Ab initio calculations demonstrate that both Ni vacancy formation and migration energies at the coherent Ni/α-Al2O3 interface are much smaller than in bulk Ni, suggesting that the activation energy for diffusion along coherent Ni/α-Al2O3 interfaces is comparable to that along (incoherent/high angle) grain boundaries. Based on these results, we develop a simple model for diffusion along metal/ceramic interfaces, apply it to a wide range of metal/ceramic systems and validate it with several ab initio calculations. These results suggest that fast metal diffusion along metal/ceramic interfaces should be common, but is not universal.Interface diffusion along a metal/ceramic interface present in numerous energy and electronic devices can critically affect their performance and stability. Hole formation in a polycrystalline Ni film on an  Little is known about diffusion along metal/ceramic interfaces even though it controls the physical behavior and lifetimes of many devices . Here, the authors show that diffusion along a nickel/sapphire interface is abnormally fast due to nickel vacancies and generalise their findings to a wide-range of metal/ceramic systems. Device performance thus directly depends on the integrity of these metal/ceramic interfaces . For example, it was shown that the kinetics of electromigration-induced Cu precipitation and dissolution in Al-Cu bamboo-structure interconnects deposited on a TiN barrier layer is controlled by Cu diffusion along the metal/TiN interface4. The morphological evolution of thin passivated metal films is also commonly controlled by metal diffusion along the metal-passivation layer interface 6. These examples demonstrate that rational device design demands an improved understanding of transport along metal/ceramic interfaces7. While an extensive literature exists on the mechanical strength8, atomic structure9, and chemical composition10 of metal/ceramic interfaces, little is known about atomic diffusion along this channel. Diffusion along extended crystal defects ) is commonly much more rapid than bulk diffusion12. The widely-quoted hierarchy of diffusivities is Dbulk ≤ Ddislocation ≤ DGB ≤ Dsurface14. Here, we focus on where metal/ceramic interfaces fall within this hierarchy, to discover the features that control such diffusion, and use these to predict its magnitude.Metal/ceramic interfaces are ubiquitous building blocks for a wide range of technologies, from semiconductor devices 15 and metal/semiconductor16 interfaces have been made. We are unaware of any such direct measurements of metal diffusion along metal/ceramic interfaces. However, there is indirect evidence to suggest that metal/ceramic interfaces may be high-diffusivity paths for metal atoms. For example, Gan et al.17 showed that Cu diffusion along the /Cu interface is faster than bulk diffusion in Cu. They extracted the interface diffusivities from the kinetics of stress relaxation in the Cu films, making their results indirect and strongly model-dependent. Arnaud et al.18 demonstrated that Cu diffusion along a Cu/SiN interface plays a prominent role in electromigration and estimated the activation energy of this interface diffusion. Recently, indirect evidence of fast diffusion along metal/ceramic interfaces was found in the solid-state dewetting of metal films on oxide substrates20. It is reasonable to conjecture that diffusion along metal/ceramic interfaces will be comparable to that along other internal interfaces . Yet, metal–oxygen bonds (in oxide ceramics) may be much stronger than those between metal atoms, suggesting rather that diffusion along metal/ceramic interfaces may be suppressed . Fast diffusion along GBs is usually attributed to their lower density or higher free volume (relative to grain interiors); a concept supported by the observation that diffusion along coherent twin boundaries appears no faster than in the bulk21. In fact, Chen et al.22 showed that coherent twin boundaries slowed electromigration in Cu. Nonetheless, there is evidence that diffusion along coherent (and semicoherent) metal/ceramic interfaces is rapid24. This seems contrary to the association of fast transport with low atomic density.Ideally, bicrystal samples should be used to directly measure interface diffusivities; a diffusant is deposited on a bicrystal surface, the sample is annealed, and the composition profile is measured, leading to the determination of the interface diffusion coefficient. Only a very small set of measurements for metal/metalα-Al2O3 (sapphire) interface. We explore the origin of this effect as a function of environment  using ab initio calculations of point defects within these materials and near their interface. We establish that in this case, the dominant defects for interface transport are Ni vacancies and that the diffusivity of Ni along the interface is anomalously fast. We then present a simple model that generalizes these results to a very wide range of metal/ceramic interface systems and validate the model through examination of additional metal/ceramic interface systems.In this work, we report experimental evidence of fast diffusion along the Ni/25  surface of sapphire. All Ni grains have see Fig. .Fig. 1Ex−2 μm3, while the volume of the material removed to form the hole is ~2X larger. The “sunken” grain indicated by the magenta arrow in Fig. 26, all of the material removed around the GB is transported away from the GB into the ridge by surface diffusion. We now consider where the missing Ni atoms go and how these holes form.The presence of isolated holes, not connected to GBs, is a clear indication that isolated/embedded grains see Fig.  sink and26). For this process to continue, Ni diffusion along the Ni/sapphire interface must be rapid. In this case, the film surrounding the sunken grain must thicken by the accretion of Ni atoms at the Ni/sapphire interface25. Similar homogeneous thickening of a metal film associated with material redistribution along the metal/ceramic interface was recently reported in Al/sapphire23.We suggest that the missing Ni in the grooving/hole formation, diffuses down the GB and then along the Ni/sapphire interface see Fig. . The matR0 . These experimental observations yield Di ≈ Dgb (for a random large angle GB in Ni27) as shown in Fig. We analyze the experimental case of Fig. This interface diffusivity is surprisingly large given that this metal/ceramic interface is nearly coherent and the strong bonding between this metal and ceramic. We now investigate the cause of this large interface diffusivity and discuss whether it is a generic finding for all metal/ceramic interfaces. 28 using a combination of first-principles and classical methods for Ag/MgO. More recently, there have been studies of oxygen vacancy formation energy30 and oxygen Frenkel pair31 formation energies at metal/oxide interfaces and oxide/oxide interfaces33. However, most such studies of metal/ceramic interfaces focused on the oxide side of the interface and on the electronic behavior of the contact rather than diffusion—the primary focus of the present work. There have also been no reports on point defect migration along the interface. Here, we carry out a systematic investigation of point defect energetics near the Ni/sapphire interface as well as diffusion along it.To understand fast transport along the Ni/sapphire interface, we first focus on point defect formation energetics using density functional theory (DFT) (to account for the complex bonding at the interface). One of the earliest works that looked at the point defect formation energy near a metal/ceramic interface was carried out by Duffy et al.2O3(0001)2O3(0001)35 35. Perfect interface coherency demands that Ni is biaxially strained −2.8% for M1 and +12.3% for M2. We focus on the less strained M1 interface which is the only observed variant in our experiments (the M2 interface will have a much higher interface energy associated with a high density of misfit dislocations). The O-terminated Al2O3/Ni interface is shown in Fig. 2O3 may also be terminated by one or two Al atom planes. Examination of Fig. 2O3 interfaces will be thermodynamically stable  plane stacking of FCC materials —these 3 terminations also represent Ni crystal shifts parallel to the interface entclass1pt{minimaentclass1pt{minima37, we determine vacancy formation energies in Ni and α-Al2O3 as a function of distance d ; 40) or 1.25 eV . Similarly, since Schottky defects are more prevalent than Frenkel defects in α-Al2O342, we focus on Al and O vacancies on the sapphire side of the interface. For bulk sapphire, the neutral vacancy formation energies depend on oxygen partial pressure; on oxygen sites We first calculate neutral vacancy formation energies in bulk Ni and d from the O-terminated sapphire and 2Al-terminated sapphire/C-plane Ni interfaces. For the O-terminated sapphire interface, the Ni vacancy formation energy α-Al2O3 interfaces, implying the thermal concentration of Ni vacancies near the interface is much higher than elsewhere in Ni.Figure 29 and /HfO230 interfaces show a similar trend of decreasing O vacancy formation energy on approaching the interface. Similarly, looking at the Al vacancy in the O-terminated case, 43, even at the maximum separation from the interface in our computational cell (~6 Å).Figure While the lowest formation energy point defect near the interface is the Ni vacancy D0 accounts for crystal structure, the effective atomic vibration frequency, interatomic separation, correlation and entropy effects, kBT is the thermal energy, 44. The arrhenius terms describe the equilibrium vacancy concentration and vacancy migration, respectively. While in ceramics, the point defect density may be modified by doping, in metals it is usually dictated by equilibrium thermodynamics (vacancies are easily produced/annihilated by dislocation climb).Vacancy defect-mediated diffusion is commonly characterized as 45, we determine the barrier for Ni vacancy migration  with cohesive energy . Combining the vacancy formation and migration results reported here, these results suggest that at, for example, half the Ni melting point, the interface diffusivity should be ≥104 times faster than in bulk Ni. GB diffusivities in metals are typically (104–106) faster than lattice diffusion14, which implies that metal/ceramic interface diffusivity and GB diffusivities are comparable at the same homologous temperature. Therefore, these results demonstrate that Ni transport along the Ni/sapphire interface is extraordinarily fast (relative to bulk diffusion). This conclusion is valid for both the coherent interface case analyzed here in detail and the case where the interface is semicoherent (the misfit dislocations in semicoherent interfaces are short-circuit diffusion paths).Using nudged elastic band calculationsWad and the metal surface energy γm. We estimate the ratio of the metal vacancy formation energy at the metal/ceramic interface to that in the bulk metal in terms of a simple, heuristic bond breaking model , FCC-Cu/α-Al2O3 and HCP-Ti/α-Al2O3. As shown in Table Wad/γm) accurately predicts the ratio of To test the applicability of this simple prediction, we compare it with the DFT results for FCC-Ni/mentclass2pt{minim52. The resultant predictions are summarized in Fig. 2O3 (and Cu/Al2O3). For systems with 2O3) and for others 2O3, for which we have DFT data, is near the cusp—the interface diffusivity should be comparable with bulk diffusion in Ti. Amongst the cases shown in Fig. 2O3 and that {Au, Cu, Sn, Ag, Fe, Co, Ga}/SiO2 interfaces all show We use the obtained descriptor to predict the ratio of the vacancy formation energy at the metal/ceramic interface to its value in the bulk metal for a wide range of metal/ceramic systems based upon Eq.  and usinWe thus show both experimentally and computationally that diffusion at the Ni/sapphire interface is surprisingly fast. Our first-principles vacancy formation and migration energy calculations demonstrate that this is a result of relatively low cohesion at this interface compared with bulk Ni. This observation suggests a simple descriptor for diffusion at the interface compared with the bulk based upon readily available experimental and/or first-principles results. Based on this descriptor, we conclude that for most metal/ceramic systems , interface diffusion is fast compared with the bulk and comparable with metal grain boundary diffusivities in many cases; yet there are exceptions (as determined based on interface cohesion). Systems where the metal only weakly wets (or does not wet) the ceramic, the interface diffusivity will be high; inversely, where the tendency for wetting is strong, the interface diffusivity will be low . This suggests that alloying to modify wettability also affects interface diffusion kinetics. This simple result provides easily applicable guidance for material design in a wide range of applications; especially in the energy and microelectronics industries.α-Al2O3 (sapphire) single crystal substrates. Prior to deposition, the substrates were prepared in a clean room with soap, deionized water, acetone, methanol, isopropanol, followed by dipping in Piranha solution  and deionized water. The films were deposited at a rate of 1 Å s−1 at room temperature and a vacuum of 7 × 10−7 torr. The deposition resulted in a maze-bicrystal microstructure. The samples were then annealed for 10 min at 700 °C in forming gas flow  in a rapid thermal annealing (RTA) furnace.45 nm thick Ni films (5N purity) were deposited by electron beam deposition on (0001) c-plane-oriented The morphology of the sample was characterized by atomic force microscopy  in tapping mode using a Si tip with a typical radius of curvature of 10 nm. The scan rate was 0.2–0.7 Hz.53. The method is based on surface discretization and a variational calculation of surface chemical potentials first proposed by Dornel et al.54 for a one-dimensional surface. A detailed description of the formulation of the model specifically for these applications is presented in the Supplementary Method The evolution of the surface topography was modeled employing the axisymmetric variational scheme described in detail in ref. 58. The core electrons were described using the Projector Augmented Wave (PAW) scheme60 and exchange-correlation was described using the recently developed meta-GGA functional SCAN61, which was shown to perform better than PBE-GGA62 for diversely bonded systems. The energy cut-off for the plane wave expansion was 520 eV for all calculations except those performed on pure metallic systems of Ni, Cu, and Ti where the cut-off was 350 eV.All DFT calculations were performed using the Vienna Ab initio Simulations Package (VASP)63 K-mesh was used which was 9 × 9 × 3 for Ni and Cu, and 12 × 12 × 3 for Ti. The supercell size for Ni and Cu was 24 atoms while for Ti, a supercell of 18 atoms was used. The vacancy migration barrier calculations in pure Ni were performed using the climbing image-nudged elastic band approach45 in a supercell containing 47 atoms (containing a Ni vacancy), previously relaxed using a 3 × 9 × 3 K-mesh (Monkhorst-Pack). The bulk vacancy formation and migration barriers calculations were performed in supercells strained to match that of the metal in the metal/ceramic interface supercells. The 30-atom sapphire supercell (6 α-Al2O3 formula units) was volume relaxed . The O-terminated and 2Al-terminated sapphire were created by sequentially removing an Al-plane and an O-plane respectively. 36 Cu atoms and 21 Ti atoms were used for the Cu/sapphire and the Ti/sapphire interfaces respectively, with the same number of atoms on the sapphire side as in the Ni/sapphire case. For the Ti/sapphire interface, the bicrystallography determined from experiments64 was used. For all these interface systems, a Γ centered K-mesh of 9 × 9 × 1 was used.The Ni/sapphire supercell consisted of 6 b direction (Supplementary Fig. α-Al2O3, a supercell of 120 atoms was used with a a Γ centered K-mesh of 4 × 4 × 2.For the Ni/sapphire migration barrier calculations, the supercell was two times larger in the Supplementary InformationPeer Review File


We demonstrate acoustically triggered giant deformation of soft materials, and reveal the snap-through instability and bifurcation behavior of soft materials in nonlinear deformation regime in response to combined loading of mechanical and acoustic radiation forces. Our theoretical results suggest that acoustomechanical instability and bifurcation can be readily modulated by varying either the mechanical or acoustic force. This modulation functionality arises from the sensitivity of acoustic wave propagation to nonlinear deformation of soft material, particularly to ratio of initial geometrical size of soft material to acoustic wavelength in the material. The tunable acoustomechanical instability and bifurcation behavior of soft materials enables innovative design of programmable mechanical metamaterials. PACS numbers: 43.35.+d, 43.25.+y, 46.70.De, 61.41.+e. Particular focus has been placed upon developing soft materials with programmable behaviors, which often show non-monotonic and discontinuous responses or instabilities7. Nonetheless, a remarkable limitation to realize these programmable behaviors is that architected microstructures with beam elements are typically required.Nonlinear deformation of soft materials in response to various external stimuli is often accompanied with elastic stability and bifurcation phenomena, which are traditionally averted for they represent mechanical failure. Recently, however, there is arising interest in harnessing such elastic instability and bifurcation to enable new functionalities of soft materials, such as triggering giant deformation11. The acoustic radiation force is interpreted as a time-averaged steady force, which is generated when high-frequency acoustic wave such as ultrasound propagates in a nonlinear medium14. Harnessing the sensitivity of acoustic radiation force to material configuration, we demonstrate first the occurrence of acoustomechanical instability and bifurcation of soft material when it is subjected to combined mechanical and acoustical loads, and then realize their modulation via programmable mechanical force and acoustic input, respectively. The fast and non-contact modulation of acoustic input enables instantaneous and highly controllable nonlinear behavior of soft materials, with promising applications in medical devices, microfluidic actuators, adaptive robots, and so like.We address this deficiency by demonstrating a novel strategy for tunable instability and bifurcation of homogeneous and isotropic soft materials by manipulating the acoustic radiation forces20P is acoustic pressure, ρa medium density, ca acoustic speed in medium, ui, uj and uk velocity components, and 〈⋅〉 denotes time-average manipulation over an oscillation cycle. Therefore, the acoustical radiation stress is scaled as p0 being amplitude of input sound pressure. Commonly, as a focused acoustic pressure lies between 0.1 and 4 MPa, the corresponding acoustic radiation stress ranges from 70247 Pa to 112 MPa in air, and from 4.44 Pa to 7111 Pa in water. If one note that the elastic modulus of soft materials is generally several times kPa, the acoustic radiation stress is lager enough to cause material deformation: even large nonlinear deformation is possible24.The propagation of ultrasonic wave from surrounding medium into a soft material generates acoustic radiation force due to acoustic momentum transfer inside the medium and material as well as at the interface between the two, which manifests in the form of a second-rank stress tensor if we consider a micro-cubic element of the material. This acoustic radiation stress tensor can be expressed asρ1c1 and ρ2c2, respectively. The thickness of the sheet is considered comparable to the wavelength of acoustic wave propagating in the sheet, while its in-plane dimensions are much larger than sheet thickness. Representative dimensions of such a sheet may be ~5 mm × 100 mm × 100 mm. We further assume that the material is nearly incompressible. Along the thickness direction, two counterpropagating acoustic waves p = p0ej(k⋅x−ωt)−  strike the thin sheet, governed by momentum equation ∇⋅σ = ρ∂2u/∂t2 in Eulerian coordinates, σ being Cauchy stress tensor. The propagation of acoustic waves with wavelength Λ = 2πca/ω in the sheet induces acoustic radiation forces, causing it to deform from reference state  of Fig. l1, l2, l3) of Fig. To be more specific, we consider in Fig. W(F) is the Helmholtz free energy corresponding to nonlinear elastic deformation of material, which is a symmetric function of deformation gradient F(≡∂x/∂X). If we consider the Cauchy stress as external mechanical stress, this equation actually gives the force balance condition among the external mechanical Cauchy stress, elastic deformation Cauchy stress and acoustic radiation Cauchy stress. Although it looks no explicit coupling term between the elastic deformation stress and acoustic radiation stress in Eq. , and J = det(F). When Jm → ∞, the Gent model becomes the classical neo-Hookean model. Under symmetric acoustic fields  and exert a mechanical force f in the y-direction, as shown schematically in Fig. f fixed or altering f with acoustic wave input fixed.We further constrain the soft material sheet to be undeformed in the J = det(F) = 1 is fully enforced. For an incompressible soft material, the relationship between normalized mechanical force fn ≡ f/(μL1L3) and principal stretches  can be obtained by applying the constitutive relation of Eq.  at the specified acoustic input l2/Λ (or λ2) and the out-plane deformed dimension l3/Λ (or λ3), respectively in Figs To be more specific, the mechanical response of the soft material to the mechanical force x-axis), these intersection points can be regarded as the initial state of each acoustical loading process. Without acoustical loading, the soft material can hold at a steady deformed state due to the stretch of the pre-mechanical force. From these initial states with different fixed pre-mechanical forces, each acoustic input-stretch curve tracks different acoustical loading path with the increase of the acoustic input. If we plot a horizontal line for any given acoustic input, this line will have multiple intersection points with the acoustic input-stretch curve with a fixed pre-mechanical force. These multiple intersection points manifest that there exist multiple steady states with different deformed configuration for a fixed pre-mechanical force. This is because for different deformed configurations, the ultrasonic wave propagation can generate different acoustic fields and the corresponding different acoustic radiation stresses, these different acoustic radiation stresses accompanying with the fixed mechanical stress/force are just in balance with the elastic deformation stresses in these multiple deformed configurations. Furthermore, it is found from Fig. As shown in Fig. f/(μL1L3) = −0.9 is solely selected to plot as a function of the in-plane deformed dimension l2/Λ (or λ2) and the out-plane deformed dimension l3/Λ (or λ3), respectively in Fig. To be more specific, the acoustical response of the soft material to the acoustic input L3/Λ, we further reveal the modulated bifurcation behavior of soft material in mechanical response by exerting different acoustic inputs  of inputting acoustic waves. As shown in Fig. Harnessing the sensitivity of acoustic wave propagation to normalized initial sheet thickness uts Fig. . The ratL3/Λ, the dimension of soft material jumps discontinuously from point A to point B, accompanied by a sudden and large alteration of sheet stretching, which signifies the occurrence of snap-through instability.The branch of L3/Λ is treated as the sole loading parameter and each curve in these diagrams represents the load-stretch response or loading history. Each diagram of Fig. f/(μL1L3) = −1, −2, −3, −4, −5, implying mechanical force modulated load-stretch response. In Fig. f/(μL1L3) = −1 is individually plotted to reveal the appearance of snap-through instability when L3/Λ is gradually increased. The deformation state discontinuously jumps from point A to point B not only in the  parameter space but also in the  stretch space. This snap-through jumping realizes the direct switch from one equilibrium sate to another equilibrium state, striding over the unstable state between the two.Similar to acoustic modulation, we demonstrate that the bifurcation behavior of soft material can be modulated by varying the mechanical force when prescribing the acoustic input. Figure In summary, we demonstrate the snap-through instability and bifurcation behavior of homogeneous, isotropic soft material sheets in response to not only mechanical load but also acoustic load. Snap-through instability can even show up in bifurcation branches when the ratio of initial sheet thickness to wavelength is taken as a sole loading parameter. We further uncover the feasibility of convenient modulation of acoustomechanical instability and bifurcation by altering either the mechanical or acoustic load. This new functionality of soft material enables pre-programmable response of soft material by varying the pre-mechanical force and instant-programmable response via adjusting the frequency and amplitude of acoustic input. This work would inspire innovative designs of soft actuators, microfluidic devices, adaptive robots, etc. by harnessing the tunable and programmable acoustomechanical behavior of soft materials.Supplementary material


Pcdha) gene cluster regulates neuronal migration during cortical development and cytoskeletal dynamics in primary cortical culture through the WAVE  complex. In addition, overexpression of proline-rich tyrosine kinase 2 , a non-receptor cell-adhesion kinase and scaffold protein downstream of Pcdhα, impairs cortical neuron migration via inactivation of the small GTPase Rac1. Thus, we define a molecular Pcdhα/WAVE/Pyk2/Rac1 axis from protocadherin cell-surface receptors to actin cytoskeletal dynamics in cortical neuron migration and dendrite morphogenesis in mouse brain.Diverse clustered protocadherins are thought to function in neurite morphogenesis and neuronal connectivity in the brain. Here, we report that the protocadherin alpha ( There are hundreds of billions of neurons in a human brain, and each one can form several thousand connections with other neurons. This complex network determines our thoughts, memories, personality, and behavior, but how does it form? During brain development, specific areas give rise to new neurons, which then migrate long distances to other parts of the brain. Upon arrival, they generate several structures, called dendrites, which connect with other neurons.To distribute themselves correctly, the migrating immature neurons must be able to travel long distances and steer clear of one another. The dendrites from a single mature neuron must also avoid each other, a phenomenon known as self-avoidance. Certain membrane-spanning proteins, called clustered protocadherins, may help neurons achieve this. The portion of the protocadherins that sits on the cell surface is highly variable, and acts as a zipcode that helps cells to recognize one another. However, the section of the protein inside the cell varies little and is shared by all members of a protocadherin family. When the clustered protocadherin is ‘switched on’, this internal segment can trigger a cascade of reactions that create changes in the cell. Yet, little was known about the nature of this signaling cascade.Using gene editing in mice, Fan, Lu et al. focus on the signaling cascade of the clustered protocadherin alpha family. The experiments show that the internal portion of these proteins interacts with a protein complex called WAVE. It also inhibits an enzyme known as Pyk2, which increases the activity of another enzyme called Rac1 GTPase, that then further activates WAVE. This results in the WAVE complex also interacting with the internal skeleton inside the neurons and dendrites, which regulates the ability of these cells to migrate and of the dendrites to avoid each other.Many brain conditions, such as autism spectrum disorders or depression, result from abnormal neuronal migration and connectivity. Mutations in the genes of clustered protocadherins increase the risk of these disorders. By showing how these proteins help to regulate the migration and connectivity of neurons, Fan, Lu et al. add to our understanding of brain development in health and disease. These complex neural circuits are required for normal brain function, and inappropriate assemblies of neural circuits underlie neurodevelopmental and neuropsychiatric disorders  cell adhesion genes in the 5q31 region for various developmental and psychiatric disorders   . The Pcdclusters . In partnhancers  , recruit WAVE complex to the plasma membrane  motif of Pcdhα constant domain (CD). In addition, Pyk2 regulates cortical neuron migration by inactivating the small GTPase Rac1. Given that actin cytoskeletal dynamics are central for neurite morphogenesis and neuronal migration, our findings have interesting implications for mechanisms of Pcdhα by using a GFP knockin mouse line (PcdhαGFP) (Pcdhα cluster is expressed in the developing brain (We mapped the embryonic expression pattern of cdhαGFP)  and founcdhαGFP) . ImmunoscdhαGFP) . RT-PCR ng brain . Pcdhα kng brain . The αKDng brain . Live ceng brain . In adding brain . This su+ cells (+intermediate progenitor cells (IPCs) (Pcdhα cluster (αKO)  , nor thes (IPCs) . Moreoves (IPCs) . Finallys (IPCs)  in mice er (αKO) . The pheer (αKO) .To rescue the migration defect, we constructed shRNA-resistant forms of α6 (α6*), which represents members of the alternate α1-α12, and of the two divergent c-types (αc1* and αc2*) . Indeed To investigate whether the extracellular domain and transmembrane segment play a role in cortical neuron migration, we replaced them with a myristoylation signal to attach the shRNA-resistant intracellular domain (ICD) to the plasma membrane  . We founWAVE2 (Wasf2) or Abi2 in vivo rescues the cortical neuron migration defect of αKD neurons (Recent studies linked Pcdhα6 to the WAVE complex through the WIRS (WAVE interacting receptor sequence) motif within the Pcdhα constant domain . We thus neurons  although neurons . Consist neurons . In addi neurons . As cont neurons . Thus, PPyk2 (Ptk2b) knockdown (Pyk2KD) per se or CRISPR knockout of Pyk2 (Pyk2KO) does not affect cortical neuron migration  could recapitulate αKD cortical neuron migration defects. We found that the majority of Pyk2OE cells are stalled in the middle intermediate zone (mIZ) , a stageWe next examined the orientation of the Golgi apparatus of cells in mIZ, which is essential for transporting vesicles for oriented motility , by immuTo rule out the potential nonspecific effect of the CAG promoter, which is active in both progenitors and postmitotic neurons, we ectopically overexpressed Pyk2 at E15.5 only in postmitotic neurons using the NeuroD promoter . We founQ61L) in Pyk2OE neurons. We found that Rac1Q61L rescues defects of multipolar migration and morphology of Pyk2OE neurons  impairs cortical neuron migration  domain  Chen et. We systs of αKD . Howeverase-dead , cannot ase-dead . This suRemarkably, overexpression of the Pyk2 FERM domain alone recapitulates the blocking activity of Pyk2OE , whereasQ61L rescues the blocking effect of Pyk2OE  light and 12 hr (19:00–7:00) dark schedule. The day of vaginal plug was considered to be embryonic day 0.5 (E0.5). All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of the Shanghai Jiao Tong University.The Pcdhαescribed . Pyk2KO Y402F were generated by using CRISPR/Cas9. Briefly, sgRNA scaffold sequences were constructed in the pLKO.1 plasmid. The construct was then used as template for amplifying a PCR product containing T7 promoter and sgRNA target sequence. The PCR product was gel-purified and used as templates for in vitro transcription of sgRNA . Cas9 mRNA was transcribed in vitro from linearized pcDNA3.1-Cas9 plasmid . Both Cas9 mRNA and sgRNAs were purified , mixed in M2 (Millipore) at the concentration of 100 ng/μl, and then injected into the cytoplasm of fertilized eggs of C57BL/6 mice. For Pyk2Y402F mice, single-stranded oligo-donor nucleotides (ssODN) with mutation at Y402 residue and nonsense mutation at PAM sequence were co-injected together with the Cas9 mRNA and sgRNA. After equilibration for 30 min, 15–25 injected fertilized eggs were transferred into fallopian tube of pseudopregnant ICR mouse females. Offspring of these mice were genotyped by PCR, restriction endonuclease digestion, and Sanger sequencing. All oligos used are listed in Mouse lines of Pyk2KO and Pyk2The following antibodies were used for biochemistry experiments: mouse anti-β-actin , mouse anti-Myc , rabbit anti-Pyk2 . The following antibodies were used for immunocytochemistry and immunohistochemistry: mouse anti-Tuj1 , rabbit anti-αCD , rabbit anti-GFP , rabbit anti-BLBP (Brain lipid binding protein) , mouse anti-GM130 , rat anti-BrdU , rabbit anti-activated caspase 3 , rabbit anti-Tbr2 , rabbit anti-WAVE2 , goat anti-rabbit Alexa Fluor 488 , goat anti-rabbit Alexa Fluor 568 , goat anti-mouse Alexa Fluor 568 , goat anti-mouse Alexa Fluor 647 .Pcdha6, Pcdhac1, Pcdhac2, WAVE2 (GenBank AY135643.1), Abi2 (GenBank NM_198127.2) were cloned from mouse brain total RNA preparations by reverse transcriptase PCR (RT-PCR). The cDNAs of Myr-αCD, Rac1 and G12VRac1, Pyk2 and Pyk2 mutations , Pyk2 fragments  were cloned from previously published plasmids , Q61LRac1, were constructed from the above plasmids. Constructs used in IUE for overexpression were cloned into the pCAG-Myc vector or pNeuroD-IRES-GFP vector  using restriction enzyme sites. For knockdown, short-hairpin RNA (shRNA) coding sequences were cloned into the pLKO.1 vector. All oligo sequences with corresponding restriction enzyme sites are listed in Full-length cDNAs of plasmids . WIRS-muIUE was performed as previously described with modifications . Briefly5) were plated into four-well chamber or 35-mm glass-bottom Petri dish precoated with 100 μg/ml poly-L-lysine (Sigma) and 5 μg/ml laminin (Invitrogen). The cells were incubated with 5% CO2 at 37°C for 4 hr. The plating medium was then replaced with a serum-free culture medium . For immunocytochemistry, cells were cultured for additional 20 hr in vitro (hiv).For cortical neuron primary culture, electroporated cortices were collected from E17.5 embryos in Hanks’ Balanced Salt Solution (HBSS) with 0.5% glucose, 10 mM Hepes, 100 μg/ml penicillin/streptomycin. The cortices were then digested with 0.25% trypsin for 10 min at 37°C. The reaction was terminated with 0.5 mg/ml trypsin inhibitor for 3 min at room temperature (RT). The cortical tissues were gently triturated in the plating medium  until fully dissociated. Cell viability and density were determined using 0.4% trypan blue and a hemocytometer. The dissociated cells . Images were taken at 3 μm steps with 10–15 optical sections and were captured every 15 min for up to 16 hr with the Nikon A1 confocal laser microscope system.For cortical organotypic slice culture, the head of E17.5 embryos were briefly placed in 70% ethanol and the brains were carefully dissected. The brains were embedded in 3% low-melting agarose and glued to the chuck of a water-cooled vibratome (Leica). The 250-μm-thick whole-brain coronal sections were cut and collected in the sterile medium. The organotypic slices were carefully placed in a 0.4 μm membrane cell culture insert (Millipore) in a six-well plate. Slices were cultured in slice culture medium: 67% Basal Medium Eagle (BME), 25% HBSS, 5% FBS, 1% N2, 1% penicillin/streptomycin/glutamine (Invitrogen) and 0.66% glucose (Sigma). Slices (three per well) were cultured in six-well plates at 37°C and 5% COFor single-cell time-lapse imaging, cortical neurons were plated into a 35-mm glass-bottom Petri dish. Images were taken at 1 μm steps with 10–15 optical sections and were captured every 5 min for up to 10 hr with Nikon A1 confocal laser microscope system.Primary cultured cortical neurons were washed once with PBS, fixed in 4% PFA for 20 min at RT, washed and permeabilized with 0.2% Triton X-100 for 10 min. After blocking with 5% BSA, cells were incubated with primary antibodies at 4°C overnight followed by incubation of secondary antibodies for 1–2 hr at RT. F-actin was labeled by Alexa-546 phalloidin (Sigma). For immunohistochemistry, the dams were sacrificed, and embryonic brains were fixed in 4% PFA overnight at 4°C. The brains were then sectioned at 50 μm with a vibratome (Leica). Sections were washed three times in PBS, blocked in 3% BSA, 0.1% Triton X-100 in PBS for 1 hr at RT, and then incubated with primary antibodies at 4°C overnight and secondary antibodies at RT for 1–2 hr. Cell nuclei were visualized with DAPI. Images were collected with a confocal microscope (Leica) under a 10x objective for brain sections. High-resolution images were collected under a 60x oil objective with a 3x digital zooming factor for primary cultured neurons.g at 4°C for 30 min. The lysates were subjected to Western blot analyses.HEK293T cells were maintained in DMEM with 10% FBS and 100 μg/ml penicillin/streptomycin. Cultured cells were transfected using Lipofectamine 2000 (Invitrogen). Total protein of HEK293T cells was extracted by lysis buffer  with protease inhibitors and then centrifuged at 12,000 × Total RNA was extracted from embryonic mouse brain tissues with TRIzol (Ambion). The reverse-transcription reaction was performed with 1 μg total RNA preparations. All oligos used are listed in IUE experiments were performed using at least three pregnant female mice, by which we usually harvested at least six embryonic brains. We obtained 15 ~ 20 sections from each electroporated brain, and quantified one typical section per brain. Nearly identical areas in the presumptive somatosensory cortices of anatomically matched brain sections were chosen for imaging and quantification. For bin analysis, the cortices were divided into ten equal bins and all GFP+ neurons in each bin were counted. In total, about 150 ~ 300 cells were counted per section. Statistical significance was assessed using one-way ANOVA, followed by a post hoc Tukey’s multiple comparisons test.For each group, the In primary culture experiments, the development stage of cultured neurons were defined as in Dotti’s paper: at stage 1, the cell body was surrounded by flattened lamellipodia; at stage 2, the lamellipodia transformed into neural processes with growth cones . We immut tests. One-way ANOVA was used for multiple comparisons by the GraphPad software.The significance of differences between two groups was analyzed using unpaired Student’s  In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.eLife. Your article has been reviewed by a Senior Editor, a Reviewing Editor, and three reviewers. As you will see, all of the reviewers were impressed with the importance and novelty of your work.Thank you for submitting your manuscript "A Pcdhα/WRC/Pyk2/Rac1 Axis for Cortical Neuron Migration and Lamellipodial Dynamics" to In the reviews and the follow-up discussion, the major points regarding new experimental data that emerged are: (1) it would be useful to see comparison of the Pcdhα KO vs. the Pcdhα KD (knockdown) phenotypes; (2) likewise, a comparison between Pcdhα KD vs. Pyk2-OE (over-expression) phenotypes to determine the extent to which the defects are similar and thus consistent with misregulation of a Pcdhα-WRC-Pyk2 axis in neuronal migration and cytoskeletal dynamics; (3) assessing whether the mutant versions of Pcdhα go to the plasma membrane; and (4) describe sample sizes and consider other statistical tests such as ANOVA for comparing multiple groups.We are including the three reviews at the end of this letter, as there are many specific and useful suggestions in them. We appreciate that the reviewers' comments cover a broad range of suggestions for improving the manuscript. Please use your best judgment in deciding which of these can be accommodated in a reasonable period of time. We look forward to receiving your revised manuscript.Reviewer #1:Cortical neurons born from the proliferative ventricular zone and subventricular zones migrate radially to reach the appropriate laminar positions during development. Wu et al. have investigated the role of protocadherin cell surface molecules, and their downstream signaling through the WAVE complex and Pyk2 kinase, in radial migration. The authors carried out an extensive set of experiments utilizing in vivo genetic manipulation, live imaging, immunohistochemistry, and primary cell culture to address this question. Using electroporation of shRNA targeting the common intracellular domain of the protocadherin-α cluster (Pcdhα), they find a dramatic effect on migration: Pcdhα knockdown sequesters neurons within the lower intermediate zone and inhibits them from arriving at the cortical plate. This effect is then shown to involve signaling through Pyk2, WAVE, and Rac1. Knockdown of Pcdhα elevates Pyk2 function; the deleterious effects of increased Pyk2 are shown to require Pyk2 kinase activity as well as its scaffolding FERM domain.Overall, this is an interesting study. Little is known about signaling downstream of protocadherins, so this is a nice contribution. The authors provide a thorough and convincing picture of how Pcdhα/WRC/PKY2/Rac1 axis affects cortical neuron migration. The findings are generally well supported by the data presented. However, additional data may help make this study more impactful and is needed to support some of the conclusions.Specific comments:1) The authors demonstrated a clear lamellipodial phenotype in culture. However, its link to neuron phenotype in vivo was fuzzy. For one thing, the "stunted neurites"  morphology was not well documented. I had trouble appreciating the neuronal morphology in Figure 1E. The authors should include drawings of individual cells, as was done in Figure 3C (Pyk2 overexpression), because it is hard to tell the morphological detail in these panels.2). Related to this: Based on the images provided in Figure 1E and Video 1 and Video 2, the Pyk2 overexpressing cells seem more branchy than Pcdhα knockdown cells. The authors' model requires both manipulations to affect the same underlying actin cytoskeletal biology. How do the authors explain the difference in morphology?3) Last point on the morphology of migrating cells: It is unclear whether the "stunted neurite" morphology of Pcdhα-knockdown cells is due to altered lamellipodia. Same is true for extra branches in Pyk2 overexpressing cells. I thought I could discern some lamellipodia-like structures in Video 1 and Video 2, although without actin labeling it's hard to be sure. To connect the in vivo phenotype to the cultured cell phenotype, the authors should use an actin marker (e.g. lifeact) to find out if lamellipodia are affected in vivo.4) The authors state that the WIRS motif is located in the common intracellular domain shared by most Pcdhα family members. They cite Chen et al., 2014 for this information. This is the paper that shows interaction of WAVE complex with the WIRS domain of Pcdhα6. I could not find documentation in that paper of where WIRS is located (i.e. is it in the variable or common cytoplasmic region). The authors should clarify how they determined that WIRS is in the common region. If it were in the variable region, they would need to show biochemical interactions with WAVE for other Pcdhα isoforms, to demonstrate that their point mutants are indeed disrupting this particular interaction. As long as it's in the common region I think the interaction shown in Chen et al. is sufficient.5) There is not enough information provided on how the bin analysis was done to measure neuronal migration. The authors state "n=6." I assume that is the number of animals (please confirm). But how many sections were analyzed per animal, and how many cells were analyzed per section? How were particular sections/cells/fields of view selected for analysis? Also, since statistical comparisons were presumably made for multiple bins, the authors should justify the use of a T-test (rather than ANOVA with post-hoc comparisons) and/or explain how they controlled for multiple comparisons.6) The authors concluded that Rac1Q61L cannot rescue the blocking activity of FERM domain alone. However, from the images shown in Figure 4—figure supplement 1D, this looks to me like a partial rescue compare to the control (FERM domain only) condition. In the absence of further data I don't necessarily agree with the authors' conclusion that FERM domain acts independently of Rac1. A related point: It would be helpful to have at least brief consideration in the Discussion of how the FERM domain could function without the kinase. This would help guide readers who don't know much about FERM domains.7) The authors refer to cultured neurons at "Stage 1" and "Stage 2" but these terms are not defined. Please elaborate. Also, it should be stated in the methods how cultured cells were selected for analysis . Finally, the Materials and methods section should include more detail on how lamellipodia were counted – i.e. what criteria were used to distinguish them from other protrusions.eLife has no length limits, the authors should endeavor to get rid of as many of these as is practical. Everything that is a two-letter abbreviation (e.g. IZ for intermediate zone) for sure, and hopefully others.8) There are many abbreviations throughout the manuscript. These abbreviations can make it hard for readers to follow the flow of the story. Since 9) While the effects of downstream signaling from Pcdhα are demonstrated to be quite important, I was less clear on the authors' model for when this pathway might be activated. Do they think it is constitutively required? Or activated under certain circumstances – i.e. when Pcdhα mediates cell adhesion in a particular context? One possibility raised by the use of electroporation for the loss of function studies is that cells lacking Pcdhα might be at a relative disadvantage compared to the surrounding wild-type cells. This is a phenotype that has been observed for other cell adhesion molecules in other contexts. The authors have previously studied germline Pcdhα knockout mice; it would be quite informative if these animals had a phenotype that was less severe than the sparse knockdown.10) Finally, a suggestion: Protocadherins have remarkably diverse extracellular domains that allow them to function in many cellular contexts. The authors argue that they are studying the common downstream output of this diverse protein family. It is a plausible argument based on the shared common intracellular domain. However, if they could show that the Pcdhα/WRC/PKY2/Rac1 axis generalizes to other Pcdhα functions – e.g. dendrite morphology, as they have previously studied, or self-avoidance – this would bolster their claim that they are in fact studying a common downstream pathway. Such a finding would, in my opinion, substantially increase the impact of the study. For example, the authors could test whether the WIRS domain point mutants can rescue Pcdhα loss-of-function effects on pyramidal cell dendrite branching.For the title, all the abbreviations are probably going to stifle accessibility to a broad audience. "Protocadherin" and "WAVE complex" should substitute for their abbreviations, at minimum.Reviewer #2:Prior studies have demonstrated interaction between representative clustered and non-clustered protocadherins (Pcdhs) and the WAVE complex. Nevertheless, the biological relevance of Pcdh-VAVE interactions have not been demonstrated. In the current paper, Wu and colleagues report experiments in mice demonstrating that knockdown of α Pcdh leads to defects in neuronal migration, which can be rescued by reintroduction of a single a-Pcdh isoform. Mapping of the molecular regions responsible show that the cytoplasmic region can rescue alone, and this effect is lost by mutation of the a-Pcdh WAVE-binding WIRS sequence. Additional knockdown and overexpression experiments of non-receptor tyrosine kinase Pyk2, known to interact with Pcdh cytoplasmic regions, suggest that Pyk2 inhibits migration. Dissection of Pyk2 identifies the FERM domain as a key actor. Pyk2 appears to exert its effects by modulating the activation state of Rac1. Altogether, the authors define a putative pathway by which a-Pcdh interaction recruits the WAVE complex to the membrane to regulate neuronal migration. Concomitantly, a-Pcdh down-regulates Pyk2, leading to activation of WAVE through Rac1 activation.Overall, this is a fascinating paper, which adds biological context to the previously reported interaction between Pcdhs and the WAVE complex. It should be of high interest to people in the field.There are a number of questions raised by the findings, which are not addressed in the Discussion section:1) How could ABI2 rescue if there's no anchor to the membrane?2) Why is there no effect on brightness of GFP in αKD cells?3) Two different Rac1 mutations have different effects, but potential interpretations are not discussed.4) Overexpression of Pyk2 leads to a defect, which is further along than for αKD. Overexpression of the Pyk2 FERM domain recapitulates this phenotype, and overexpression of Pyk2 lacking the FERM domain appears wild-type. Since defects seem to be associated with FERM domain overexpression. It is hard to understand why the overexpression of a full-length kinase-dead Pyk2, containing the FERM domain, yields a wild-type phenotype. The authors should comment on this observation.5) It would help to improve the description of the differences between the α-Pcdhs that give different phenotypes. These different phenotypes are presumably due to differences in the juxtamembrane "variable" cytoplasmic domain region. A sequence alignment showing differences between the α-Pcdhs would be useful. Also, it would help to mark the location of the WAVE-binding WIRS peptide.Reviewer #3:This is an interesting manuscript reporting on a pathway in which α-Pcdhs influence neuronal migration through regulation of WAVE-Pyk2-RAC1 signaling. The clustered Pcdhs regulate diverse aspects of neuronal patterning, but little is known of the pathways by which Pcdhs transduce signals and regulate cytoskeletal dynamics. This group and others have shown that Pcdhs influence neurite patterning by negatively regulating Pyk2 and FAK, and indirectly promoting activation of Rac1. Another group identified a WAVE-interacting sequence motif present in α-Pcdhs, suggesting that Pcdhαs may also signal through the WAVE complex .Here, the authors test whether α-Pcdhs functionally interact with a WAVE-Pyk2-RAC1cascade by interrogating them in the context of cortical neuronal migration. They show that knock-down of Pcdhαs causes cortical migration defects, and then further manipulate Pcdhα and WAVE-Pyk2-RAC1 components through in utero electroporation and neuronal culture studies to determine their functional relationships. Their main conclusions are: (1) knockdown of Pcdhα specifically affects the extent and rate of migration, and this phenotype cannot be rescued by αc2 or by Pcdhα with mutations in the putative WAVE interacting motif (WIRS); (2) migration defects are observed when its downstream inhibitory target Pyk2 is overexpressed; and (3) the influence of α-Pcdhs on actin remodeling is also illustrated in lamellipodia formation and rescued by overexpression of Wave and Abl1 kinase.Overall, the study is important and provides the first demonstration of functional interactions between WAVE and Pcdhαs. However, there are several concerns that need to be addressed (see below), which would require further experiments and analyses. The major weaknesses are the omission of studies using Pcdhα-KO tissue and the lack of biochemical data showing interactions between Pcdhα and WAVE components. Moreover, the study manipulates different components of the pathway, but it fails to compare the phenotypes to each other. And the manipulations on Pyk2, Rac1 etc., are not verified in the context of Pcdh-a KD-induced migration defects. Therefore, the studies fall short of demonstrating a Pcdhα-WAVE-Pyk2-Rac1 axis in the regulation of cortical migration and cytoskeletal dynamics.1) The study is limited to shRNA-mediated knockdowns of Pcdhα, and does not extend to Pcdhα-KO brains. The omission is perplexing as the authors have generated and studied Pcdhα-KO mice in previous work . Data from Pcdhα-KO mice would significantly improve the quality of the results and, support the findings from the knockdown approaches. If they no longer carry these mice, they could obtain KO or conditional Pcdhα brains from other groups to report whether migration phenotypes are detected in fixed mutant tissue. If migration defects are not detected, they could further investigate if developmental delays, redundancy among Pcdhs, or possibly differential adhesion resulting from Pcdh mosaicism contributes to the mutant knockdown phenotype. Moreover, they could include an additional control for the shRNA by IUE Pcdhα-6 shRNA into Pcdh-aKO mice.2) The images showing Pcdhα protein expression and co-localization with WAVE are not informative. In Figure 1, the panels are low magnification. High power images of Pcdhαs localized along processes of migrating neurons (in WT and IUE tissue with GFP+ labeled neurons) should be shown. In Figure 2, the localization of Pcdhα and WAVE appear to cytosolic rather than at the membranes. Have the authors detected their co-localization in neuronal membranes in migrating cells in IUE tissue, or in growth cones or lamellipodia , which would be relevant to this study? Note that other groups have used membrane-targeted GFP for IUE-mediated labeling of migrating neurons to better resolve neurite structures .3) The 'WIRS' sequence in Pcdhα6 proposed by Chen et al., (2014) resides in the 3rd constant exon, which is shared by all Pcdhαs, including αc2. Interestingly, the authors show that, in contrast to α6 and αc1, full-length αc2 does not rescue the phenotype. However, deleting the αc2 variableCD leads abolishes this effect, suggesting that the αc2 variableCD distinguishes the activity of αc2 from the other Pcdhαs. This is potentially important and could advance the idea Pcdhα isoforms have different functions through their VCDs. But follow-up is needed, especially given that Pcdhαs also differ in their extracellular domains. The authors could test if chimeric forms of full-length Pcdhα can rescue the migration phenotype (i.e. α6 ECD-αc2 VCD-αCD).4) The finding in Figure 2G that mutating the WIRS motif in Pcdhαs fails to rescue migration is very interesting. However, it is premature to conclude: "Thus, Pcdhα regulates cortical neuron migration through the WRC complex". Additional data are needed: (1) Control experiments showing that this mutant Pcdhα-WIRS (AA) variant reaches the cell surface. There are reports in in vitro models suggesting that Pcdhαs do not traffic well to the cell-surface, and so it would be good to distinguish between alternate possibilities; and (2) Biochemical data showing interactions between Pcdhα and WAVE, such as pulldowns, preferably using cortical tissue. At the very least, pulldown assays in cell lines  could be done to show that interactions are abolished with this mutant form.5) In Figure 3, Pyk2OE also leads to neuronal migration phenotypes, but these mutant neurons are multipolar with increased branching, and their Golgi are misoriented. Were these phenotypes analyzed in Pcdhα-KD tissue? Migrating Pcdhα-KD neurons look bipolar in Figure 1, but there are no Lucida drawings. Likewise, does electroporating the constitutive Rac1 mutant rescue cortical defects in Pcdhα-KD? Again, comparing the same phenotypes across the manipulations would strengthen study's objective that a Pcdhα-WRC-Pyk2 axis regulates neuronal migration and cytoskeletal dynamics.6) In subsection “Dissection of Pyk2 domain in cortical neuron migration”, the authors describe the Pyk2 structure-function analyses in terms 'recapitulating the migration defects of αKD'. 'Recapitulate' is misused here. Do they mean phenocopy? If that is the case, there is not sufficient evidence for this. As stated in point 5, the phenotypes were not evaluated in the same way, and Pyk2OE leads to multipolar phenotypes that is not shown for αKD. While the Pyk2 structure function analyses do reveal relevant domains, I fail to see how they inform on αKD regulation of Pyk2 activity. Moreover, these manipulations were limited to Pyk2, and were not coupled with αKD manipulations .7) The migration defects are vaguely described in the Results section and are not sufficiently discussed in the context of previous studies. Many phenotypes are described, but the biological significances of these effects and whether the components produce the same effects are unclear.For example, in subsection “A role of Pyk2 in cortical neuron migration”, the authors note that Pyk2OE leads to a multipolar phenotype. They interpret the results:"Thus, Pyk2OE blocks multipolar migration by disrupting proper localization of the Golgi apparatus", but this statement is not fully supported by the data, nor are relevant citations given. Jossin et al., showed that Golgi orientation is important for orientating the direction of migration, but does not affect the speed. Here, Pyk2OE also affects migration speed.Regarding this point: "Finally, early born Pyk2OE neurons are also stalled at IZ, suggesting that Pyk2 also plays a role in somal translocation ."The relevant example is presented in Figure 3H, in the live imaging. But no quantifications are presented.Xie et al., 2013 showed that during the radial migration phase, cortical neurons undergo a multipolar-bipolar transition in their morphology for glia-guided locomotion, which is dependent on WAVE2, Abi2 . Is this relevant to the Pcdhα-WAVE-Pyk2 pathway? This could be expanded in the Discussion section.8) The sequence of results are disconnected and the rationales are not clear.For example, the transitions between Pcdhα to Pyk2 and back to Pcdhα are disconnected, and the two seem like separate studies.Subsection “A role of Pyk2 in cortical neuron migration”“We previously found that Pcdhα regulates dendritic and spine morphogenesis through inhibiting Pyk2 kinase activity . To this end, we investigated whether Pyk2 was involved in Pcdhs-regulated cortical neuron migration.”The authors could better articulate their goal to test whether there is increased Pyk2 in αKD tissue, and provide experiments combining KD and Pyk2 manipulations.Another example: It's not clear why the authors chose to further analyze the functional interactions between Pcdhα and Abi/Wave in lamellipodia using cultures of naïve, non-polarized neurons. Why not revisit this more mature lamellipodia structures relevant to migration, such as leading processes. The data on 'primary neurites' in Figure 5—figure supplement 1 are more convincing than those in Figure 5.9) The method used for quantifying lamellipodia should be described. Which fluorescent structure was used? (GFP? F-Actin?). The GFP appears to be cytosolic and may not fully resolve the membrane structures.10) Samples sizes are not properly described. Ns are given, but it's not clear what they comprise. The authors should report the numbers of cells, sections analyzed, animals per litter, numbers of litter/replicates.11) Statistical tests are limited to t-tests. But in many instances, multiple groups are analyzed and thus ANOVA and post-hoc multiple comparisons would be more suitable.12) I think it is also premature to exclude effects on survival, since quantification of numbers of GFP labeled cells in each IUE manipulation are not given. Cleaved-caspase 3 might only be detected during a small window, and labeled cells are cleared rapidly. In the reviews and the follow-up discussion, the major points regarding new experimental data that emerged are: (1) it would be useful to see comparison of the Pcdhα KO vs. the Pcdhα KD (knockdown) phenotypes;Thanks for your insightful synthesis. We have performed the experiments suggested by the three reviewers. The resulting new data are presented as new figures or figure panels. The new Figure1—figure supplement 1I shows new data of the Pcdhα knockout mice by IUE experiments with GFP plasmids. Quantification revealed that there is no significant difference between heterozygous and homozygous Pcdhα KO mice. Thus, there appears to be no cortical neuron migration defect in embryonic Pcdhα KO mice. This is consistent with the normal cortical layering of the adult Pcdhα KO mice reported in our original paper . In addition, there are not uncommon for the different phenotypes between acute gene inactivation by RNAi and constitutive germline knockout by gene targeting .doublecortin and doublecortin-like in cortical neuron migration. Knockdown of either doublecortin or doublecortin-like results in clear and convincing cortical neuron migration defects. By contrast, knockout of either doublecortin or doublecortin-like results in no cortical neuron migration defects . In our case of the Pcdhα, we have two independent shRNA targeting different subregions of the common constant region of all members of the Pcdhα family. In addition, we also have clear rescue experiments by shRNA-resistant constructs. These data provide strong and convincing evidence for a role of Pcdhα in cortical neuron migration. We have added the following sentence to the Results section: “Finally, there is no cortical migration defect  in mice with deletion of the entire Pcdhα cluster (αKO) . The phenotypic discrepancy may be due to known genetic compensation mechanisms induced by deletion but not knockdown .”Perhaps the most famous and relevant examples are the established role of (2) likewise, a comparison between Pcdhα KD vs. Pyk2-OE (over-expression) phenotypes to determine the extent to which the defects are similar and thus consistent with misregulation of a Pcdhα-WRC-Pyk2 axis in neuronal migration and cytoskeletal dynamics;We have performed extensive in-vitro and in-vivo (with Lifeact) experiments to compare αKD vs. Pyk2OE phenotypes in cytoskeletal dynamics and neuronal migration. The resulting data are presented in the new Figure 6 and Figure 6—figure supplement 1. We renamed the old Figure 6 as the new Figure 7 accordingly. We added a new subsection to the Results section: “A comparison between PcdhαKD and Pyk2OE in cytoskeletal dynamic”:“Consistent with that Pyk2KD rescues cortical neuron migration defects of PcdhαKD , we found that knockdown of Pyk2 in αKD cells results in a significant increase of lamellipodial sizes of stage1 neurons as well as of the percentage of primary neurites with lamellipodia of stage2 neurons . In addition, Pyk2OE results in a significant decrease of lamellipodial sizes, consistent with that of αKD . […] In the upper intermediate zone, Pyk2OE neurons exhibit branchy morphology with multiple aberrant processes; however, the control neurons have normal bipolar morphology with single leading processes and growth cones .”Although both α knockdown and Pyk2 overexpression result in cortical neuron migration defects, they do display subtle differences. We added the following two sentences to the Discussion section: “We noted that αKD neurons stall in the lower intermediate zone and Pyk2OE neurons stall in the middle intermediate zone. In other words, αKD phenotype is more severe than that of Pyk2OE. In addition, αKD neurons display stunted processes while Pyk2OE neurons have branchy morphology.”(3) assessing whether the mutant versions of Pcdhα go to the plasma membrane;Indeed, these control experiments are important as previous transfection experiments have suggested that cell-surface delivery of Pcdhα to neuronal membrane requires Pcdhγ . To this end, we constructed C-terminal Myc-tagged mutant versions of α6*-AA, αc1*-AA, Myr-αCD*-AA, as well as their corresponding wildtypes α6*, αc1*, Myr-αCD*, and transfected each mutant version or their corresponding wildtype into primary cultured neurons, which are most likely expressing Pcdhγ. We then immunostained these primary cultured neurons with Myc antibody to show the location of transfected Pcdhα. Both wildtype and WIRS motif mutant versions are detected at the very tip of neurite ends, so we concluded that they go to the cell-surface. We presented the new data as Figure 2—figure supplement 1F and added the following sentence to the Results section: “As controls, these WIRS mutated isoforms as well as wildtypes appears to reach the plasma membrane .”and (4) describe sample sizes and consider other statistical tests such as ANOVA for comparing multiple groups.We have gone through the relevant legends and methods to make corrections. In addition, we have described sample sizes and used ANOVA for comparing multiple groups. Finally, we have added the following subsection of detailed description to the Materials and methods section: “Statistical analysis/ Image analysis and quantification”Reviewer #1:[…] Overall, this is an interesting study. Little is known about signaling downstream of protocadherins, so this is a nice contribution. The authors provide a thorough and convincing picture of how Pcdhα/WRC/PKY2/Rac1 axis affects cortical neuron migration. The findings are generally well supported by the data presented. However, additional data may help make this study more impactful and is needed to support some of the conclusions.Indeed, the novelty of Pcdhα in cortical neuron migration supported by thorough and convincing evidence is the main finding of the work. We have performed additional experiments suggested by Reviewer #1 and obtained new data to make the study more impactful and to support some of the conclusions.Specific comments:1) The authors demonstrated a clear lamellipodial phenotype in culture. However, its link to neuron phenotype in vivo was fuzzy. For one thing, the "stunted neurites"  morphology was not well documented. I had trouble appreciating the neuronal morphology in Figure 1E. The authors should include drawings of individual cells, as was done in Figure 3C (Pyk2 overexpression), because it is hard to tell the morphological detail in these panels.We thank Reviewer #1 for this suggestion and we have added Lucida drawings of typical cells as in new Figure 1E, to show the morphological differences between control and Pcdhα knockdown neurons. We have changed the sentence from: “The αKD multipolar neurons in IZ display stunted neurites ” to: “The αKD multipolar neurons in IZ display stunted processes, as shown by lucida drawings ”.2) Related to this: Based on the images provided in Figure 1E and Video 1 and Video 2, the Pyk2 overexpressing cells seem more branchy than Pcdhα knockdown cells. The authors' model requires both manipulations to affect the same underlying actin cytoskeletal biology. How do the authors explain the difference in morphology?We agree that Pyk2OE and αKD neurons have different morphology. As stated above, we have performed additional experiments to compare them and the resulting data are presented in new Figure 6. Our new data show that Pyk2OE results in a significant increase of filopodial number per stage1 neuron and of primary neurite number per stage2 neuron, consistent with more branchy phenotype. Pyk2OE leads to the inhibition of Rac1 activity . As Rac1 is thought to provide the spatial information for actin polymerization , loss of Rac1 activity leads to aberrant actin polymerization at many sites with no controlled spatial information, resulting in more aberrant filopodia. This explains that immature Pyk2OE neurons display more branching while wildtype neurons are bipolar.We therefore added the following sentence to the Results section: “Pyk2OE leads to the inhibition of Rac1 activity . As Rac1 is thought to provide the spatial information for actin polymerization , loss of Rac1 activity leads to aberrant actin polymerization at many sites with no controlled spatial information, resulting in more branchy phenotype.”3) Last point on the morphology of migrating cells: It is unclear whether the "stunted neurite" morphology of Pcdhα-knockdown cells is due to altered lamellipodia. Same is true for extra branches in Pyk2 overexpressing cells. I thought I could discern some lamellipodia-like structures in Video 1 and Video 2, although without actin labeling it's hard to be sure. To connect the in vivo phenotype to the cultured cell phenotype, the authors should use an actin marker (e.g. lifeact) to find out if lamellipodia are affected in vivo.We thank Reviewer #1 for this insightful suggestion. To address this question, we performed the suggested experiment using the Lifeact to see whether lamellipodia are affected in αKD and Pyk2OE cells. Lifeact was constructed into pCAG plasmid with C-terminal mCherry in-frame fusion, so the Lifeact labeled actin can be observed by red fluoresce. We added a paragraph at the end of the Results section: “To see whether growth cones with lamellipodia and filopodia are affected in vivo, we co-electroporated Lifeact, an actin marker, with either αKD or Pyk2OE plasmids into the developing mouse cortex. […] In the upper intermediate zone, Pyk2OE neurons exhibit branchy morphology with multiple aberrant processes; however, the control neurons have normal bipolar morphology with single leading processes and growth cones .”4) The authors state that the WIRS motif is located in the common intracellular domain shared by most Pcdhα family members. They cite Chen et al., 2014 for this information. This is the paper that shows interaction of WAVE complex with the WIRS domain of Pcdhα6. I could not find documentation in that paper of where WIRS is located (i.e. is it in the variable or common cytoplasmic region). The authors should clarify how they determined that WIRS is in the common region. If it were in the variable region, they would need to show biochemical interactions with WAVE for other Pcdhα isoforms, to demonstrate that their point mutants are indeed disrupting this particular interaction. As long as it's in the common region I think the interaction shown in Chen et al. is sufficient.We appreciate these comments and apologize for the confusion. As seen in the new sequence alignments in 5) There is not enough information provided on how the bin analysis was done to measure neuronal migration. The authors state "n=6." I assume that is the number of animals (please confirm). But how many sections were analyzed per animal, and how many cells were analyzed per section? How were particular sections/cells/fields of view selected for analysis? Also, since statistical comparisons were presumably made for multiple bins, the authors should justify the use of a T-test (rather than ANOVA with post-hoc comparisons) and/or explain how they controlled for multiple comparisons.We have added a paragraph to provide detailed information on how the bin analysis was done to measure neuronal migration: “For each group, the IUE experiments were performed using at least three pregnant female mice, by which we usually harvested at least 6 embryonic brains. We obtained 15~20 sections from each electroporated brain and quantified 1 typical section per brain. Nearly identical areas in the presumptive somatosensory cortices of anatomically matched brain sections were chosen for imaging and quantification. For bin analysis, the cortices were divided into ten equal bins and all GFP+ neurons in each bin were counted. In total, about 150~300 cells were counted per section. Statistical significance was assessed using one-way ANOVA, followed by a post hoc Tukey’s multiple comparisons test.”t tests are not appropriate for multiple comparisons. We re-performed all the significance tests using ANOVA with post-hoc comparisons. We added one sentence to the end of the above paragraph.We thank Reviewer #1 for pointing out that some of the statistical analyses in manuscript are not correctly used. We agree that Student’s 6) The authors concluded that Rac1Q61L cannot rescue the blocking activity of FERM domain alone. However, from the images shown in Figure 4—figure supplement 1D, this looks to me like a partial rescue compare to the control (FERM domain only) condition. In the absence of further data I don't necessarily agree with the authors' conclusion that FERM domain acts independently of Rac1. A related point: It would be helpful to have at least brief consideration in the Discussion of how the FERM domain could function without the kinase. This would help guide readers who don't know much about FERM domains.Q61L in old Figure 4—figure supplement 1D. However, as shown in (Author response figure 2) of all six sections from six mouse brains, overall there does not appear to be significant different between FERM+Rac1Q61L and FERM alone. We have nevertheless replaced the FERM+Rac1Q61L image in old Figure 4—figure supplement 1D with a new one  to avoid potential confusion.We thank Reviewer#1 for this comment. Because there appears to be more neurons migrated into the cortical plate than FERM in this particular section, we agree that it looks like partial rescue of FERM+Rac1It was previously reported that the FERM domain of Pyk2 has a role in malignant glioma cell migration , and Pyk2 FERM domain is involved in the regulation of Pyk2 activity by acting to regulate the formation of Pyk2 oligomers which is critical for Pyk2 activity . Overwhelming evidence in literature shows that the FERM domain of FAK, a close homolog of Pyk2, has clear kinase-independent scaffolding activity by interacting with numerous partners and regulating downstream signaling . As suggested we have added the following sentence to the Discussion section to help guide readers about the FERM domain scaffolding: “Moreover, our data suggest that Pyk2 also has kinase-independent scaffolding activity through its FERM  domain, similar to the FERM domain of FAK, which binds to numerous interacting partners and connects cell cortex to diverse downstream intracellular pathways .7) The authors refer to cultured neurons at "Stage 1" and "Stage 2" but these terms are not defined. Please elaborate. Also, it should be stated in the methods how cultured cells were selected for analysis . Finally, the Materials and methods section should include more detail on how lamellipodia were counted – i.e. what criteria were used to distinguish them from other protrusions.Thanks for the suggestion. We added the following to subsection “Pcdh in lamellipodial formation and cytoskeletal dynamics”: “The early development of primary cultured neurons can be divided into two stages: stage1, in which the cell body is surrounded by flattened lamellipodia, and stage2, in which the lamellipodia transform into definitive processes with growth cones .”We selected neurons with typical stage1 or stage2 morphology based on the GFP signal, which labels transfected cells, and the phalloidin signal, which stains F-actin.We immunostained the cultured cells with a Tuj1 (Neuron-specific class III β-tubulin) antibody, which is regarded as a neuron-specific marker, to exclude differentiated glia or radial glia.For stage1 neurons, we selected the lamellipodia region by the wand tool in the ImageJ software, and measured the area’s size. For stage2 neurons, the neurite tips with F-actin-enriched protrusions two folds larger than the neurite width are defined as neurites with lamellipodia.8) There are many abbreviations throughout the manuscript. These abbreviations can make it hard for readers to follow the flow of the story. Since eLife has no length limits, the authors should endeavor to get rid of as many of these as is practical. Everything that is a two-letter abbreviation (e.g. IZ for intermediate zone) for sure, and hopefully others.Thanks for the suggestion and we have gone through the entire text carefully to get rid of as many as abbreviations as is practical.9) While the effects of downstream signaling from Pcdhα are demonstrated to be quite important, I was less clear on the authors' model for when this pathway might be activated. Do they think it is constitutively required? Or activated under certain circumstances – i.e. when Pcdhα mediates cell adhesion in a particular context? One possibility raised by the use of electroporation for the loss of function studies is that cells lacking Pcdhα might be at a relative disadvantage compared to the surrounding wild-type cells. This is a phenotype that has been observed for other cell adhesion molecules in other contexts. The authors have previously studied germline Pcdhα knockout mice; it would be quite informative if these animals had a phenotype that was less severe than the sparse knockdown.Cell motility, in particular cortical neuron migration, is enormously complex . The two highly-similar mammalian cell-adhesion kinases, Pyk2 and FAK, are likely central for cell motility. Our data suggest that Pcdhα and Pyk2 function in cortical neuron migration through WAVE complex to regulate actin fiber dynamics. We think that our working model is dynamic, undulating between activated and inactivated states through coordinated cycles in the cell leading edge and trailing edge. We do not know whether these activities are dependent on the cell-adhesion activity of Pcdhα or not. Nevertheless, in conjunction with the germline knockout experiments, our data suggest that cells lacking Pcdhα might be indeed at a relative disadvantage compared to the surrounding wild-type cells.As stated above, we have performed the α germline knockout experiments and the new data are presented in Figure 1—figure supplement 1I. Indeed, the knockout mice appear to have no defect in cortical neuron migration. This is strikingly similar to members of the DOUBLECORTIN protein family as discussed extensively above.10) Finally, a suggestion: Protocadherins have remarkably diverse extracellular domains that allow them to function in many cellular contexts. The authors argue that they are studying the common downstream output of this diverse protein family. It is a plausible argument based on the shared common intracellular domain. However, if they could show that the Pcdhα/WRC/PKY2/Rac1 axis generalizes to other Pcdhα functions – e.g. dendrite morphology, as they have previously studied, or self-avoidance – this would bolster their claim that they are in fact studying a common downstream pathway. Such a finding would, in my opinion, substantially increase the impact of the study. For example, the authors could test whether the WIRS domain point mutants can rescue Pcdhα loss-of-function effects on pyramidal cell dendrite branching.We thank Reviewer #1 for this good suggestion and we have performed suggested dendritic experiment and Sholl analysis to address whether Pcdhα WIRS motif play a general role in dendrite development and branching. We previously showed that Myr-αCD function as a dominant-negative construct and lead to dendrite developmental defect in cultured hippocampal neurons . Our new experiments with primary cultured cortical neurons using Myr-αCD reproduced the dendrite developmental defect . Strikingly, mutation of the WIRS (from FITFGK to FIAAGK) abolished the dendrite developmental defect, suggesting that other Pcdhα functions such as dendrite development are also dependent on the signaling through the WAVE complex . We added the following sentence to the Discussion section: “Sholl analysis demonstrated that the WIRS domain point mutation can rescue the Pcdhα dominant-negative effects on dendrite outgrowth and branching of primary cultured cortical neurons, suggesting that the Pcdhα/WAVE/Pyk2/Rac1 signaling axis indeed functions in dendrite morphogenesis .”For the title, all the abbreviations are probably going to stifle accessibility to a broad audience. "Protocadherin" and "WAVE complex" should substitute for their abbreviations, at minimum.We have considered carefully and changed the title to: “Αlpha Protocadherins and Pyk2 kinase Regulate Cortical Neuron Migration and Cytoskeletal Dynamics via Rac1 GTPase and WAVE complex”.Reviewer #2:[…] Overall, this is a fascinating paper, which adds biological context to the previously reported interaction between Pcdhs and the WAVE complex. It should be of high interest to people in the field.Indeed, this work provides strong evidence for novel function of the previously reported interactions between Pcdhs and the WAVE complex in the brain.There are a number of questions raised by the findings, which are not addressed in the Discussion section:1) How could ABI2 rescue if there's no anchor to the membrane?We thank Reviewer#2 for this insightful question. There are four PXXP motifs within the common Pcdhα cytoplasmic constant domains which are anchors for ABI2 to the membrane. We have added the following two sentences to the Discussion section: “The WIRS motif of members of the Pcdhα family binds to a composite surface formed by Abi2 and Sra1 of WAVE . In addition, the Pcdhα proteins may also recruit WAVE through the direct binding of Abi2 C-terminal SH3 domain to the four PXXP motifs, which are specific to the constant domain of the Pcdhα but not Pcdhγ family .”2) Why is there no effect on brightness of GFP in αKD cells?IUE experiments, we co-electroporated two separate plasmids: one plasmid expresses shRNA and the other one expresses GFP. The two plasmids should not influence each other. Thus, the GFP expression level is not decreased in αKD neurons.We are sorry for the confusion. For all the α knockdown The GFP is used to label transfected cells in the IUE experiments. If two plasmids are co-electroporated into the mouse brain by IUE, almost all of the transfected cells are co-transfected. This is proved by GFP-mCherry co-electroporation experiments .3) Two different Rac1 mutations have different effects, but potential interpretations are not discussed.Q61L.”The two Rac1 mutations are likely have different constitutive activities because the Q61L mutant has a higher affinity for GTP than the G12V mutant . We have added the following at the end of the relevant sentence: “likely because it has a lower affinity for GTP and thus lower constitutive activity than Rac14) Overexpression of Pyk2 leads to a defect, which is further along than for αKD. Overexpression of the Pyk2 FERM domain recapitulates this phenotype, and overexpression of Pyk2 lacking the FERM domain appears wild-type. Since defects seem to be associated with FERM domain overexpression. It is hard to understand why the overexpression of a full-length kinase-dead Pyk2, containing the FERM domain, yields a wild-type phenotype. The authors should comment on this observation.We thank Reviewer #2 for the comment. As discussed above, Pyk2 has both tyrosine kinase signaling and FERM domain scaffolding activity under physiological conditions. According to the structure of FAK , which is a very close homolog of Pyk2 and likely shares similar structures, the FERM and kinase domains are sequestered in an autophosphorylation-closed inactive state. With the kinase-dead mutation, there is no conformational change for opening and phosphorylation, and subsequent Pyk2 activation. Therefore, it is understandable that the overexpression of a full-length kinase-dead Pyk2, despite containing the FERM domain, because of its closed state, still yields a wild-type phenotype of cortical neuron migration.5) It would help to improve the description of the differences between the α-Pcdhs that give different phenotypes. These different phenotypes are presumably due to differences in the juxtamembrane "variable" cytoplasmic domain region. A sequence alignment showing differences between the α-Pcdhs would be useful. Also, it would help to mark the location of the WAVE-binding WIRS peptide.We appreciate Reviewer #2 for this good suggestion. We performed the suggested sequence alignment to demonstrate the differences in the juxtamembrane "variable" cytoplasmic domain region . Pcdhα1-Pcdhα12 have relatively short VCDs with high similarity, while Pcdhαc1 and Pcdhαc2 have longer and divergent VCDs. These structural differences may underlie the functional differences between diverse Pcdhα isoforms. We have added the following sentence to the Results section: “Consistently, sequence analysis revealed that αc2 VCD is the longest and most divergent among those of αc1 as well as of α1-α12 .”The WIRS motif is in the common cytoplasmic constant region of the Pcdhα proteins as described above in addressing reviewer #1’s comments .Reviewer #3:[…] 1) The study is limited to shRNA-mediated knockdowns of Pcdhα and does not extend to Pcdhα-KO brains. The omission is perplexing as the authors have generated and studied Pcdhα-KO mice in previous work . Data from Pcdhα-KO mice would significantly improve the quality of the results and, support the findings from the knockdown approaches. If they no longer carry these mice, they could obtain KO or conditional Pcdhα brains from other groups to report whether migration phenotypes are detected in fixed mutant tissue. If migration defects are not detected, they could further investigate if developmental delays, redundancy among Pcdhs, or possibly differential adhesion resulting from Pcdh mosaicism contributes to the mutant knockdown phenotype. Moreover, they could include an additional control for the shRNA by IUE Pcdhα-6 shRNA into Pcdh-aKO mice.We appreciate Reviewer #3 for these comprehensive constructive suggestions. As described above, we have performed experiments with Pcdhα knockout mice and there appears no cortical neuron migration defect. We discussed the reasons extensively above. Because of limited time to revise the manuscript, we have not investigated the reasons experimentally. We appreciate the additional control experiment for the shRNA by IUE Pcdh α6 shRNA into α knockout mice. However, we have used two independent shRNAs targeting distinct subregions of the Pcdhα constant region. Most importantly, we have solid rescue results. The consilience of all of these results demonstrates that the αKD defect is not due to off-target.2) The images showing Pcdhα protein expression and co-localization with WAVE are not informative. In Figure 1, the panels are low magnification. High power images of Pcdhαs localized along processes of migrating neurons (in WT and IUE tissue with GFP+ labeled neurons) should be shown. In Figure 2, the localization of Pcdhα and WAVE appear to cytosolic rather than at the membranes. Have the authors detected their co-localization in neuronal membranes in migrating cells in IUE tissue, or in growth cones or lamellipodia , which would be relevant to this study? Note that other groups have used membrane-targeted GFP for IUE-mediated labeling of migrating neurons to better resolve neurite structures .IUE tissues because there are too many cells compact together. The WAVE2 and Abi2 proteins co-localize with F-actin in lamellipodia enriched growth cones . We have performed GFP staining using the PcdhαGFP mice. As shown in We thank Reviewer #3 for this comment. It’s hard to detect endogenous Pcdhα localization along processes of migrating neurons in WT and 3) The 'WIRS' sequence in Pcdhα6 proposed by Chen et al., (2014) resides in the 3rd constant exon, which is shared by all Pcdhαs, including αc2. Interestingly, the authors show that, in contrast to α6 and αc1, full-length αc2 does not rescue the phenotype. However, deleting the αc2 variableCD leads abolishes this effect, suggesting that the αc2 variableCD distinguishes the activity of αc2 from the other Pcdhαs. This is potentially important and could advance the idea Pcdhα isoforms have different functions through their VCDs. But follow-up is needed, especially given that Pcdhαs also differ in their extracellular domains. The authors could test if chimeric forms of full-length Pcdhα can rescue the migration phenotype (i.e. α6 ECD-αc2 VCD-αCD).As can be seen from the VCD sequence alignment, each member of the Pcdhα family proteins has a unique VCD sequences, with the αc2 having the longest and most divergent VCD sequences. To test the VCD function, we performed experiments to delete the VCD domain of the α6, αc1, and αc2 protein . Because4) The finding in Figure 2G that mutating the WIRS motif in Pcdhαs fails to rescue migration is very interesting. However, it is premature to conclude: "Thus, Pcdhα regulates cortical neuron migration through the WRC complex". Additional data are needed: (1) Control experiments showing that this mutant Pcdhα-WIRS (AA) variant reaches the cell surface. There are reports in in vitro models suggesting that Pcdhαs do not traffic well to the cell-surface, and so it would be good to distinguish between alternate possibilities; and (2) Biochemical data showing interactions between Pcdhα and WAVE, such as pulldowns, preferably using cortical tissue. At the very least, pulldown assays in cell lines  could be done to show that interactions are abolished with this mutant form.This has been addressed above. We have not done the pulldown because it will not provide stronger evidence than the Chen et al., 2014 paper.5) In Figure 3, Pyk2OE also leads to neuronal migration phenotypes, but these mutant neurons are multipolar with increased branching, and their Golgi are misoriented. Were these phenotypes analyzed in Pcdhα-KD tissue? Migrating Pcdhα-KD neurons look bipolar in Figure 1, but there are no Lucida drawings. Likewise, does electroporating the constitutive Rac1 mutant rescue cortical defects in Pcdhα-KD? Again, comparing the same phenotypes across the manipulations would strengthen study's objective that a Pcdhα-WRC-Pyk2 axis regulates neuronal migration and cytoskeletal dynamics.We have added Lucida drawings of αKD neurons, showing the stunted morphology. As stated above, we have changed the sentence from: “The αKD multipolar neurons in IZ display stunted neurites ” to: “The αKD multipolar neurons in IZ display stunted processes, as shown by lucida drawings ”. During migration, cortical neurons reach the lower IZ and become multipolar, starting “multipolar migration” at IZ. The multipolar neurons later reorient the Golgi towards the pia at the IZ/CP and establish a dominant pia-directed leading process, this is known as “multipolar-bipolar transition”, leading to radial migration at CP . Since αKD neurons accumulate in the lower IZ with stunted multipolar morphology, not look bipolar, as shown by Lucida drawings. By contrast, Pyk2OE neurons are stalled in the middle IZ (mIZ) with branchy morphology as discussed above, suggesting defects of multipolar-bipolar transition. The Golgi orientation experiment is designed to address the “multipolar-bipolar transition” defect, so we didn’t analyze the Golgi orientation in αKD neurons.Q61L and of Pyk2OE by electroporating WAVE2. As shown in We have performed the suggested rescue experiments of αKD by electroporating Rac16) In subsection “Dissection of Pyk2 domain in cortical neuron migration”, the authors describe the Pyk2 structure-function analyses in terms 'recapitulating the migration defects of αKD'. 'Recapitulate' is misused here. Do they mean phenocopy? If that is the case, there is not sufficient evidence for this. As stated in point 5, the phenotypes were not evaluated in the same way, and Pyk2OE leads to multipolar phenotypes that is not shown for αKD. While the Pyk2 structure function analyses do reveal relevant domains, I fail to see how they inform on αKD regulation of Pyk2 activity. Moreover, these manipulations were limited to Pyk2, and were not coupled with αKD manipulations [I initially expected co-transfection experiments but found no description of this approach].As discussed extensively above, although both αKD and Pyk2OE have cortical neuron migration defect, they are not exactly the same. αKD neurons stalled at lower IZ and have stunted multipolar morphology. Pyk2OE neurons stalled at middle IZ and have branchy morphology. Thus, we do not mean “recapitulate” as “phenocopy”. It’s known that Pyk2 is negatively regulated by Pcdhα . Thus, αKD results in increased endogenous Pyk2 activity. The suggested overexpression of various Pyk2 dissection experiments would not provide additional new information.7) The migration defects are vaguely described in the Results section and are not sufficiently discussed in the context of previous studies. Many phenotypes are described, but the biological significances of these effects and whether the components produce the same effects are unclear.For example, in subsection “A role of Pyk2 in cortical neuron migration”, the authors note that Pyk2OE leads to a multipolar phenotype. They interpret the results:"Thus, Pyk2OE blocks multipolar migration by disrupting proper localization of the Golgi apparatus", but this statement is not fully supported by the data, nor are relevant citations given. Jossin et al., showed that Golgi orientation is important for orientating the direction of migration, but does not affect the speed. Here, Pyk2OE also affects migration speed.As stated above, during the multipolar-bipolar transition, the multipolar cell reorients the Golgi apparatus towards the pia, establishes a dominant pia-directed leading process. This is essential for radial migration. We agree that the statement “Pyk2OE blocks multipolar migration by disrupting proper localization of the Golgi apparatus” is not fully supported by the data. However, Pyk2OE leads to dysregulated Rac1 activity , resulting in aberrant Arp2/3 and actin assembly. The Golgi apparatus thus cannot reorient. In the end, this results in blocked multipolar-to-bipolar transition. Although Golgi misorientation per se does not affect the speed, the overall radial migration speed is still reduced by Pyk2OE because the cycling of actin fibers at the leading and trailing edges may also be disrupted (in addition to misoriented Golgi).Regarding this point: " Finally, early born Pyk2OE neurons are also stalled at IZ, suggesting that Pyk2 also plays a role in somal translocation ."The relevant example is presented in Figure 3H, in the live imaging. But no quantifications are presented.The quantification of early born Pyk2OE neurons stalling at IZ is shown in Figure 3—figure supplement 1D in the original manuscript. In addition, the quantifications of live imaging of Figure 3H is shown in Figure 3I in the original manuscript.Xie et al., 2013 showed that during the radial migration phase, cortical neurons undergo a multipolar-bipolar transition in their morphology for glia-guided locomotion, which is dependent on WAVE2, Abi2 . Is this relevant to the Pcdhα-WAVE-Pyk2 pathway? This could be expanded in the Discussion section.We appreciate Reviewer #3 for this insightful suggestion. We have expanded the following text in the Discussion section: “The WIRS motif of members of the Pcdhα family binds to a composite surface formed by Abi2 and Sra1 of WAVE . In addition, the Pcdhα proteins may also recruit WAVE through the direct binding of Abi2 C-terminal SH3 domain to the four PXXP motifs, which are specific to the constant domain of the Pcdhα but not Pcdhγ family . Consistently, WAVE2 and Abi2 are required for growth cone activity during cortical neuron migration .”8) The sequence of results are disconnected and the rationales are not clear.For example, the transitions between Pcdhα to Pyk2 and back to Pcdhα are disconnected, and the two seem like separate studies.Subsection “A role of Pyk2 in cortical neuron migration”“We previously found that Pcdhα regulates dendritic and spine morphogenesis through inhibiting Pyk2 kinase activity . To this end, we investigated whether Pyk2 was involved in Pcdhs-regulated cortical neuron migration.”The authors could better articulate their goal to test whether there is increased Pyk2 in αKD tissue, and provide experiments combining KD and Pyk2 manipulations.Thanks for the good suggestion. We have changed the relevant text to: “Pcdhα physically interacts with and negatively regulates the Pyk2 kinase . In addition, we previously found that Pcdhα regulates dendritic and spine morphogenesis through inhibiting Pyk2 activity . To this end, we investigated whether knockdown of Pyk2 could rescue cortical neuron migration defects of αKD.”Another example: It's not clear why the authors chose to further analyze the functional interactions between Pcdhα and Abi/Wave in lamellipodia using cultures of naïve, non-polarized neurons. Why not revisit this more mature lamellipodia structures relevant to migration, such as leading processes. The data on 'primary neurites' in Figure 5—figure supplement 1 are more convincing than those in Figure 5.Thanks for this suggestion. We agree that “The data on 'primary neurites' in Figure 5—figure supplement 1 are more convincing than those in Figure 5.” The WAVE2 and Abi2 data in Figure 5—figure supplement 1 are really good. However, the paper is too long. We would be happy to change Figure 5—figure supplement 1 to a main figure if your esteemed journal requires us to do so.9) The method used for quantifying lamellipodia should be described. Which fluorescent structure was used? (GFP? F-Actin?). The GFP appears to be cytosolic and may not fully resolve the membrane structures.We used both GFP (labeling transfection) and phalloidin (F-actin) for quantifying lamellipodial structure. We have added the following sentences to Materials and methods section to address these comments: “For quantification, we selected neurons with typical stage1 or stage2 morphology based on GFP and phalloidin signals. For stage1 neurons, we selected the lamellipodia region by the wand tool of the ImageJ software (NIH) and measured the area size. For stage2 neurons, the neurite tips with F-actin-enriched protrusions two folds larger than its width were defined as “neurite with lamellipodia”.10) Samples sizes are not properly described. Ns are given, but it's not clear what they comprise. The authors should report the numbers of cells, sections analyzed, animals per litter, numbers of litter/replicates.We have added the detailed information as described above.11) Statistical tests are limited to t-tests. Bu in many instances, multiple groups are analyzed and thus ANOVA and post-hoc multiple comparisons would be more suitable.We have now used ANOVA and post-hoc multiple comparisons to analyze multiple groups.12) I think it is also premature to exclude effects on survival, since quantification of numbers of GFP labeled cells in each IUE manipulation are not given. Cleaved-caspase 3 might only be detected during a small window, and labeled cells are cleared rapidly.We have quantified the numbers of GFP labeled cells in each IUE manipulation. As shown in