unexplored, because the knockout mice show no major pathology in these organs. reelin's role in the growing central nervous system has been extensively characterized. it promotes the differentiation of progenitor cells into radial glia and affects the orientation of its fibers, which serve as the guides for the migrating neuroblasts. the position of reelin-secreting cell layer is important, because the fibers orient themselves in the direction of its higher concentration. for example, reelin regulates the development of layer-specific connections in hippocampus and entorhinal cortex. reelin controls the direction of radial glia growth. a fragment of an illustration from nomura t. et al., 2008. reelin-expressing cells (red) on c stimulate the growth of green glial fibers, while on b, where the red cells do not express reelin, radial glia is more disarrayed. mammalian corticogenesis is another process where reelin plays a major role. in this process the temporary layer called preplate is split into the marginal zone on the top and subplate below, and the space between them is populated by neuronal layers in the inside-out pattern. such an arrangement, where the newly created neurons pass through the settled layers and position themselves one step above, is a distinguishing feature of mammalian brain, in contrast to the evolutionary older reptile cortex, in which layers are positioned in an "outside-in" fashion. when reelin is absent, like in the mutant reeler mouse, the order of cortical layering becomes roughly inverted, with younger neurons finding themselves to be unable to pass the settled layers. subplate neurons fail to stop and invade the upper most layer, creating the so-called superplate in which they mix with cajal-retzius cells and some cells normally destined for the second layer. increased reelin expression changes the morphology of migrating neurons: unlike the round neurons with short branches (c) they assume bipolar shape (d) and attach themselves (e) to the radial glia fibers that are extending in the direction of reelin-expressing cells. nomura t. et al., 2008. there is no agreement concerning the role of reelin in the proper positioning of cortical layers. the original hypothesis, that the protein is a stop signal for the migrating cells, is supported by its ability to induce the dissociation, its role in asserting the compact granule cell layer in the hippocampus, and by the fact that migrating neuroblasts evade the reelin-rich areas. but an experiment in which murine corticogenesis went normally despite the malpositioned reelin secreting layer, and lack of evidence that reelin affects the growth cones and leading edges of neurons, caused some additional hypotheses to be proposed. according to one of them, reelin makes the cells more susceptible to some yet undescribed positional signaling cascade. reelin may also ensure correct neuronal positioning in the spinal cord: according to one study, location and level of its expression affects the movement of sympathetic preganglionic neurons. the protein is thought to act on migrating neuronal precursors and thus controls correct cell positioning in the cortex and other brain structures. the proposed role is one of a dissociation signal for neuronal groups, allowing them to separate and go from tangential chain-migration to radial individual migration. dissociation detaches migrating neurons from the glial cells that are acting as their guides, converting them into individual cells that can strike out alone to find their final position. top: representative image of somatic reelin immunoreactivities found in 12-day-in-vitro hippocampal neurons. bottom: reelin immunofluorescence (red) overlaid with map2 counterstain (green). a fragment of an illustration from campo et al., 2009. reelin takes part in the developmental change of nmda receptor configuration, increasing mobility of nr2b-containing receptors and thus decreasing the time they spend at the synapse. it has been hypothesized that this may be a part of the mechanism behind the "nr2b-nr2a switch" that is observed in the brain during its postnatal development. ongoing reelin secretion by gabaergic hippocampal neurons is necessary to keep nr2b-containing nmda receptors at a low level. in adults in the adult nervous system, reelin plays an eminent role at the two most active neurogenesis sites, the subventricular zone and the dentate gyrus. in some species, the neuroblasts from the subventricular zone migrate in chains in the rostral migratory stream (rms) to reach the olfactory bulb, where reelin dissociates them into individual cells that are able to migrate further individually. they change their mode of migration from tangential to radial, and begin using the radial glia fibers as their guides. there are studies showing that along the rms itself the two receptors, apoer2 and vldlr, and their intracellular adapter dab1 function independently of reelin, most likely by the influence of a newly proposed ligand, thrombospondin-1. in the adult dentate gyrus, reelin provides guidance cues for new neurons that are constantly arriving to the granule cell layer from subgranular zone, keeping the layer compact. reelin also plays an important role in the adult brain by modulating cortical pyramidal neuron dendritic spine expression density, the branching of dendrites, and the expression of long-term potentiation as its secretion is continued diffusely by the gabaergic cortical interneurons those origin is traced to the medial ganglionic eminence. in the adult organism the non-neural expression is much less widespread, but goes up sharply when some organs are injured. the exact function of reelin upregulation following an injury is still being researched. evolutionary significance cajal-retzius cells, as drawn by cajal in 1891. the development of a distinct layer of these reelin-secreting cells played a major role in brain evolution. neuronal development: mammals (left) and avians (right) have different patterns of reelin expression (pink). nomura t. et al., 2008. reelin-dab1 interactions could have played a key role in the structural evolution of the cortex that evolved from a single layer in the common predecessor of the amniotes into multiple-layered cortex of contemporary mammals. research shows that reelin expression goes up as the cortex becomes more complex, reaching the maximum in the human brain in which the reelin-secreting cajal-retzius cells have significantly more complex axonal arbour. reelin is present in the telencephalon of all the vertebrates studied so far, but the pattern of expression differs widely. for example, zebrafish have no cajal-retzius cells at all; instead, the protein is being secreted by other neurons. these cells do not form a dedicated layer in amphibians, and radial migration in their brains is very weak. as the cortex becomes more complex and convoluted, migration along the radial glia fibers becomes more important for the proper lamination. the emergence of a distinct reelin-secreting layer is thought to play an important role in this evolution. there are conflicting data concerning the importance of this layer, and these are explained in the literature either by the existence of an additional signaling positional mechanism that interacts with the reelin cascade, or by the assumption that mice that are used in such experiments have redundant secretion of reelin compared with more localized synthesis in the human brain. cajal-retzius cells, most of which disappear around the time of birth, coexpress reelin with the har1 gene that is thought to have undergone the most significant evolutionary change in humans compared with chimpanzee, being the most "evolutionary accelerated" of the genes from the human accelerated regions. there is also evidence of that variants in the dab1 gene have been included in a recent selective sweep in chinese populations. mechanism of action the main reelin signaling cascade (apoer2 and vldlr) and its interaction with lis1. zhang et al., 2008 sfk: src family kinases.jip: jnk-interacting protein 1 receptors reelin's control of cell-cell interactions is thought to be mediated by binding of reelin to the two members of low density lipoprotein receptor gene family: vldlr and the apoer2. the two main reelin receptors seem to have slightly different roles: vldlr conducts the stop signal, while apoer2 is essential for the migration of late-born neocortical neurons. it also has been shown that the n-terminal region of reelin, a site distinct from the region of reelin shown to associate with vldlr/apoer2 binds to the alpha-3-beta-1 integrin receptor. the proposal that the protocadherin cnr1 behaves as a reelin receptor has been disproven. as members of lipoprotein receptor superfamily, both vldlr and apoer2 have in their structure an internalization domain called npxy motif. after binding to the receptors reelin is internalized by endocytosis, and the n-terminal fragment of the protein is re-secreted. this fragment may serve postnatally to prevent apical dendrites of cortical layer ii/iii pyramidal neurons from overgrowth, acting via a pathway independent of canonical reelin receptors. reelin receptors are present on both neurons and glial cells. furthermore, radial glia express the same amount of apoer2 but being ten times less rich in vldlr. beta-1 integrin receptors on glial cells play more important role in neuronal layering than the same receptors on the migrating neuroblasts. reelin-dependent strengthening of long-term potentiation is caused by apoer2 interaction with nmda receptor. this interaction happens when apoer2 has a region coded by exon 19. apoer2 gene is alternatively spliced, with the exon 19-containing variant more actively produced during periods of activity. according to one study, the hippocampal reelin expression rapidly goes up when there is need to store a memory, as demethylases open up the reln gene. the activation of dendrite growth by reelin is apparently conducted through src family kinases and is dependent upon the expression of crk family proteins, consistent with the interaction of crk and crkl with tyrosine-phosphorylated dab1. moreover, a cre-loxp recombination mouse model that lacks crk and crkl in most neurons was reported to have the reeler phenotype, indicating that crk/crkl lie between dab1 and akt in the reelin signaling chain. signaling cascades reelin activates the signaling cascade of notch-1, inducing the expression of fabp7 and prompting progenitor cells to assume radial glial phenotype. in addition, corticogenesis in vivo is highly dependent upon reelin being processed by embryonic neurons, which are thought to secrete some as yet unidentified metalloproteinases that free the central signal-competent part of the protein. some other unknown proteolytic mechanisms may also play a role. it is supposed that full-sized reelin sticks to the extracellular matrix fibers on the higher levels, and the central fragments, as they are being freed up by the breaking up of reelin, are able to permeate into the lower levels. it is possible that as neuroblasts reach the higher levels they stop their migration either because of the heightened combined expression of all forms of reelin, or due to the peculiar mode of action of the full-sized reelin molecules and its homodimers. the intracellular adaptor dab1 binds to the vldlr and apoer2 through an npxy motif and is involved in transmission of reelin signals through these lipoprotein receptors. it becomes phosphorylated by src and fyn kinases and apparently stimulates the actin cytoskeleton to change its shape, affecting the proportion of integrin receptors on the cell surface, which leads to the change in adhesion. phosphorylation of dab1 leads to its ubiquitination and subsequent degradation, and this explains the heightened levels of dab1 in the absence of reelin. such negative feedback is thought to be important for proper cortical lamination. activated by two antibodies, vldlr and apoer2 cause dab1 phosphorylation but seemingly without the subsequent degradation and without rescuing the reeler phenotype, and this may indicate that a part of the signal is conducted independently of dab1. reelin stimulates the progenitor cells to differentiate into radial glia, inducing the expression of radial glial marker blbp by affecting the notch1 cascade. a fragment of an illustration from keilani et al., 2008. a protein having an important role in lissencephaly and accordingly called lis1 (pafah1b1), was shown to interact with the intracellular segment of vldlr, thus reacting to the activation of reelin pathway. complexes reelin molecules have been shown to form a large protein complex, a disulfide-linked homodimer. if the homodimer fails to form, efficient tyrosine phosphorylation of dab1 in vitro fails. moreover, the two main receptors of reelin are able to form clusters that most probably play a major role in the signaling, causing the intracellular adaptor dab1 to dimerize or oligomerize in its turn. such clustering has been shown in the study to activate the signaling chain even in the absence of reelin itself. in addition, reelin itself can cut the peptide bonds holding other proteins together, being a serine protease, and this may affect the cellular adhesion and migration processes. reelin signaling