complex picture emerged over longer time periods of imaging. At later stages of development, Col IV appeared to spread at a distance from hemocytes and fill the hemocoel. Therefore Col IV was imaged within embryos over a longer period of approximately 12 hr, which represents the time frame just prior to embryonic hatching. Inducing hemocyte aggregation in the anterior of the embryo through overexpression of Pvf2 or RacN17/RacV12 revealed an accumulation of Col IV around hemocytes approximately 6 hr after Col IV induction. However, by 12 hr the fluorescence of Col IV was distributed throughout the embryo despite a continued aggregation of hemocytes. These data suggest that Col IV is eventually capable of spreading within the hemocoel but suffers from very slow effective diffusion (Matsubayashi, 2017).

Whether hemocyte migration and even BM deposition are functionally important for embryogenesis was subsequently tested. Therefore VNC condensation, a known morphogenetic event that requires hemocytes and BM, was examined. As the BM surrounds the outer surface of the VNC, it is readily accessible to ultrastructural analysis. Fillet preparations of the embryonic VNC were generated, and the developing BM was examined by scanning electron microscopy (SEM). At stage 14 of development, the matrix surrounding the VNC was surprisingly fibrillar in appearance. However, by stage 15 these matrix fibrils were rapidly remodelled into a contiguous sheet containing holes that progressively closed during VNC condensation. Next the distribution of the BM surrounding the VNC was examined after inhibition of hemocyte migration, which severely affected the condensation process and led to a reduced embryonic viability. This revealed that, while the wild-type VNC showed a relatively even distribution of BM, Pvf2 overexpression led to a dense matrix in the head region with a sparse matrix surrounding the VNC in the tail. This highlights that uniform hemocyte dispersal is indeed essential for even incorporation of BM and that the catching up in fluorescence levels upon the inhibition of hemocyte migration is likely the result of diffusing Col IV within the hemocoel rather than proper incorporation (Matsubayashi, 2017).

Whether the severity of hemocyte migration defects correlated with embryonic lethality was examined. Hemocytes are completely essential for embryogenesis, as killing off hemocytes led to 100% lethality as measured by the frequency of embryonic hatching. Varying degrees of hemocyte migration defects were examined. Expression of a dominant-negative Myosin II specifically in hemocytes led to minor clumping defects but no obvious effects on embryonic lethality. In contrast, Pvf2 overexpression or hemocyte-specific expression of RacN17 led to intermediate migration defects and resulted in approximately 50% embryonic lethality. Finally, hemocyte-specific expression of RacV12, which induced severe migration defects with hemocytes failing to disperse from their origin in the head, led to the most severe embryonic phenotype with 96% lethality. Importantly, these differences in lethality were not correlated with levels of Col IV expression, indicating that the lethality was not related to a change in Col IV levels. These data show that hemocyte migration is indeed essential for embryonic viability (Matsubayashi, 2017).

Finally, whether a genetic interaction could be observed between hemocyte migration defects and BM mutant alleles was examined. Causing aberrant hemocyte migration in the presence of a heterozygous colIV mutant allele, which led to a 50% reduction in Col IV expression, abolished VNC condensation and induced a synergistic effect on embryonic lethality with 100% of embryos failing to hatch. This lethality was higher than homozygous colIV mutants, showing that the synergy between hemocyte migration and Col IV reduction is not simply the result of a loss of Col IV expression; it also suggests that uneven Col IV deposition may be worse for the embryo than a complete loss of Col IV. In contrast, combining hemocyte migration defects with heterozygous laminin mutants led to a slight increase in lethality, which was similar to homozygous laminin mutant embryos. These data further show that Col IV deposition is more dependent on hemocyte migration than other BM components, such as Laminin (Matsubayashi, 2017).

This study has shown that during Drosophila embryogenesis, a subset of BM components requires local deposition by migrating hemocytes. This highlights that the ability of hemocytes to evenly spread throughout the embryo, part of a wider mechanism to uniformly deliver ECM. Therefore, as is increasingly realized for vertebrate macrophages, which are also involved in morphogenetic processes that involve matrix remodelling, hemocytes have important non-immune roles critical for development. Interestingly, mammalian macrophages have recently been revealed to produce various ECM components; along with the current data, this suggests that a critical role for macrophage-derived ECM may be more ubiquitous than previously recognized (Matsubayashi, 2017).

It is unclear why embryonic BM components like Col IV require local delivery by hemocytes, while in larvae they are thought to diffuse from the fat body. This may be related to physiological differences between embryo and larva. In larvae, the heart pumps hemolymph around the animal, which may aid in the spreading of BM proteins. In contrast, the embryonic heart does not begin beating until stage 17, which is after the start of Col IV deposition; in lieu of flowing hemolymph, BM factors with low effective diffusion may therefore require a moving source. Interestingly, recent work has revealed that at least one larval tissue, the developing ovary, requires hemocyte-specific production of Col IV, and it is possible that tissues not in direct contact with hemolymph require other mechanisms of BM deposition. However, it is unclear whether hemocytes associated with the ovary plaster Col IV in a manner similar to embryonic hemocytes or shed soluble Col IV similarly to the larval fat body (Matsubayashi, 2017).

It is also likely that there are differences between the mechanisms of de novo BM formation in the embryo versus homeostatic mechanisms involved in BM growth in the larva; when Col IV is first deposited in the embryo, its binding sites in the nascent Laminin matrix will be completely unsaturated leading to its rapid capture, thus preventing it from spreading far from its source. As Col IV saturates the BM at later stages of development, this would allow for its subsequent long-distance diffusion in older embryos and larvae. The larva may also have specific mechanisms that aid in Col IV solubility. Indeed, Sparc mutant larvae have abnormal extracellular BM deposits, and recent data from both Drosophila and C. elegans suggest that Sparc is a carrier for components like Col IV. It is interesting to note that there is no embryonic phenotype in Drosophila in the absence of Sparc, suggesting that embryonic Col IV does not need to be solubilized, which is hypothesized to be due to its specific hemocyte-dependent mechanism of delivery during de novo BM formation (Matsubayashi, 2017).


GENE STRUCTURE

cDNA clone length - 14155 bases

Exons - 15


PROTEIN STRUCTURE

Amino Acids - 3712

Structural Domains

Laminin is a heterotrimer consisting of three chains, A, B1 and B2. B1 and B2 subunits show similarity to their vertebrate homologs in both the arrangement and sequence of their multidomain structures. Laminin forms a cruciform structure in which the N-terminal end region (domain I/II) of each of the three chains forms one of the short arms. The C-terminal ends are joined together in the long arm of the cross as coiled-coil amphipathic alpha helices linked by interchain disulfide bonds. The mature protein has 35 consensus sites for N-linked glycosylation. It lacks any Arg-Gly-Asp (RGD) sequence which has been implicated in binding of cells to mouse laminin. The overall level of amino acid similarity is 29% between fly and mouse, compared with 78% between mouse and human. The short arm of the Drosophila A chain is made up of the N-terminal signal sequence followed by the VI globular domain and two other globular domains (IVb and IVa) separated by cysteine-rich, thread-like segments called laminin repeats. Domain VI is very similar to domains VI of all chareacterized laminin chains. The globular domain IVb is homologous to domain IVb of the mouse laminin A chain. It is also similar to the domains IV of laminin B2 chains and to some globular domains of Perlecan. Between the globular domains of the short arm Laminin A chain are the thread-like domains V, IIIb and IIIa, consisting of laminin repeats. These repeats are structural motifs related to EGF repeats, consisting of 50 amino acid residues, but containing eight Cys residues rather than the six residues found in EGF repeats. They occur in all laminin chains and also in Perlecan and Agrin. The number of EGF repeats differs between the fly and the mouse. The C-terminal G domain, distinguishing Laminin A chains from B chains, is made up of G-loops. These are sequence motifs found in a variety of secreted and cell surface proteins. Mouse and fly G-loops show 26% amino acid identity, whereas the G domains of mouse Laminin A and human Merosin show 41% identity, suggesting that mouse Laminin A and human Merosin may have evolved by means of gene duplication after the evolutionary split leading to chordates and arthropods (Kusche-Gullberg, 1992 and Henchcliffe, 1993).


Laminin A: Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References

date revised: 10 August 97  

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