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).

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).

date revised: 10 August 97  

Home page: The Interactive Fly © 1995, 1996 Thomas B. Brody, Ph.D.

The Interactive Fly resides on the
Society for Developmental Biology's Web server.