Cell migration in invertebrates: clues from border and distal tip cells.

Recent studies in two invertebrate systems, border cells in Drosophila melanogaster and distal tip cells in Caenorhabditis elegans, have provided important insight into the mechanisms of directed cell migration. These migrating cells are guided by extracellular signals, such as EGF, TGF-beta and netrin. In addition, metalloproteases alter the extracellular matrix of the tissue through which these cells migrate. Along the migratory path, migrating cells respond to changes in guidance signals by altering the expression of receptor signaling pathways. Finally, Dock180, CrkII and the GTPase Rac link the extracellular signals to the cellular machinery that controls cell motility.     

Control of tangential/non-radial migration of neurons in the developing cerebral cortex

Projection neurons in the developing cerebral cortex of rodents are basically born near the ventricle and migrate radially to beneath the marginal zone, whereas their cortical interneurons are generated in the ventral telencephalon and migrate tangentially to the cortex. The origins and migratory profiles of each interneuron subtype have been studied extensively in the last decade, and an enormous effort has been made to clarify the cellular and molecular mechanisms that regulate interneuron migration. More recently, the interaction between projection neurons and migrating interneurons, including how they are incorporated into their proper layers, has begun to be analyzed. In this review, I outline the most recent findings in regard to these issues and discuss the mechanisms underlying the development of cortical cytoarchitecture.     

Cellular and molecular guidance of GABAergic neuronal migration from an extracortical origin to the neocortex.

Formation of the normal mammalian cerebral cortex requires the migration of GABAergic inhibitory interneurons from an extracortical origin, the lateral ganglionic eminence (LGE). Mechanisms guiding the migratory direction of these neurons, or other neurons in the neocortex, are not well understood. We have used an explant assay to study GABAergic neuronal migration and found that the ventricular zone (VZ) of the LGE is repulsive to GABAergic neurons. Furthermore, the secreted protein Slit is a chemorepellent guiding the migratory direction of GABAergic neurons, and blockade of endogenous Slit signaling inhibits the repulsive activity in the VZ. These results have revealed a cellular source of guidance for GABAergic neurons, demonstrated a molecular cue important for cortical development, and suggested a guidance mechanism for the migration of extracortical neurons into the neocortex.     

Short- and long-range attraction of cortical GABAergic interneurons by neuregulin-1

Most cortical interneurons arise from the subcortical telencephalon, but the molecules that control their migration remain largely unidentified. Here, we show that different isoforms of Neuregulin-1 are expressed in the developing cortex and in the route that migrating interneurons follow toward the cortex, whereas a population of the migrating interneurons express ErbB4, a receptor for Neuregulin-1. The different isoforms of Neuregulin-1 act as short- and long-range attractants for migrating interneurons, and perturbing ErbB4 function in vitro decreases the number of interneurons that tangentially migrate to the cortex. In vivo, loss of Neuregulin-1/ErbB4 signaling causes an alteration in the tangential migration of cortical interneurons and a reduction in the number of GABAergic interneurons in the postnatal cortex. These observations provide evidence that Neuregulin-1 and its ErbB4 receptor directly control neuronal migration in the nervous system.     

Ephrins guide migrating cortical interneurons in the basal telencephalon

Cortical interneurons are born in the proliferative zones of the ganglionic eminences in the subpallium and migrate to the developing cortex along well-defined tangential routes. The mechanisms regulating interneuron migration are not completely understood. Here we examine the role of class-A members of the Eph/ephrin system in directing the migration of interneurons. In situ hybridizations demonstrated that ephrin-A3 is expressed in the developing striatum, an area that is strictly avoided by migrating cortical interneurons in vivo, which express the EphA4 receptor. We then examined interneuron migration in grafting experiments, where explants of the medial ganglionic eminence (MGE) from enhanced green fluorescent protein-expressing transgenic mice were homotopically grafted into host slices from wild-type littermate embryos. After blocking ephrin-A ligands, many interneurons invaded the striatal anlage. Moreover, stripe assay experiments revealed that ephrin-A3 acts as a repellent cue for neurons from the medial ganglionic eminence. Downregulation of the EphA4 receptor via siRNA transfection reduced the repulsive effect of ephrin-A3, indicating that EphA4 mediates at least in part the repulsive effect of ephrin-A3 on these cells. Together, these results suggest that ephrin-A3 acts as a repulsive cue that restricts cortical interneurons from entering inappropriate regions and thus contributes to define the migratory route of cortical interneurons.Key words: interneuron migration, cortical development, neuronal guidance cues, ephrin, Eph receptors, organotypic slice cultures     

The COUP-TF nuclear receptors regulate cell migration in the mammalian basal forebrain

Cells migrate via diverse pathways and in different modes to reach their final destinations during development. Tangential migration has been shown to contribute significantly to the generation of neuronal diversity in the mammalian telencephalon. GABAergic interneurons are the best-characterized neurons that migrate tangentially, from the ventral telencephalon, dorsally into the cortex. However, the molecular mechanisms and nature of these migratory pathways are only just beginning to be unravelled. In this study we have first identified a novel dorsal-to-ventral migratory route, in which cells migrate from the interganglionic sulcus, located in the basal telencephalon between the lateral and medial ganglionic eminences, towards the pre-optic area and anterior hypothalamus in the diencephalon. Next, with the help of transplantations and gain-of-function studies in organotypic cultures, we have shown that COUP-TFI and COUP-TFII are expressed in distinct and non-overlapping migratory routes. Ectopic expression of COUP-TFs induces an increased rate of cell migration and cell dispersal, suggesting roles in cellular adhesion and migration processes. Moreover, cells follow a distinct migratory path, dorsal versus ventral, which is dependent on the expression of COUP-TFI or COUP-TFII, suggesting an intrinsic role of COUP-TFs in guiding migrating neurons towards their target regions. Therefore, we propose that COUP-TFs are directly involved in tangential cell migration in the developing brain, through the regulation of short- and long-range guidance cues.     

Early specification of GAD67 subventricular derived olfactory interneurons

Olfactory bulb interneurons are continuously generated in the subventricular zone (SVZ) and migrate along the rostral migratory stream (RMS) into the olfactory bulb (OB) where the majority becomes local GABAergic interneurons. We previously showed that SVZ-derived progenitor cells expressed glutamic acid decarboxylase 65 kDa (GAD65) very early in the migratory pathway. However, only approximately half of OB GABAergic interneurons use GAD65, an equal number express the 67 kDa GAD enzyme. To investigate the differentiation of these GABAergic interneurons we examined their migration in a transgenic mouse expressing green fluorescent protein (GFP) under the control of the GAD67 promoter. In adult, GFP was expressed by a subpopulation of migratory cells in the SVZ and along the RMS. Using Doublecortin (DCX) as a marker of migrating neuroblasts and bromodeoxyuridine (BrdU) incorporation, we show that these GAD67-GFP neurons co-express DCX and incorporate BrdU indicating they are newly born migratory neuroblasts. This is similar to GAD65 transgene expression, and in contrast to dopaminergic interneuron transgene expression which occurs only after cells reach the olfactory bulb. Although the GAD65/67 transgenes are expressed early in migration, there is minimal protein production in the cells prior to reaching the OB. These results suggest that migrating SVZ-derived neuroblasts acquire GABAergic identity prior to reaching their final location in the olfactory bulb.     

RGMa Regulates Cortical Interneuron Migration and Differentiation

The etiology of neuropsychiatric disorders, including schizophrenia and autism, has been linked to a failure to establish the intricate neural network comprising excitatory pyramidal and inhibitory interneurons during neocortex development. A large proportion of cortical inhibitory interneurons originate in the medial ganglionic eminence (MGE) of the ventral telencephalon and then migrate through the ventral subventricular zone, across the corticostriatal junction, into the embryonic cortex. Successful navigation of newborn interneurons through the complex environment of the ventral telencephalon is governed by spatiotemporally restricted deployment of both chemorepulsive and chemoattractive guidance cues which work in concert to create a migratory corridor. Despite the expanding list of interneuron guidance cues, cues responsible for preventing interneurons from re-entering the ventricular zone of the ganglionic eminences have not been well characterized. Here we provide evidence that the chemorepulsive axon guidance cue, RGMa (Repulsive Guidance Molecule a), may fulfill this function. The ventricular zone restricted expression of RGMa in the ganglionic eminences and the presence of its receptor, Neogenin, in the ventricular zone and on newborn and maturing MGE-derived interneurons implicates RGMa-Neogenin interactions in interneuron differentiation and migration. Using an in vitro approach, we show that RGMa promotes interneuron differentiation by potentiating neurite outgrowth. In addition, using in vitro explant and migration assays, we provide evidence that RGMa is a repulsive guidance cue for newborn interneurons migrating out of the ganglionic eminence ventricular zone. Intriguingly, the alternative Neogenin ligand, Netrin-1, had no effect on migration. However, we observed complete abrogation of RGMa-induced chemorepulsion when newborn interneurons were simultaneously exposed to RGMa and Netrin-1 gradients, suggesting a novel mechanism for the tight regulation of RGMa-guided interneuron migration. We propose that during peak neurogenesis, repulsive RGMa-Neogenin interactions drive interneurons into the migratory corridor and prevent re-entry into the ventricular zone of the ganglionic eminences.     

adrift, a novel bnl-induced Drosophila gene, required for tracheal pathfinding into the CNS

Neurons and glial cells provide guidance cues for migrating neurons. We show here that migrating epithelial cells also contact specific neurons and glia during their pathfinding, and we describe the first gene required in the process. In wild-type Drosophila embryos, the ganglionic tracheal branch navigates a remarkably complex path along specific neural and glial substrata, switching substrata five times before reaching its ultimate target in the CNS. In adrift mutants, ganglionic branches migrate normally along the intersegmental nerve, but sporadically fail to switch to the segmental nerve and enter the CNS; they wind up meandering along the ventral epidermis instead. adrift encodes a novel nuclear protein with an evolutionarily conserved motif. The gene is required in the trachea and is expressed in the leading cells of migrating ganglionic branches where it is induced by the branchless FGF pathway. We propose that Adrift regulates expression of tracheal genes required for pathfinding on the segmental nerve, and FGF induction of adrift expression in migrating tracheal cells promotes the switch from the intersegmental to the segmental nerve.     

The LIM protein Ajuba influences p130Cas localization and Rac1 activity during cell migration

Cell migration requires extension of lamellipodia that are stabilized by formation of adhesive complexes at the leading edge. Both processes are regulated by signaling proteins recruited to nascent adhesive sites that lead to activation of Rho GTPases. The Ajuba/Zyxin family of LIM proteins are components of cellular adhesive complexes. We show that cells from Ajuba null mice are inhibited in their migration, without associated abnormality in adhesion to extracellular matrix proteins, cell spreading, or integrin activation. Lamellipodia production, or function, is defective and there is a selective reduction in the level and tyrosine phosphorylation of FAK, p130Cas, Crk, and Dock180 at nascent focal complexes. In response to migratory cues Rac activation is blunted in Ajuba null cells, as detected biochemically and by FRET analysis. Ajuba associates with the focal adhesion-targeting domain of p130Cas, and rescue experiments suggest that Ajuba acts upstream of p130Cas to localize p130Cas to nascent adhesive sites in migrating cells thereby leading to the activation of Rac.     

Autonomous modes of behavior in primordial germ cell migration

Zebrafish primordial germ cells (PGCs) are guided toward their targets by the chemokine SDF-1a. PGCs were followed during three phases of their migration: when migrating as individual cells, while remaining in a clustered configuration, and when moving as a cell cluster within the embryo. We found that individually migrating PGCs alternate between migratory and pausing modes. Pausing intervals are characterized by loss of cell polarity and correlate with subsequent changes in the direction of migration. These properties constitute an intrinsic behavior of PGCs, enabling erasure of prior polarity and re-sampling of the environment. Following migration arrest at a site of high SDF-1a levels, PGCs resume migration as a cluster. The seemingly coordinated cluster migration is a result of single-cell movement in response to local variations in SDF-1a distribution. Together, these behavioral modes allow the cells to arrive at specific destinations with high fidelity and remain at their target site.     

Crossing the ventral midline causes neurons to change their response to floor plate and alar plate attractive cues during transmedian migration

Neuronal migration is required for the establishment of specific neural structures, such as layers and nuclei. Neurons migrate along specific migratory routes toward their final destinations, sometimes across long distances. However, the cellular and molecular interactions that control neuronal migration are largely unknown. Here, we examined the mechanism underlying the transmedian migration of precerebellar neurons using a flat whole-mount preparation of the rat embryo. These neurons were initially attracted by the floor plate (FP) at the ventral midline. However, after crossing the midline, they lost their responsiveness to the FP and became attracted by the alar plate (AP). Although the loss of responsiveness to FP cues was caused by an encounter of migrating cells with the FP, the gain of responsiveness to AP cues occurred irrespective of their encounter with the FP. These results identify a crucial change in the response of migrating cells to attractive guidance cues during the transmedian migration of precerebellar neurons.     

UNC-71, a disintegrin and metalloprotease (ADAM) protein,regulates motor axon guidance and sex myoblast migration in C. elegans

The migration of cells and growth cones is a process that is guided by extracellular cues and requires the controlled remodeling of the extracellular matrix along the migratory path. The ADAM proteins are important regulators of cellular adhesion and recognition because they can combine regulated proteolysis with modulation of cell adhesion. We report that the C. elegans gene unc-71 encodes a unique ADAM with an inactive metalloprotease domain. Loss-of-function mutations in unc-71 cause distinct defects in motor axon guidance and sex myoblast migration. Many unc-71 mutations affect the disintegrin and the cysteine-rich domains, supporting a major function of unc-71 in cell adhesion. UNC-71 appears to be expressed in a selected set of cells. Genetic mosaic analysis and tissue-specific expression studies indicate that unc-71 acts in a cell non-autonomous manner for both motor axon guidance and sex myoblast migration. Finally, double mutant analysis of unc-71 with other axon guidance signaling molecules suggests that UNC-71 probably functions in a combinatorial manner with integrins and UNC-6/netrin to provide distinct axon guidance cues at specific choice points for motoneurons.     

APC and Smad7 link TGFβ type I receptors to the microtubule system to promote cell migration

Cell migration occurs by activation of complex regulatory pathways that are spatially and temporally integrated in response to extracellular cues. Binding of adenomatous polyposis coli (APC) to the microtubule plus ends in polarized cells is regulated by glycogen synthase kinase 3β (GSK-3β). This event is crucial for establishment of cell polarity during directional migration. However, the role of APC for cellular extension in response to extracellular signals is less clear. Smad7 is a direct target gene for transforming growth factor-β (TGFβ) and is known to inhibit various TGFβ-induced responses. Here we report a new function for Smad7. We show that Smad7 and p38 mitogen-activated protein kinase together regulate the expression of APC and cell migration in prostate cancer cells in response to TGFβ stimulation. In addition, Smad7 forms a complex with APC and acts as an adaptor protein for p38 and GSK-3β kinases to facilitate local TGFβ/p38-dependent inactivation of GSK-3β, accumulation of β-catenin, and recruitment of APC to the microtubule plus end in the leading edge of migrating prostate cancer cells. Moreover, the Smad7-APC complex links the TGFβ type I receptor to the microtubule system to regulate directed cellular extension and migratory responses evoked by TGFβ.     

Multidirectional and multizonal tangential migration of GABAergic interneurons in the developing cerebral cortex

Most GABAergic interneurons originate from the basal forebrain and migrate tangentially into the cortex. The migratory pathways and mode of interneuron migration within the developing cerebral cortex, however, previously was largely unknown. Time-lapse imaging and in vivo labelling with glutamate decarboxylase (GAD)67-green fluorescence protein (GFP) knock-in embryonic mice with expression of GFP in gamma-aminobutyric acid (GABA)ergic neurons indicated that multidirectional tangential (MDT) migration of interneurons takes place in both the marginal zone (MZ) and the ventricular zone (VZ) of the cortex. Quantitative analysis of migrating interneurons showed that rostrocaudally migrating neurons outnumber those migrating mediolaterally in both of these zones. In vivo labelling with a lipophilic dye showed that the MDT migration in the MZ occurs throughout the cortex over distances of up to 3 mm during a period of a few days. These results indicate that MZ cortical interneurons undergo a second phase of tangential migration in all directions and over long distances, after reaching the cortex by dorsomedial tangential migration. The MDT migration in the MZ may disperse and intermix interneurons within the cortex, resulting in a balanced distribution of interneuron subtypes.     

Ephrins guide migrating cortical interneurons in the basal telencephalon

Cortical interneurons are born in the proliferative zones of the ganglionic eminences in the subpallium and migrate to the developing cortex along well-defined tangential routes. The mechanisms regulating interneuron migration are not completely understood. Here we examine the role of class-A members of the Eph/ephrin system in directing the migration of interneurons. In situ hybridizations demonstrated that ephrin A3 is expressed in the developing striatum, an area that is strictly avoided by migrating cortical interneurons in vivo, which express the EphA4 receptor. We then examined interneuron migration in grafting experiments, where explants of the medial ganglionic eminence (MGE) from enhanced green fluorescent protein-expressing transgenic mice were homotopically grafted into host slices from wild-type littermate embryos. After blocking ephrin-A ligands, many interneurons invaded the striatal anlage. Moreover, stripe assay experiments revealed that ephrin-A3 acts as a repellent cue for neurons from the medial ganglionic eminence. Downregulation of the EphA4 receptor via siRNA transfection reduced the repulsive effect of ephrin-A3, indicating that EphA4 mediates at least in part the repulsive effect of ephrin A3 on these cells. Together, these results suggest that ephrin-A3 acts as a repulsive cue that restricts cortical interneurons from entering inappropriate regions and thus contributes to define the migratory route of cortical interneurons.     

Patterns of fibronectin gene expression and splicing during cell migration in chicken embryos

A variety of evidence suggests that fibronectin (FN) promotes cell migration during embryogenesis, and it has been suggested that the deposition of FN along migratory pathways may also play a role in cell guidance. In order to investigate such a role for FN, it is important to determine the relative contribution of migrating and pathway-forming cells to the FN in the migratory track, as any synthesis of FN by the migrating cells might be expected to mask guidance cues provided by the exogenous FN from pathway-forming cells. We have therefore used in situ hybridization to determine in developing chicken embryos the distribution and alternative splicing of FN mRNA during three different cell migrations known to occur through FN-rich environments; neural crest cell migration, mesenchymal cell migration in the area vasculosa and endocardial cushion cell migration in the heart. Our results show that trunk neural crest cells do not contain significant FN mRNA during their initial migration. In contrast, migrating mesenchymal cells of the area vasculosa and endocardial cushion cells both contain abundant FN mRNA. Furthermore, the FN mRNA in these migrating mesenchymal and endocardial cells appears to be spliced in a manner identical with that present in the cells adjacent to their pathways. This in vivo evidence for FN synthesis by migrating and pathway cells argues against a generalized role for exogenously produced FN as a guidance mechanism for cell migration.