Roles of Integrins and Intracellular Molecules in the Migration and Neuritogenesis of Fetal Cortical Neurons: MEK Regulates Only the Neuritogenesis

The roles of integrin subunits and intracellular molecules in regulating the migration and neuritogenesis of neurons isolated from 16.5 gestation days rat fetal cortices were examined using in vitro assays. Results showed that laminin supported the migration of fetal cortical neurons better than fibronectin and that the fetal cortical neurons migrated on laminin using β1 and α3 integrin subunits which make up the α3β1 integrin receptor. On fibronectin, the migration was mediated by β1 integrin subunit. Perturbation of src kinase, phospholipase C, or protein kinase C activity, inhibition of IP3 receptor mediated calcium release, or chelation of intracellular calcium inhibited both migration and neuritogenesis, whereas inhibition of growth factor signaling via MEK inhibited only the neuritogenesis. The detection of α1 and α9 transcripts suggested that the migration of fetal cortical neurons may also be mediated by α1β1 and α9β1 integrin receptors. Results showed that calcium may regulate migration and neuritogenesis by maintaining optimum levels of microtubules in the fetal cortical neurons. It is concluded that the fetal cortical neurons are fully equipped with the integrin signaling cascade required for their migration and neuritogenesis, whereas crosstalk between the integrin and growth-factor signaling regulate only the neuritogenesis.


Introduction
During brain development, postmitotic neurons migrate from their site of origin to distant places, differentiate, and make connections forming different layers of the cortex. Defects in this process results in abnormal neuronal positioning and connections in the brain, which may cause neurobehavioral problems later in life [1]. Neuritogenesis, an early step of neuron differentiation, is the synthesis of multiple growth cone tipped extensions (neurites) that ultimately form the axons and dendrites of neurons [2]. Mechanisms that regulate the migration and differentiation of neurons are not fully understood.
Cell surface integrin receptors, each consisting of an and a subunit, play critical roles in the glial-guided migration of neurons in the brain [3]. e extracellular domains of the receptor subunits bind with extracellular matrix (ECM) proteins (such as �bronectin and laminin) and the cytosolic domains of subunits interact with kinases, adaptor molecules, and the cytoskeleton [4]. ese interactions facilitate the "outside-in" and the "inside-out" signaling across the cell membrane by the integrin heterodimers [5] that may lead to cell migration and neuritogenesis [6].
Integrin receptors with explicit combinations of and subunits interact with speci�c ECM proteins [7]. Developing brains express 1, 1, 3, 4, 5, 6, and v integrin subunits which may form 1 1, 3 1, 4 1, 5 1, 6 1, and v 1 integrin receptors [8,9]. Of all these receptors, the 1 1, 3 1, and 6 1 receptors interact with laminin, whereas 4 1, 5 1, and v 1 receptors interact with �bronectin [7]. Manipulation of integrin subunits 3 and 5 genes in mice causes abnormal positioning of neurons in the cerebral cortex, and nullifying the 6 gene results in cortical laminar defects [3,[10][11][12]. ese studies suggest that integrin receptors consisting of 1 subunit ( 3 1, 5 1, and 6 1) are involved in the migration of neurons in the brain. is is also in accordance to a large number of reports describing the role of integrin receptors consisting of 1 subunit in neuron migration and neuritogenesis [13][14][15][16]. However, studies involving the ablation of 1 integrin subunit gene in mice [17][18][19][20] suggest that this integrin subunit is required for the glial end feet anchorage and is not essential for neuron-glia interactions and glial-guided migration of neurons during brain development. Moreover, additional studies indicate that 1 integrin subunit is involved not in the migration but the differentiation of glia and neurons [2,[20][21][22]. erefore, to date, the studies on the role of 1 subunit containing integrins ( 3 1, 5 1, and 6 1) in the migration of fetal brain neurons have been largely inconclusive. Experiments with antibodies against integrin subunits or inhibitors of integrin signaling in slice cultures [23][24][25][26] are also not free from the indirect effects that may in�uence the migration and differentiation of neurons.
erefore, in the present study, neurons were isolated from 16.5 gestation days fetal cerebral cortices of rats for the direct examination of the role of molecules that are known to regulate the integrin-mediated cell migration. Because different ECM molecules are secreted by radial glial cells during development [27,28], initially different concentrations of laminin and �bronectin were examined on the migration of fetal cortical neurons. e propensity of subunits forming integrin receptor/s with the 1 subunit to support migration was examined by using the same concentration of antibodies against different subunits at which the migration of neurons was signi�cantly inhibited by antibody against 1 subunit. Roles of different intracellular molecules (src kinase, phospholipase C, calcium, protein kinase C, MEK kinase, and microtubules) that are known to support integrin-mediated migration of different cell types [29][30][31] were examined on neuron migration by using pharmacological inhibitors or intracellular calcium chelator. e roles of 1 and 3 integrin subunits, the intracellular molecules, and the calcium chelator were also examined on neuritogenesis. Monoclonal antibodies against the extracellular domains of integrin subunits were obtained from different commercial sources in no-azide formulations or freed from sodiumazide using Nanosep centrifugal devices (PALL Life Sciences, Ann Arbor, MI). Additional information on these antibodies as well as those used in the immuno�uorescent staining and western blotting experiments (see below) is provided in Table 1. Inhibitors against different enzymes that are known to regulate integrin signaling in different cell types and modulators of intracellular calcium levels are mentioned in Table 2.

Preparation of Dissociated
Neurons. Dissociated neurons were prepared from cortices as described previously [39] with some modi�cations. Brie�y, on gestation days 16.5, pregnant rats were deeply anesthetized with iso�urane, fetuses were removed from uteri, and cerebral cortices of fetal brains were dissected out as described previously [40]. Cerebral cortices were incubated in HBSS containing 0.1% Trypsin for 15 min at 37 ∘ C. Cortices were triturated in HBSS containing 0.025% DNase I, 0.2% Trypsin inhibitor, 0.2% BSA, and 12 mM MgSO 4 and centrifuged at 160 g. e resulting pellet was suspended in HBSS and centrifuged. Supernatant was discarded and the pellet was suspended in the culture medium DMEM/F-12 (1 : 1) containing 0.2% B-27 to estimate the number using a hemacytometer (Hausser Scienti�c, Horsham, PA). e neurons were used for migration assays and also cultured on laminin-coated wells of 8 chambered glass slides (ermo Fisher Scienti�c, Houston, T�) for immuno�uorescent microscopy and neuritogenesis assays or in �asks (Fisher Scienti�c) for western blotting and RNA isolation.

In Vitro Neuron Migration
Assay. e Boyden Chamber assay that has been successfully used for estimating the migration of fetal brain neurons previously by different laboratories [39,[41][42][43] was used for this study. Speci�cally, the Boyden chamber assay for studying fetal cerebral cortical neurons of Maeda and Noda (1998) [39] was used with some modi�cations. Transwell inserts (diameter 6.5 mm), each consisting of polycarbonate membranes with 3.0 m pore size with pore density 2 × 10 6 /cm 2 (Fisher Scienti�c� Pittsburgh, PA) were used for migration assays. Undersurface of membranes of Transwell inserts was �rst coated with Poly D Lysine (10 g/mL) overnight and then with solution (100 L) of laminin, �bronectin, or only water (diluent of laminin or �bronectin solutions) and dried overnight in sterile conditions prior to use. In all migration assays, the lower chamber contained 500 L of culture medium (controls) or medium containing antibody against integrin subunit, inhibitor, or calcium chelator (see below) and the upper chamber contained 200 L of medium containing 150,000 dissociated neurons. Plates containing the Boyden chamber assemblies were incubated for 18 h in an incubator (37 ∘ C, 5% CO 2 , humidity 99%). At the end of incubation, membranes were treated with chilled 4% paraformaldehyde solution for 20 min and the neurons on the upper surface of the membranes were removed by rubbing with cotton swabs twice. Preliminary experiments were conducted to ensure that rubbing technique removes all neurons from the upper surface of membrane and the incubation time that provided adequate number of neurons for migration assays. Membranes were cut and processed for the immuno�uorescent detection of neuronal nuclei and neuronal marker MAP2 expression as described below (see Section 2.10). All migration assays were performed at least three times with two Boyden chamber assemblies per experimental conditions. Images of di�erent �elds vertically and across (total 9) of each membrane were captured at 10x magni�cation. Total numbers of nuclei from each image of controls and experimental groups were counted using the counting tool of the Metamorph soware to determine the number of nuclei per membrane for analysis.

ECM Effects on the Migration of Neurons.
Relative roles of ECM proteins (laminin and �bronectin) on the migration of dissociated neurons were examined by coating Poly-D lysine treated membranes with different concentrations of laminin (1, 5, 10, 20, and 50 g/mL) or �bronectin (10, 20, 50, 100, and 200 g/mL) in sterile water. Medium containing neurons (150,000) were added in the upper chamber and the lower chambers contained medium only. Chambers were incubated for 18 h and the neurons on the undersurface of the membranes were counted as described above.

Antibody Effects on the Migration of Neurons.
Relative roles of different integrin subunits with respect to 1 subunit on the migration of neurons were examined using functional blocking antibodies. Azide-free monoclonal antibody against different integrin subunits, control IgG, or IgM were added in the medium of lower chamber. Control chambers contained only medium in both upper and lower chambers. Upper chamber contained neurons (150,000) in the medium (200 L). Neurons on the lower surface of membranes were counted at the end of 18 h incubations as described above. Initial experiments were conducted with monoclonal antibodies against 1 integrin subunit to determine the concentration (50 nMole) that signi�cantly inhibited the migration of neurons at on laminin (10 g/mL). Relative effects of monoclonal antibodies against additional integrin subunits at (50 nMole) were tested on the migration of neurons on laminin (10 g/mL) or �bronectin (100 g/mL) coated membranes. ese antibodies (see Table 1) are tested for their efficacy by the supplier and also reported for their interaction with integrin subunits on cell surface and inhibition of cell adhesion [44][45][46][47][48][49][50].

Effects of Inhibitors or Calcium Chelator on the Migration of Neurons.
Involvements of different intracellular signaling molecules in the migration of neurons were examined using pharmacological inhibitors. is included inhibitors PP2, U-73122 (U2), PD98059 (PD), Calphostin C, 2-APB, and Ruthenium Red against Src family of tyrosine kinases, PLC activation, MAP kinase kinase, Protein kinase C, IP3induced calcium release, and calcium induced calcium release from Ryanodine receptors, respectively. Role of intracellular calcium on migration was examined using the chelator BAPTA-AM. Inhibitors (PP2, U2, 2-APB, and PD) or control compounds U-73343 (U3) and PP3 and intracellular calcium regulators (BAPTA-AM and Ruthenium Red) were added in the medium of lower chamber. Upper chamber contained 150,000 neurons suspended in the medium only. e concentrations of these pharmacological compounds, their target and relevant references, are mentioned in Table  2.

Effects of Antibodies, Inhibitors, or Calcium Chelator on Neuritogenesis.
Roles of integrin subunits, intracellular signaling molecules and calcium on neuritogenesis were examined using antibodies, pharmacological inhibitors, and calcium chelators. Wells of 8 chambered glass slides were coated with gelatin (12 h at 4 ∘ C) followed by laminin (10 g/mL) (12 h at 4 ∘ C). Dissociated neurons (100,000) were cultured in wells in the absence (control) or presence of monoclonal antibody (Table 1), inhibitor, or calcium modulators (Table 2) for 20 h and bright-�eld images (6/well = 3 vertical and 3 across) of neurons were captured. High contrast images were captured for visualization and tracing of neurites with the tools of Metamorph soware. Experiments were repeated to collect data from at least 3 wells per control and treatment conditions (18 images/treatment condition).  Membranes or wells were rinsed with PBS three times (15 min each) and then incubated with Alexa dye conjugated secondary antibody (10 g/mL) and nuclear stain Hoechst 33342 (dilution 1 : 200). Fluorescent images were captured using a Nikon Eclipse (TE-2000 U) Microscope.

Reverse Transcription and Polymerase Chain Reactions (RT-PCR).
Expression of transcripts representing different integrin subunits in the fetal cortical neurons, cultured on laminin-coated �asks for 7 h, was examined using RT-PCR in three occasions. In each experiment, the total RNA (1 g) isolated from the neurons using Trizol-reagent (Invitrogen, Carlsbad, CA) was subjected to complementary DNA (cDNA) synthesis using a Reverse transcription III kit (Invitrogen). Equal amounts (50 ng equivalent) of cDNA samples were ampli�ed by PCR. e primer sequences (Table 3) were either obtained from the literature [51][52][53] or subjecting the rat or homologous regions of mice and human cDNA sequences (GenBank) to Primer3 soware (http://�ypush.imgen.bcm.tmc.edu/primer/primer3� www.cgi/). Authenticity of these primers was tested by PCR using cDNA from rat and human cell lines (rat PC12 and cardiac myoblasts; human trophoblast and colon cancer cell lines) that produced expected size amplicons with no spurious bands. Identity of amplicons representing 3 and 9 Neuroscience Journal 5 T 3: Primers used for the ampli�cation of integrin cDNA from rat fetal brain neurons.

Western Blotting.
A previously described western blotting methodology [40] was used to examine the expressions of phosphorylated PLC-1 in the dissociated neuronal preparations cultured on laminin-coated �asks for 6 h. Clear lysatefrom neurons containing 10 g proteins were denatured by boiling in the presence of lane marker buffer (Pierce) and subjected to electrophoresis on a 7.5% Polyacrylamide gel containing sodium dodecyl sulphate. Molecular weight markers (Bio-Rad) were also loaded in adjacent lanes. At the end of electrophoresis separated proteins were transferred on to nitrocellulose membranes and probed with primary antibodies against phosphorylated PLC-1 and -Actin (Table 1). Membranes were washed and exposed with Peroxidase conjugated secondary antibodies. Bands were detected using Amersham Hyper �lm �CL (G� Healthcare Ltd., Buckinghamshire, UK). e experiment for the detection of phosphorylated PLC-was tested using 3 different neuron preparations.

Statistical Analysis.
Analysis of the neuronal migrations and neuritogenesis was conducted by ANOVA using SPSS soware (SPSS, Chicago, IL). Neuron migrations were assayed by comparing the mean ± standard errors of mean values of the number of neuronal nuclei from 9 �elds under each Boyden membrane of total 6 to 9 membranes per treatment group. e neuritogenesis was examined by comparing the mean ± standard errors of mean values of the neurite lengths from 6 �elds per well (total 3 wells of each control and treated groups). Differences in Mean ± Standard errors of mean values at were considered signi�cant.

Isolation of Fetal Cortical
Neurons. e number of neurons isolated per pair of cortices by the described method was approximately × 1 7 neurons (  7). ese neurons attached with the laminin-coated wells and majority (approximately 95%) expressed neuronal marker NeuN  (Figure 3). Migration of cortical neurons was higher on membranes coated with 5, 10, 20, or 50 g/mL laminin than those coated with only 1 g/mL laminin or no laminin. Highest migration of neurons occurred on membranes coated with 10 g/mL laminin. e migration of neurons on membranes coated with 20, 50, 100, or 200 g/mL �bronectin was higher than those coated with 10 g/mL �bronectin or no �bronectin. e migration of neurons on membrane coated with 100 or 200 g/mL �bronectin was not signi�cantly different (

ECM Effects on the Migration of Neurons
). Further studies on the migration of neurons were conducted with membranes coated with Poly-D lysine followed by 10 g/mL laminin or 100 g/mL �bronectin (see below). Figure  4). Antibody against 1 or integrin subunit signi�cantly decreased migration of neurons on laminin-coated membranes ( ). Antibody against 6 subunit did not alter the migration of neurons on laminin-coated membranes. Moreover, the migration of neurons was not altered by control antibody (IgG or IgM) or the antibody against the v subunit (negative control on laminin) on laminin-coated membranes. On �bronectin-coated membranes, only antibody against 1 subunit inhibited the migration ( ). e migration of neurons on �bronectin-coated membranes was not altered by control antibodies (IgG or IgM), and antibodies against 3 (negative control since not expressed), 4, 5, or v subunits.  Kinase Kinase (MAPK/ERK kinase or MEK) inhibitor PD, control compounds U3 or PP3 on laminin, or �bronectincoated membranes ( ). e inhibitor of IP3 receptorinduced calcium release from intracellular stores (2-APB) and the intracellular calcium chelator BAPTA-AM inhibited the migration both on laminin-and �bronectin-coated membranes. Ruthenium Red, the inhibitor of intracellular calcium-induced calcium release from Ryanodine receptor containing calcium stores, did not alter the migration of neurons.

Effects of Antibodies, Inhibitors, and Calcium Modulators on Neuritogenesis.
Antibodies against 1 or 3 subunit inhibited neuritogenesis of fetal cortical neurons. Neuritogenesis was not inhibited in the presence of control antibodies (IgG) at 50 nM (Figure 6(a)) and even at higher concentration (100 nM), (Figure 6(b)). BAPTA-AM completely abolished the neurite formation at higher concentration (10 M), (Figure 6

Expression of Phosphorylated PLC 1 and Integrin Subunit mRNA Species in Dissociated Neurons.
Western blotting experiments with lysate prepared from neurons at 6 h of culture provided evidence for the presence of PLC-1 in its phosphorylated state at tyrosine 783 ( Figure 8). Neurons at 6 h of culture expressed mRNA for 1, 1, 3, 4, 5, v, and 9 integrin subunits ( Figure 9).

Discussion
Cortical neurons isolated from rat fetal (GD16.5) brains expressed subunits for integrin receptors and responded to cues of both laminin and �bronectin during their migration in vitro. ese neurons utilized the intracellular signaling molecules and cytoskeletal elements that are known to transmit integrin receptor signaling during cell migration and differentiation. At the same concentrations, the effects of inhibitors and intracellular calcium modulators were more robust on neuritogenesis than on migration. Results showed that growth factor signaling via MEK is only required for neuritogenesis. e connotations of these �ndings are discussed below. Membranes coated with laminin or �bronectin enhanced the migration of fetal cortical neurons (Figure 3). It was evident that laminin at a 10-fold lower concentration supported the migration better than �bronectin suggesting that laminin may be a better substrate for the migration of fetal brain neurons. is observation is consistent with the migration pattern of neuronal precursors derived from embryonic stem cells [14]. Optimum adhesion with the ECM is critical for the normal migration of cells [54,55]. erefore, the enhanced migration of neurons with the increased concentration of laminin and its downregulation with further increase in laminin (Figure 3(a)) indicates that the pace and the direction of neuron migration in the fetal brain may be regulated by the amount of speci�c ECM molecule secreted by glial cells on the migratory route. Inhibition studies with monoclonal antibodies to examine the relative roles of different integrin subunits on migration suggested that the receptor consisting of 3 and 1 subunits ( 3 1) primarily regulates the migration of fetal cortical neurons on laminin (Figure 4(a)). is is also in accordance with previous observations [3,10,15]. In addition, the lack of effects of antibody against the 6 integrin subunit on the 6 1 mediated migration is also consistent with the absence 6 subunit in fetal cortical neurons ( Figure 9) and the lack of any direct evidence for its role in the migration of fetal cortical neurons [11]. �n �bronectin-coated membranes, only the antibody against 1 subunit inhibited the migration of neurons and antibodies against the subunits ( 3, 4, 5, or v) that are the constituent of �bronectin-binding receptors ( 3 1, 4 1, 5 1, and v 1) did not inhibit the migration (Figure 4(b)) although the RT-PCR (Figure 9) results con�rmed their expression. is �nding indicates that the migration of fetal cortical neurons on �bronectin may be regulated by 1 subunit containing integrin receptor/s other than 3 1, 4 1, 5 1, and v 1. It may include the �bronectin binding receptor 8 1 that also interacts with ECM tenascin. e role of tenascin in the glial guided migration of fetal cortical neurons is further supported by the fact that tenascin is expressed by the radial glia in the developing brain [56] and transcripts of 9 subunits are  expressed in fetal cortical neurons (Figure 9), which also forms the tenascin interacting 9 1 integrin receptor. While the antibody against v subunit is reported to inhibit the migration of fetal cortical neurons in an imprint assay [3�, it failed to inhibit the migration on �bronectincoated membranes in the Boyden assay (Figure 4(b)). e role of integrin receptors consisting of v subunits ( v 1, v 5, or v 8) in the migration of fetal cortical neurons is further lessened by the fact that vitronectin, the substrate for these receptors, is primarily expressed in the proximity of blood  Table 2 capillaries [57] and the ablation of v subunit expression by gene manipulation only disturbs the development of blood vessels and axonal survival but not neuronal positioning [58]. It is surprising, however, to �nd no effect of antibody against 5 subunit on migration of neurons (Figure 4(b)) because its genetic manipulation at GD15.5 in mice causes laminar defects [12]. e exact reason for this discrepancy is yet to be known.
Application of different pharmacological inhibitors revealed that the intracellular molecules that mediate integrin signaling ( Figure 10) and known to regulate migration of various cell types [30] are fully functional in the fetal cortical neurons. ese included Src Kinase, that is activated by focal adhesion kinase following the interaction of integrin receptors with the ECM [59]; Phospholipase C , that is activated by the activated Src Kinase, and hydrolyses membrane protein PIP2 (phosphotidal inositol biphosphate) into Inositol (1,4,5)-triphosphate (IP3) and Diacylglycerol (DAG) [60,61]; Protein Kinase C, that is activated by the DAG [62], and the calcium released from the IP3 receptor containing stores [63].
e PLC-1 interacts directly with the cytoplasmic tail of the 1 integrin subunit and induces cytoskeletal organization and integrin mediated migration of cells [61]. Integrin mediated cell adhesion promotes rapid autophosphorylation [64] of Tyr-397 residue in focal adhesion kinase (FAK) promoting its interaction with the C-terminal SH2 domain of PLC-1 and associating with the Src kinases at the site of integrin-ECM interactions [64]. is causes phosphorylation of Tyrosine residue 783 (Tyr783) and the activation of PLC-1 required for the integrin mediated upregulation of intracellular calcium and migration of cells [65]. Western blotting data (Figure 8) con�rmed the presence of the activated form of PLC-1 isoform in the fetal cortical neurons suggesting its possible interaction with the cytoplasmic tail of the 1 integrin subunit in fetal cortical neurons.
Previous studies report that PKC and atypical PKC regulate migration of neuronal precursors in the fetal cerebral cortex [66,67] and different PKC isoforms (PKC , PKC I, PKC II, PKC , PKC , and PKC ) support migration of cells by associating with 1 integrin subunit [68]. Robust reduction of migration in the presence of PKC inhibitor (Calphostin C) ( Figure 5) shows that the activated state of PKC isoform/s in fetal cortical neurons is required for their migration on both laminin and �bronectin.
Even though the integrin and growth factor receptor cross-talk is reported to play a signi�cant role in the migration of various cell types [31], the MEK inhibitor PD98059 did not alter the migration of fetal cortical neurons signi�cantly ( Figure 5) but rigorously reduced neuritogenesis (approximately 14 fold) (Figure 7(c)). is suggests that the migration of fetal cortical neurons is primarily regulated by integrin mediated pathways, whereas the differentiation of these neurons is regulated by both integrin and growth factor signaling ( Figure 10). e sustained expression of certain adaptor/s (such as ILK, PINCH, and Paxillin) that are required for cross-talk between integrin and growth factor receptors during differentiation but not migration of fetal cortical neurons may instruct such effects [69]. Interestingly, ILK is required for the differentiation of Bergmann glia during the cerebellar development [22].
e reduced migration of neurons in the presence of BAPTA-AM ( Figure 5) con�rmed previous observations that intracellular calcium is an indispensable mediator for the migration of fetal brain neurons [63]. On the other hand, no effects of high concentration (50 M) of Ruthenium Red on migration are in contrast to the �nding that integrinmediated signaling mobilizes calcium from the Ryanodine Receptor gated stores [70].
e neuritogenesis data showed that the integrin receptor consisting of 3 and 1 subunits also regulates neuritogenesis in rat fetal cortical neurons. is is in accordance to previous reports [2,14]. e present study further shows that the signaling molecules that mediate the migration of fetal cortical neurons (Src kinase, PLC-, Protein kinase C, IP-3 induced calcium release, and the intracellular calcium itself) also regulate the process of neuritogenesis albeit more robustly than migration ( Figures 5 and 6(c)). Furthermore, inhibition of neuritogenesis by BAPTA at a moderate concentration (2.5 M), its total abolition at higher concentration (10 M) ( Figure 6(a)), and reduction in microtubule expression (Figure 7) suggest that intracellular calcium not only regulates the dynamics of microtubule dynamics [71] but may also maintain its intracellular levels.

Conclusions
Rat fetal cortical neurons (isolated at GD16.5) are fully equipped with the integrin receptor and the classical intracellular signaling cascade ( Figure 10) to maneuver their migration and neuritogenesis in vitro. Precisely, the lamininbinding 3 1 integrin receptor and intracellular signaling molecules drive both the migration and neuritogenesis while the cross-talk of integrin with growth factor signaling may add to more robust signaling towards the differentiation process. e detection of 9 subunit transcripts in fetal cortical neurons indicates that migration of fetal cortical neurons on radial glia may be supported by tenascin via the 9 1 integrin receptor. ese results suggest that the composition of extracellular matrix in the path of migration and speci�c repertoire of integrin receptors regulates the dynamics of migration of neuronal precursors in the developing brains. e present study also indicates that calcium may regulate the migration of fetal cortical neurons also by maintaining the optimum supply of microtubules.
Additional studies are now required to delineate the exclusive role of each downstream molecule in the "outsidein" and "inside-out" integrin signaling that mediates the migration and neuritogenesis of fetal cortical neurons. is includes estimating the surface expression of each integrin subunit by �ow cytometry and to examine the relevant concentrations of each antibody in the presence or absence of different combinations of pharmacological inhibitors as well as siRNA manipulation of components of integrin and growth factor signaling. Moreover, restoring the migration and neuritogenesis of neurons by activating different kinases following their inhibition by antibodies and/or pharmacological inhibitors may also be required to further ascertain their role.
Additional studies are also required to identify the PKC isoform/s that regulates the migration and neuritogenesis of fetal cortical neurons, the roles of 1 and 9 integrin subunits, that are the constituent of laminin-binding 1 1 and tenascin-binding 9 1 integrin receptors respectively, the manipulation of molecules that regulate cross-talk between integrin and growth factor signaling (such as ILK, Paxillin, and PINCH), and the Semaphorins (speci�cally of Semaphorin 7A) that in�uence axon outgrowth and activation of MAPK [72].
Finally, in the perspective of results obtained by experimenting directly with fetal cortical neurons in culture and those describing no changes in the migration of neurons in 1 integrin mutant (in vivo studies) mice [18,19], it is conceivable that the acute effects of monoclonal antibody on the neurons in culture (an in vitro test) may not be compensated which may occur in gene manipulation (in vivo) studies. It is also possible that the migration machinery of fetal cortical neurons in mice, which was used for gene manipulation studies by other investigators, may not be exactly similar to rat that was used in the current study. Even though the �ndings of in vitro studies presented here have their limitations, conclusions of previous gene manipulation studies [17][18][19] indicating that the 1 subunit is involved in glial differentiation but not in the glial-guided migration of neurons may not be stringent as data presented here show that fetal cortical neurons do utilize 1 and 3 integrin subunits and the intracellular molecules, that are known to support integrin signaling, for their migration.
Con�ict o� �nte�ests �. K. Rout has no con�ict of interests pertinent to the work presented in this paper.