Netrin-1–␣3␤1 integrin interactions regulate the migration of interneurons through the cortical marginal zone

Amelia Stancoa, Charles Szekeresb, Nikhil Patela, Sarada Raoa, Kenneth Campbellc, Jordan A. Kreidbergd, Franck Polleuxe, and E. S. Antona,1

aUniversity of North Carolina Neuroscience Center and the Department of Cell and Molecular Physiology, The University of North Carolina School of Medicine, Chapel Hill, NC 27599; bCollege of Medicine, University of South Florida, Tampa, FL 33620; cDivision of Developmental Biology, Cincinnati Children’s Hospital Medical Center, University of Cincinnati College of Medicine, Cincinnati, OH 45229; dDepartment of Medicine, Children’s Hospital and Department of Pediatrics, Harvard Medical School, Boston, MA 02115; and eDepartment of Pharmacology, The University of North Carolina School of Medicine, Chapel Hill, NC 27599

Edited by Pasko Rakic, Yale University School of Medicine, New Haven, CT, and approved February 27, 2009 (received for review November 22, 2008) Cortical GABAergic interneurons, most of which originate in the The prominent expression of netrin-1 in the ganglionic eminence ganglionic eminences, take distinct tangential migratory trajecto- (GE), where interneurons originate (9), as well as the expression of ries into the developing cerebral cortex. However, the ligand– ␣3␤1 integrin in migrating cortical neurons (5), suggests that receptor systems that modulate the tangential migration of dis- netrin-1–␣3␤1 integrin interactions may influence specific aspects tinct groups of interneurons into the emerging cerebral wall of the tangential migration of GABAergic interneurons. Recent remain unclear. Here, we show that netrin-1, a diffusible guidance evidence that ␣3␤1 integrin serves as a receptor for netrin-1 in cue expressed along the migratory routes traversed by GABAergic epithelial cells and evidence for cross-talk between integrins and interneurons, interacts with ␣3␤1 integrin to promote interneuro- classical netrin receptors have opened an avenue for investigating nal migration. In vivo analysis of interneuron-specific ␣3␤1 this possibility (10). Netrins serve as secreted guidance cues, integrin, netrin-1–deficient mice (␣3lox/؊Dlx5/6-CIE, netrin-1؊/؊) regulating axonal outgrowth and neuronal migration by binding to reveals specific deficits in the patterns of interneuronal migration Deleted in Colon Cancer (DCC), Down’s Syndrome Cell Adhesion NEUROSCIENCE along the top of the developing cortical plate, resulting in aberrant Molecule (DSCAM), or UNC5 receptors (11, 12). Netrin-1 interacts interneuronal positioning throughout the cerebral cortex and with DCC and DSCAM to induce growth cone attraction (12, 13), hippocampus of conditional ␣3lox/؊Dlx5/6-CIE, netrin-1؊/؊ mice. whereas its association with UNC5 receptors promotes axonal repulsion (14). Recently, Yebra and colleagues (10) have demonstrated that These results indicate that specific guidance mechanisms, such as ␣ ␤ ␣ ␤ netrin-1–␣3␤1 integrin interactions, modulate distinct routes of 6 4 and 3 1 integrins regulate the migration of epithelial cells interneuronal migration and the consequent positioning of groups on netrin-1 through a direct interaction of these integrins with the of cortical interneurons in the developing cerebral cortex. C-terminal domain of netrin-1, although the significance of these interactions is yet to be determined in vivo. Here, we have inves- tigated the contribution of netrin-1–␣3␤1 integrin–mediated signal he development of the six-layered cerebral cortex, composed transduction to the migration of GABAergic interneurons from the Tpredominantly of glutamatergic projection neurons and MGE into the developing cerebral wall. GABAergic interneurons, depends on the appropriate migration of neurons from ventricular zones of the dorsal and ventral telen- Results cephalon to their final position in the emerging cortical plate. Netrin-1 Is Localized Along the Migratory Route of Cortical GABAergic Neuronal progenitors from the dorsal telencephalon migrate radi- Interneurons. To evaluate the potential involvement of netrin-1– ally as cohorts along the radial glial scaffold, pass their predecessors ␣3␤1 integrin signaling in interneuronal migration, we mapped the in the developing cortical plate, and coalesce into layers, giving rise expression pattern of netrin-1 and ␣3␤1 integrin in embryonic to glutamatergic projection neurons (1). In contrast, neuronal cortex during interneuronal migration (Fig. 1 and Fig. S1). Sections precursors from the ventral telencephalon, principally the medial from netrin-1 gene trap indicator mice (Netrin-1LacZ/ϩ), in which ganglionic eminence (MGE), migrate tangentially through the cortical ␤-galactosidase expression faithfully indicates endogenous netrin-1 marginal zone and intermediate zone and enter the cortical plate, using gene expression (15), were co-labeled with ␤-galactosidase and ␣3 the radial glial scaffold, before differentiating into GABAergic inter- integrin antibodies. Prominent netrin-1–LacZ expression is evident neurons (2–4). A subset of these interneurons initially migrates radially in the ventricular zone of the GE and in the marginal zone of the inwards, toward the cortical ventricular zone, before turning back developing cortex (Fig. 1 B, E, and H). ␣3 integrin is present in radially up toward the cortical plate (4). These different modes of neuronal and tangentially oriented neurons in the cerebral wall (Fig. 1 A, J, and migration are coordinated temporally to achieve the ‘‘inside-out’’ L and Fig. S1). Both ␣3 integrin and netrin-1 are prominently co- laminar organization of neurons in the neocortex. expressed in the marginal zone region (Fig. 1 C, F, and I). Specific cell adhesion interactions between neurons, glia, and the Although LacZ expression is indicative of netrin-1 gene expres- surrounding ECM are critical for the appropriate migration and sion patterns, it may not reveal fully the localization of secreted placement of cortical neurons. These adhesive interactions are netrin-1 in the developing cortex. To examine the expression pattern mediated in large part by integrins, heterodimeric cell-surface ECM receptors, which serve as structural links between extracellular Author contributions: A.S. and E.S.A. designed research; A.S., N.P., and S.R. performed ligands and the internal cytoskeleton. The functional significance of research; A.S., C.S., K.C., J.A.K., and F.P. contributed new reagents/analytic tools; A.S. integrin signaling for cerebral cortical development is demonstrated analyzed data; and A.S. and E.S.A. wrote the paper. by the distinct types of cortical malformations exhibited by mice The authors declare no conflict of interest. deficient in ␣v, ␣3, ␣6, ␤1, and ␤4 integrins (1, 5–8). However, the This article is a PNAS Direct Submission. selective adhesive interactions needed to coordinate the migration 1To whom correspondence should be addressed. E-mail: [email protected]. of distinct groups of interneurons and projection neurons into the This article contains supporting information online at www.pnas.org/cgi/content/full/ developing cerebral cortex remain poorly understood. 0811343106/DCSupplemental.

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Fig. 1. Expression of netrin-1 and ␣3␤1 integrin in the developing cortex. (A–C) Coronal sections of the forebrain at E13.5 labeled with ␣3 integrin (A) and ␤-galactosidase (B) antibodies demonstrate that tangentially migrating interneurons express ␣3␤1 integrin (arrowheads in A) and indicate that ne- trin-1 is present at the cortical marginal zone (arrows in B) and ventricular zone of the GE (asterisk in B). C is a merge of A and B.(D–F) High magnification of the developing cerebral wall boxed in C shows colocalization (arrow in F)of netrin-1 (E) and ␣3␤1 integrin (D) at the marginal zone. (G–I) Tangentially migrating interneurons at the marginal zone express ␣3␤1 integrin (arrow- heads in G and I) and netrin-1 (arrowheads in H and I). (J–L) Radially migrating neurons in the intermediate zone express ␣3 integrin (arrowheads in J and L), but not netrin-1 (K and arrowheads in L). (M) Coronal section of E13.5 cortex Fig. 2. Netrin-1–␣3␤1 integrin interactions. To verify netrin-1–␣3␤1 integrin labeled with netrin-1 antibodies demonstrates that secreted netrin-1 is ex- interactions, E13.5 forebrain extracts (A), netrin-1–expressing 293 cell lysates (B), pressed in the developing forebrain during tangential neuronal migration. (N) or a mixture of purified netrin-1 (ntn1) and ␣3␤1 integrin proteins (C) were Netrin-1 expression was not detected in the forebrain of E13.5 netrin-1–null immunoprecipitated (IP) with ␣3 integrin or netrin-1 antibodies and Western mice by immunohistochemistry. (O–P) Immunoblot of netrin-1 in the GE, blotted (WB) with antibodies to ␣3 integrin or netrin-1. (A–C) In each assay, lateral pallium, and dorsal pallium of E14.5 forebrain (P) indicate that netrin-1 netrin-1 co-immunoprecipitated with ␣3 integrin and vice versa. (D) Netrin-1 or is expressed in each of these regions (schematized in O). (Scale bar, 15 ␮m.) ␣3 integrin was not detected when ␣3 integrin-deficient or netrin-1–deficient forebrain extracts were used to co-immunoprecipitate netrin-1 or ␣3 integrin. (E) The co-immunoprecipitation of ␣3 integrin and laminin-1, a known ␣3 integrin of functional, secreted netrin-1 in the developing telencephalon during ligand, and of netrin-1 and DCC, a known netrin-1 receptor, from E14.5 forebrain tangential migration, embryonic day 13.5 (E13.5) cortices were immu- lysates serves as positive controls. (F) Robust ␤1 integrin receptor activation is nolabeled with netrin-1 antibodies. Expression of secreted protein was observed following netrin-1 supernatant (sup), recombinant mouse netrin-1 (rm), and ECM stimulation of MGE neurons. detected in marginal zone and intermediate zone regions traversed by tangentially migrating interneurons (Fig. 1M). This immunolo- calization pattern was not detected in netrin-1–null mice (Fig. 1 N blotting with anti-netrin-1 antibodies shows that ␣3 integrin co- and P). The presence of netrin-1 protein in the ventral and dorsal immunoprecipitates with netrin-1 in vitro (Fig. 2B). To establish telencephalon was verified further by immunoblotting performed whether netrin-1 and ␣3␤1 integrin associate directly, netrin-1– on microdissected portions of the telencephalon (Fig. 1P). To- containing supernatants from stably transfected 293 cells were gether, these results reveal ␣3␤1 integrin expression in interneurons incubated with purified ␣3␤1 integrin protein. Netrin-1 or ␣3 and the presence of netrin-1 in the migratory route of these integrin bound to each other was analyzed following immunopre- interneurons in the developing cerebral cortex. cipitation with either anti-␣3 integrin or netrin-1 antibodies, re- ␣ ␣ ␣ spectively. 3 integrin co-immunoprecipitates with netrin-1 in these Netrin-1– 3 Integrin Interactions. To determine if 3 integrin and assays, thus further indicating a direct association between ␣3 netrin-1 interact with each other in the developing cerebral cortex, integrin and netrin-1 (Fig. 2C). we first performed co-immunoprecipitations of netrin-1 and ␣3 integrin. Immunoprecipitation of E13.5 forebrain lysates with anti- Netrin-1 Activates ␤1 Integrin Receptors. To establish whether the netrin-1 antibodies and immunoblotting with anti-␣3 integrin an- physical interaction between netrin-1 and ␣3␤1 integrin has func- tibodies or immunoprecipitation of lysates with anti-␣3 integrin tional relevance, we examined the effects of netrin-1 on ␤1 integrin antibodies and immunoblotting with anti-netrin-1 antibodies dem- receptor activation. E14.5 MGE cells were incubated with netrin- onstrates that ␣3 integrin co-immunoprecipitates with netrin-1 in 1–containing supernatant, recombinant mouse netrin-1 (50 ng/ml), vivo (Fig. 2A). Previously known ligands for ␣3 integrin (i.e., medium alone, or ECM (10 mg/ml). The medium-alone condition laminin-1) or other known receptors for netrin-1 (i.e., DCC) were serves as a negative control, and ECM stimulation functions as a ␤ co-immunoprecipitated in these assays and served as positive positive control for 1 integrin receptor activation. The activation ␣ of ␤1 integrin in netrin-1–treated MGE cells was assessed by controls (Fig. 2E). Netrin-1 or 3 integrin was not co- ␤ immunoprecipitated when ␣3 integrin-deficient or netrin-1– application of a 1 integrin antibody (9EG7), which specifically deficient forebrain extracts were used to co-immunoprecipitate recognizes its active conformation. Supernatants containing se- netrin-1 or ␣3 integrin, respectively (Fig. 2D). To verify further the creted netrin-1 or mouse recombinant netrin-1 markedly increased ␤1 integrin receptor activation in MGE cells when compared with interaction between ␣3 integrin and netrin-1, we performed co- controls (Fig. 2F), thus demonstrating a functional, physiologically immunoprecipitations of netrin-1 and ␣3 integrin using netrin-1– relevant interaction between netrin-1 and ␣3␤1 integrin. expressing 293 cells transfected with ␣3 integrin. Immunoprecipi- tation of these cell lysates with anti-netrin-1 antibodies and Disruption of Netrin-1–␣3␤1 Integrin Interactions Reduces the Num- immunoblotting with anti-␣3 integrin antibodies or immunopre- ber of Interneurons Migrating Through the Cortical Marginal Zone. In cipitation of lysates with anti-␣3 integrin antibodies and immuno- vitro observations suggest that retrin-1–modulated interneuro-

7596 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0811343106 Stanco et al. Downloaded by guest on September 26, 2021 nal migration from the MGE involves ␣3 integrin function (see Fig. S2 and SI Text). To evaluate the significance of netrin-1–␣3 integrin interactions during interneuronal migration in vivo, we generated interneuron-specific ␣3 integrin-deficient mice in a netrin-1–null background. Dlx5/6-Cre-IRES-EGFP (Dlx5/6- CIE) mice, in which interneurons generated from the ganglionic eminence express Cre and EGFP, were used to inactivate ␣3 integrin in interneurons. Generation of mice with a floxed and null Itga3 allele (␣3lox/Ϫ) in a netrin-1–null background, which also contain the Dlx5/6-Cre-IRES-EGFP transgene, enables the inactivation of netrin-1–␣3 integrin interactions in interneurons. Dlx5/6-Cre induces recombination throughout the GE and ven- tral telencephalon from E12.5 onwards (16), and loss of ␣3 integrin is evident in the interneurons of ␣3lox/ϪDlx5/6-CIE mice (Fig. S1). We compared the patterns of tangential interneuronal migration in double-mutant mice with ␣3 integrin (␣3lox/ϪDlx5/ 6-CIE, netrin-1ϩ/ϩ) or netrin-1 (␣3ϩ/ϩDlx5/6-CIE, netrin-1Ϫ/Ϫ) single-mutant mice and in control (␣3lox/ϩDlx5/6-CIE, netrin- 1ϩ/ϩ) mice. Migration patterns were evaluated first at E13.5, during early stages of cortical tangential migration, immediately following Dlx5/6-Cre–mediated inactivation of ␣3 integrin. We determined the extent of interneuronal migration in the dorsal telencephalon. Because tangential migration through the mar- Fig. 3. Real-time assessment of interneuronal migration patterns through the ␣ lox/Ϫ ginal zone precedes migration through the intermediate zone or cortical marginal zone reveals abnormal migration in 3 Dlx5/6-CIE, netrin- 1Ϫ/Ϫ double mutants. (A–D) Live imaging of E15.5 cortices reveals an increase in subventricular zone, we measured the extent of migration as the the number of interneurons transitioning from tangential migration along the distance from the corticostriatal junction to the tip of the leading marginal zone to ventricular surface-directed radial migration in ␣3lox/ϪDlx5/6- process of the foremost tangentially migrating neuron in the CIE, netrin-1Ϫ/Ϫ double mutants (B, cells highlighted in blue) compared with lox/Ϫ ϩ ϩ Ϫ cortical marginal zone. ␣3 integrin (␣3 Dlx5/6-CIE, netrin- ␣3lox/ Dlx5/6-CIE, netrin-1 / controls (A, cells highlighted in red). The number of NEUROSCIENCE ϩ ϩ ϩ ϩ Ϫ Ϫ 1 / ) and netrin-1 (a3 / Dlx5/6-Cre, netrin-1 / ) single mutants interneurons reversing their tangential migratory orientations in the marginal as well as double-mutant (a3lox/ϪDlx5/6-Cre, netrin-1Ϫ/Ϫ) mice zone region is increased in ␣3lox/ϪDlx5/6-CIE, netrin-1Ϫ/Ϫ double-mutant cortices displayed an 8% (SDϮ1.6), 9% (SDϮ1.7), and 10% (SDϮ1.3) (D, cells highlighted in blue) compared with controls (C, cells highlighted in red). reduction in the extent of migration through the marginal zone, (E) Quantification of the number of interneurons migrating toward the dorsal respectively, compared with controls (Fig. S3 A–E). Further, telencephalon in controls and double-mutant mice. (F–G) Quantification of the number of interneurons transitioning from tangential to radial migration (F) and binning analysis of interneurons across the developing cerebral reversing tangential trajectories (G) through the marginal zone region. Time wall (i.e., from pial to ventricular surface) indicates a 50% elapsed since the onset of observations is indicated in minutes. (Scale bar, 50 ␮m.) (SDϮ0.02) reduction in interneurons in the marginal zone of Data shown are mean Ϯ SEM (n ϭ 5); *, significant when compared with controls double mutants (Fig. S3 A–D and F-I). We also examined the at P Ͻ 0.05 (Student’s t test). ps, pial surface. number of interneurons migrating into the cortex by dividing the dorsal telencephalon, from the corticostriatal junction to the top of the dorsal telencephalon, into 10 equal bins and quantifying modulate distinct patterns of interneuronal migration within the interneurons in each of these bins. A significant decrease in the developing cerebral cortex, especially the interneuronal migration number of EGFPϩ interneurons migrating into the dorsal tel- through the marginal zone region of the developing cerebral cortex. encephalon in single- and double-mutant mice was evident (data Real-Time Assessment of Interneuronal Migration Patterns Through the not shown). These results suggest that netrin-1 and ␣3␤1 integrin ,Cortical Marginal Zone Reveals Abnormal Migration in ␣3lox/؊Dlx5/6-Cre may regulate very early stages of tangential neuronal migration ؊/؊ through the cortical marginal zone. Netrin-1 Double Mutants. To characterize further these altered To explore further the relevance of netrin-1–␣3 integrin inter- patterns of interneuronal migration in vivo, real-time imaging of actions to cortical tangential migration, we examined migration interneuron migration was performed on control and double- patterns at E16.5, at the height of interneuronal migration into the mutant E15.5 embryonic cortices. In the cerebral wall, the leading developing cerebral wall. Quantification of interneuron distribution processes of migrating interneurons are oriented toward the direc- within distinct compartments of the cerebral wall indicates that, tion of their movement. We found a significant decrease in the compared with control mice, ␣3lox/ϪDlx5/6-CIE, netrin-1Ϫ/Ϫ dou- number of interneurons migrating toward the dorsal cortex, away ϩ ␣ lox/Ϫ Ϫ/Ϫ ble-mutant mice exhibit a substantial reduction in EGFP inter- from the GE, in 3 Dlx5/6-CIE, netrin-1 double mutants ␣ lox/ϩ ϩ/Ϫ neurons migrating through the marginal zone region of the cortical when compared with 3 Dlx5/6-CIE, netrin-1 controls (con- Ϫ Ϫ Ϫ Ϯ ␣ lox/Ϫ Ϫ/Ϫ plate (control, 27% [SDϮ1.06]; ␣3lox/ Dlx5/6-CIE, netrin-1 / , trol, 41% [SD 2.3]; 3 Dlx5/6-CIE, netrin-1 , 34% 19% [SDϮ0.52]) (Fig. S4 A and C). Correspondingly, double [SDϮ2.7]) (Fig. 3E and Movies S1 and S2). Furthermore, com- mutants display an increased number of EGFPϩ cells ectopically pared with controls, a 2.7-fold increase in the number of interneu- migrating through the ventricular zone (control, 28% [SDϮ1.04]; rons switching from tangentially oriented migration along the ␣3lox/ϪDlx5/6-CIE, netrin-1Ϫ/Ϫ, 37% [SDϮ0.47]) (Fig. S4 B and D). marginal zone to ventricular surface–directed radial migration is Both ␣3 integrin and netrin-1 single-mutant mice exhibited an evident in double-mutant mice (Fig. 3 A, B, and F and Movies S3 increase in EGFPϩ interneurons ectopically placed in the ventric- and S4). This increase in ventricular surface-directed radial migra- ular zone (control, 28% [SDϮ1.04]; ␣3lox/ϪDlx5/6-CIE, netrin-1ϩ/ϩ, tion at the cortical marginal zone may underlie the reduction of Ϫ Ϫ Ϫ 32% [SDϮ0.58]; ␣3ϩ/ϩDlx5/6-CIE, netrin-1Ϫ/Ϫ, 34% [SDϮ0.91]) interneurons at the marginal zone of ␣3lox/ Dlx5/6-CIE, netrin-1 / (Fig. S4 B and D). The ratio of interneurons in the marginal zone double mutants. Last, double-mutant mice also displayed a 5.6-fold to those in the ventricular zone indicates the significance of this increase in the number of interneurons undergoing local reversals misplacement and reveals that a substantial number of interneurons in the orientation of tangential migration (i.e., interneurons migrate in netrin-1–␣3 integrin–deficient mice are misrouted (Fig. S4E). tangentially toward the dorsal cortex, then turn around and move These data suggest that netrin-1–␣3 integrin interactions may in the opposite direction) in the marginal zone region (Fig. 3 C, D,

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FGH I J J′ Fig. 4. Loss of netrin-1–␣3␤1 integrin interactions decreases the number and alters the positioning of interneurons in cortex at P0. (A–D, F–I, K–N) Compared with controls (A, F, K), ␣3 integrin mutants (B, G, L), or netrin-1 mutants (C, H, M), ␣3lox/ϪDlx5/6-CIE, netrin- 1Ϫ/Ϫ double-mutant mice (D, I, N), display a significant reduction in the number of EGFPϩ, calbindinϩ, and GABAϩ cells at the marginal zone (D, I, N, arrowhead) K L MNO O′ and throughout the P0 cortex (D, I, N, arrow). (E-EЈ, J-JЈ, O-OЈ) Quantification of the number of EGFPϩ, calbi- ndinϩ, and GABAϩ cells in the marginal zone (E, J, O) and cortex (EЈ,JЈ,OЈ). (P–S) No significant changes in the number or positioning of NPYϩ interneurons were no- ticed in the absence of netrin-1–␣3␤1 integrin interac- tions (S, arrow). (T-TЈ) Quantification of the number of P Q T′ NPYϩ cells in the marginal zone (T) and cortex (TЈ). Data R S T shown are mean Ϯ SEM; *, significant when compared with controls at P Ͻ 0.05 (one-way ANOVA, Tukey- Kramer posthoc test). cp, cortical plate; ; iz, intermedi- ate zone; mz, marginal zone; vz, ventricular zone. (Scale bar, 170 ␮minA–D, 105 ␮minF–I,65␮minK–N, and 100 ␮minP–S.)

and G; Movies S5 and S6). These results indicate that netrin-1–␣3 that observed in controls (Fig. 4 A, D, and EЈ). This reduction in the integrin interactions are required for interneurons to maintain number of interneurons in ␣3lox/ϪDlx5/6-CIE, netrin-1Ϫ/Ϫ double- appropriate, dorsally directed tangential migration along the cor- mutant cortices at P0 may in part be the result of increased tical marginal zone region. apoptosis of misplaced interneurons, because we detected an To investigate this deficit further, we performed a slice co-culture increase in cleaved caspase-3ϩ cells in double-mutant cortices assay in which E14.5 MGE explants from control or conditional ␣3 compared with controls (Fig. S6). The decrease in cortical inter- integrin-mutant mice were placed over the ganglionic eminences of neurons in double mutants is unlikely to be caused by reduced E14.5 wild-type or netrin-1–mutant coronal brain slices. Neuronal neurogenesis. We did not detect any significant changes in mitot- migration from the explants onto distinct domains of cortical ically active progenitors, as assessed by phospho-histone H3 label- substrates was assessed 72 h later. ␣3 integrin-deficient interneu- ing, in the GE ventricular zone of double-mutant forebrains com- rons displayed significant deficits in their ability to migrate onto pared with controls (Fig. S6). cortical slices in the absence of netrin-1 (Fig. S5 A, D, E, H, and I). To evaluate this disruption in interneuronal organization further, A 25% (SDϮ0.06) reduction in interneurons traversing the upper we immunolabeled subpopulations of interneurons with calbindin, cortical plate was noticed (Fig. S5 A, D, E, H, and J). Neuronal calretinin, GABA, somatostatin, neuropeptide Y (NPY), and dis- migration from wild-type explants onto netrin-1–deficient slices tal-less homeobox 2 (Dlx2) antibodies. The cerebral wall was was not affected (Fig. S5 A, C, E, G, and I). Together, these divided into 10 equal bins, and interneurons in each of these bins real-time interneuronal migration analyses and slice co-culture were quantified (Figs. 4 and Fig. S7). Selective changes in inter- ␣ assays suggest that netrin-1– 3 integrin signaling facilitates inter- neuronal positioning in the absence of netrin-1–␣3 integrin inter- neuronal migration in the developing cerebral cortex, specifically actions are evident. Strikingly, interneuronal placement in the the subset of interneurons migrating through the top of the cortical upper cortical plate is reduced consistently (Fig. 4 D, I, and N, plate. arrowhead). The number of calbindinϩ interneurons at the marginal Loss of Netrin-1–␣3␤1 Integrin Interactions Decrease the Number and zone (i.e., bin 10, Fig. S7) and throughout the cerebral wall were ␣ lox/Ϫ Alter the Positioning of Interneurons in Cerebral Cortex. To evaluate decreased by 58% and 66%, respectively, in 3 Dlx5/6-CIE, ␣ netrin-1Ϫ/Ϫ brains, compared with controls (Fig. 4 F, I, J, and JЈ, the outcome of disrupted netrin-1– 3 integrin interactions on ϩ interneuronal organization in vivo, we analyzed the positioning of arrowhead and arrow). Similarly, a substantial decrease in GABA Ϫ Ϫ Ϫ interneurons in the postnatal day 0 (P0) cerebral cortex, when active interneurons is evident in ␣3lox/ Dlx5/6-CIE, netrin-1 / double interneuronal migration comes to an end. Netrin-1–␣3 integrin mutants, particularly at the upper cortical plate region. The number ϩ double-null mice do not survive beyond P0. The migration deficits of GABA cells at the marginal zone and throughout the cerebral Ϫ observed in ␣3lox/ϪDlx5/6-CIE, netrin-1Ϫ/Ϫ double-mutant mice in cortex was reduced by 57% and 58%, respectively, in ␣3lox/ Dlx5/ Ϫ Ϫ vivo predict disruptions in the number and positioning of inter- 6-CIE, netrin-1 / brains, compared with controls (Fig. 4 K, N, O, neurons in the ␣3lox/ϪDlx5/6-CIE, netrin-1Ϫ/Ϫ postnatal cortex. and OЈ, arrowhead and arrow). Furthermore, similar reductions in Analysis of P0 cortices revealed that the number of EGFPϩ Dlx2ϩ interneurons of 40% and 35% were noticed at the marginal interneurons in the upper cortical plate region in double-mutant zone (Fig. S7) and throughout the cerebral cortex, respectively, in mice indeed was reduced by 64% compared with ␣3lox/ϩDlx5/6- ␣3lox/ϪDlx5/6-CIE, netrin-1Ϫ/Ϫ double mutants (Fig. S8 A and D–F). CIE, netrin-1ϩ/ϩ controls (Fig. 4 A, D, and E). Surprisingly, the total Calretininϩ interneurons in ␣3lox/ϪDlx5/6-CIE, netrin-1Ϫ/Ϫ brains number of EGFPϩ interneurons in ␣3lox/ϪDlx5/6-CIE, netrin-1Ϫ/Ϫ did not exhibit deficits at the upper cortical plate region but double-mutant cortices also was reduced substantially, to 45% of displayed 44% and 36% reductions in number at the ventricular

7598 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0811343106 Stanco et al. Downloaded by guest on September 26, 2021 AB CD Fig. 5. Disorganization and reduced numbers of hip- pocampal interneurons in ␣3lox/ϪDlx5/6-CIE, netrin-1Ϫ/Ϫ mutants at P0. (A–H) A reduction in the number and E F G H organization of EGFPϩ interneurons throughout the hip- pocampal subfields of ␣3lox/ϪDlx5/6-CIE, netrin-1Ϫ/Ϫ dou- ble mutants is evident (D and H, arrow) compared with controls (A and E). (Scale bar, 500 ␮m.)

surface and throughout the cerebral wall, respectively, compared CIE, netrin-1Ϫ/Ϫ double mutants (Fig. S4 A and C). Correspond- with controls (Fig. S7). No significant changes in the number or ingly, ␣3lox/ϪDlx5/6-CIE, netrin-1Ϫ/Ϫ double-mutant mice exhibited positioning of NPYϩ interneurons were observed in double mu- a significant increase in the number of interneurons aberrantly tants (Fig. 4 P–TЈ). Both the conditional ␣3 integrin single mutants migrating through the ventricular zone, a region largely devoid of and netrin-1 single mutants displayed deficits in the positioning of netrin-1 expression (Fig. S4 B and D). Therefore, in the absence of interneurons as well. ␣3 integrin single-mutant mice displayed a netrin-1–␣3␤1 integrin interactions, cortical interneurons may take 26% reduction in the number and ectopic positioning of calbindinϩ alternate, aberrant routes into the developing cortex. Real-time interneurons (Fig. 4 F, G, and JЈ). In netrin-1–deficient mice, a imaging analysis of tangential migration in E15.5 ␣3lox/ϪDlx5/6- ϩ general dispersal of calbindin interneurons in the cortical plate CIE, netrin-1Ϫ/Ϫ double-mutant mice reveals, at the marginal zone, ϩ was noticed (Fig. 4H). Somatostatin interneurons were detected an increase in tangential to radial transitions directed toward the only in the very lateral piriform cortex, where their numbers were ventricular zone (Fig. 3 B and F and Movie S4). This increase in Ϫ Ϫ Ϫ decreased in ␣3lox/ Dlx5/6-CIE, netrin-1 / double nulls (data not ventricular surface-directed radial migration at the cortical mar- shown). ginal zone provides an explanation for the decrease in the number Because the hippocampus represents the end point of long- of interneurons at the marginal zone of E16.5 ␣3lox/ϪDlx5/6-CIE, distance migration from the ventral telencephalon through the netrin-1Ϫ/Ϫ double-mutant mice and underscores the relevance of marginal zone region of the cerebral cortex (17, 18), analysis of netrin-1–␣3␤1 integrin interactions for appropriate, dorsal cortex interneuronal positioning in the hippocampal cortex provides an- directed tangential migration in this region. Furthermore, the NEUROSCIENCE other reliable measure of defects in long-distance interneuronal absence of netrin-1–␣3␤1 integrin interactions also disrupts the migration through the marginal zone region. We find a significant local migration of interneurons within the cortical plate (Fig. 3 D reduction in the number and organization of EGFPϩ interneurons Ϫ Ϫ Ϫ and G and Movie S6). It remains to be determined if the aberrant throughout the hippocampus in ␣3lox/ Dlx5/6-CIE, netrin-1 / dou- tangential to radial transition of interneurons in double mutants ble mutants compared with controls (Fig. 5 A, D, E, and H). In sum, affects the areal-specific distribution of interneurons in the cerebral these analyses of interneuronal organization indicate that netrin- cortex. By P0, a substantial change in the number and positioning ␣ 1– 3 integrin interactions are necessary for the appropriate migra- of interneurons within the marginal zone and cerebral wall is tion and positioning of distinct subsets of interneurons within the observed. No significant changes were evident in interneuronal developing cerebral cortex. progenitor proliferation in the GE (Fig. S6). Aberrant neuronal migration patterns resulting in abnormal neuronal positioning may Discussion lead to apoptosis of misplaced neurons. Consistently, we observed In this study, we provide genetic evidence for the significance of increased apoptosis in the developing cerebral cortex (Fig. S6). ␣ netrin-1– 3 integrin interactions in distinct aspects of interneuronal In addition, the presence of a large ectopic aggregation of ␣ ␤ migration in the developing cerebral cortex. 3 1 integrin is interneurons in the ventral telencephalon (data not shown) expressed by tangentially migrating interneurons, and netrin-1 is suggests that apoptosis and misrouting of cortical interneurons present along their migratory route. In vitro, netrin-1 activates ␤1 ␣ ␤ ␣ ␤ in the absence of netrin-1– 3 1 integrin interactions may con- integrin and modulates the migration of interneurons in an 3 1 tribute to the deficiency in the number of cortical interneurons integrin signaling-dependent manner. In vivo, interneuron-specific ␣ ␤ in double-mutant mice. Together, these data suggest that netrin- 3 1 integrin, netrin-1–deficient mice display deficits in the mi- 1–␣3␤1 integrin interactions are necessary for interneurons to gration of interneurons in the upper cortical plate region. Signifi- sustain directed tangential migration along the upper cortical cant and selective disruptions in the number and positioning of plate and for the appropriate positioning of interneurons in the subsets of interneurons, especially calbindinϩ,GABAϩ,Dlx2ϩ, and ϩ ϩ developing telencephalon. calretinin interneurons, but not NPY interneurons, were ob- Clearly, netrin-1 and ␣3␤1 integrin do not have an exclusive served in the absence of netrin-1–␣3␤1 integrin interactions. Thus, ␣ ␤ ligand-receptor relationship and the less severe deficits seen in netrin-1– 3 1 integrin interactions guide the appropriate naviga- single mutants are suggestive of redundancies in these interactions tion of subsets of GABAergic interneuronal populations through (see SI Text). Furthermore, how the loss of netrin1-␣3␤1 integrin the developing cerebral wall and are necessary for the proper interactions may lead to non-cell-autonomous changes in other placement of interneurons in the cerebral cortex. signaling pathways (e.g., CXCL12/CXCR4; 19, 20) capable of Altered Migration and Positioning of Interneurons in the Absence of influencing interneuronal also needs to be deciphered. Netrin-␣3␤1 Integrin Signaling. The heterogeneity of cortical inter- Deficits in the timing of cortical plate invasion by GABAergic neuron origins and the differential distribution of interneuron interneurons results in aberrant laminar and regional distribution of subtypes in the cerebral cortex suggest that cotical interneuron cortical interneurons. Specifically, disrupted CXCL12/CXCR4 sig- subtypes may take distinct migratory routes into the developing naling results in premature migration of GABAergic interneurons cerebral cortex (see SI Text). Our data show that netrin-1–␣3 into the cortical plate and, consequently, aberrant laminar and integrin interactions modulate interneuronal migration through the regional positioning of cortical interneurons (19, 20). Furthermore, cortical marginal zone from the beginning at E13.5. Deficits in this GABAergic interneurons migrating through the marginal zone, migration become progressively more severe in double-mutant including calbindinϩ interneurons, exhibit slow multidirectional mice. By E16.5, the migration of GABAergic interneurons through movement, a local positioning phase, before descent and integra- the upper cortical plate is significantly retarded in ␣3lox/ϪDlx5/6- tion into the cortical plate (21, 22). Disruption of netrin-1–␣3␤1

Stanco et al. PNAS ͉ May 5, 2009 ͉ vol. 106 ͉ no. 18 ͉ 7599 Downloaded by guest on September 26, 2021 integrin–mediated cell migration through the cortical marginal terneuron subtype development. Our study demonstrates that the zone may lead to temporal and spatial alterations in the local differential regulation of interneuronal subtype migration by di- positioning phase of interneurons and subsequent deficits in the rectional guidance molecules, such as netrin-1, ultimately may integration of interneurons within the cortical plate. Indeed, the modulate the placement and connectivity of interneuronal sub- calbindinϩ cell distribution of ␣3lox/ϪDlx5/6-CIE, netrin-1Ϫ/Ϫ dou- populations in distinct regions of the cerebral cortex and contribute ble-mutant mice is altered significantly compared with controls, to normal cortical function. indicating that netrin-1–␣3␤1 integrin interactions at the cortical marginal zone may modulate the laminar positioning of calbindinϩ Materials and Methods interneurons within the cortical plate. In contrast to the established Mutant Mouse Strains. The generation and characterization of ␣3 integrin ‘‘inside-out’’ gradient of cortical plate invasion, calretininϩ inter- floxed alleles is described in Liu et al. (35). See SI Text for details on the ␣ lox/Ϫ Ϫ/Ϫ neurons populate the cortex in an ‘‘outside-in’’ gradient (30). generation and characterization of 3 Dlx5/6-CIE, netrin-1 double mu- Therefore, netrin-1–␣3␤1 integrin interactions may differentially tants and related controls. ϩ influence the guidance of calretinin interneurons into the cortical Quantification of Interneuronal Cell Distribution in Cerebral Cortex. Interneu- plate compared with other interneuron subpopulations. The re- ronal cell counts in the E16.5 and P0 cortex were performed in sections duction of calretininϩ interneurons at the ventricular zone instead corresponding to the somatosensory cortex as described previously (36). See SI of the marginal zone, in the absence of netrin-1–␣3␤1 integrin Text for details. ϩ interactions, supports this idea. Furthermore, the calbindin and ␤1 Activation Assay. Netrin-1–mediated ␤1-integrin activation in MGE cells was ϩ calretinin cell distribution is affected more severely than the assayed using methods described in SI Text. NPYϩ cell distribution of ␣3lox/ϪDlx5/6-CIE, netrin-1Ϫ/Ϫ double- mutant mice, suggesting that netrin-1–␣3␤1 integrin interactions Explant Co-cultures. Explant co-cultures were prepared as described previously (14). For details, see SI Text. influence the migration and placement of specific interneuronal ϩ subpopulations. Whether the reduced expression of Dlx2 inter- Cortical Slice-MGE Explant Co-cultures. Slice co-cultures were prepared as de- neurons in the cerebral wall also is indicative of potential changes scribed previously, with minor alterations (3). For details, see SI Text. ␣ lox/Ϫ Ϫ/Ϫ in oligodendrocyte cell fate in 3 Dlx5/6-CIE, netrin-1 dou- Real-Time Imaging. Coronal slices (200 ␮m) of E15.5 ␣3lox/ϩDlx5/6-CIE, netrin-1ϩ/Ϫ ble-mutant mice remains to be determined (31). control and littermate ␣3lox/ϪDlx5/6-CIE, netrin-1Ϫ/Ϫ double-mutant brains were Disruptions in the placement and development of cortical inter- imaged repeatedly and analyzed as described earlier (4). For details, see SI Text. neuron subtypes may underlie the pathogenesis of neurodevelop- Immunohistochemical, immunoprecipitation, and immunoblotting methods mental disorders such as epilepsy, schizophrenia, and autism (see are described in the SI Text. SI Text ). This emerging evidence for interneuronal subtype–specific ACKNOWLEDGMENTS. We thank P. Maness and A. Lamantia for helpful com- dysfunction in a variety of neurodevelopmental disorders highlights ments. This research was supported by National Institutes of Health Grant the importance of understanding the mechanisms underlying in- MH060929 and by a Staglin Award from NARSAD to E.A.

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