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• W2264766091 abstract "•Cux1 couples neuronal identity and wiring through modulation of firing modes•Cux1 transcription factor determines postnatal development of the corpus callosum•Cux1 determines Kv1 expression levels and firing modes in layer II/III neurons•Firing modes determine axonal connectivity during development Neuronal subtype-specific transcription factors (TFs) instruct key features of neuronal function and connectivity. Activity-dependent mechanisms also contribute to wiring and circuit assembly, but whether and how they relate to TF-directed neuronal differentiation is poorly investigated. Here we demonstrate that the TF Cux1 controls the formation of the layer II/III corpus callosum (CC) projections through the developmental transcriptional regulation of Kv1 voltage-dependent potassium channels and the resulting postnatal switch to a Kv1-dependent firing mode. Loss of Cux1 function led to a decrease in the expression of Kv1 transcripts, aberrant firing responses, and selective loss of CC contralateral innervation. Firing and innervation were rescued by re-expression of Kv1 or postnatal reactivation of Cux1. Knocking down Kv1 mimicked Cux1-mediated CC axonal loss. These findings reveal that activity-dependent processes are central bona fide components of neuronal TF-differentiation programs and establish the importance of intrinsic firing modes in circuit assembly within the neocortex.Video AbstracteyJraWQiOiI4ZjUxYWNhY2IzYjhiNjNlNzFlYmIzYWFmYTU5NmZmYyIsImFsZyI6IlJTMjU2In0.eyJzdWIiOiJkMjNkZjFiY2Y3YzZlZWE4Yzc4MmU5NzFmZmJmN2UyZCIsImtpZCI6IjhmNTFhY2FjYjNiOGI2M2U3MWViYjNhYWZhNTk2ZmZjIiwiZXhwIjoxNjc4NDY3NTI2fQ.hK2r6SDSwULDp3aAqZFHZs24Cwyjoz5avfdB4c7y5eiavT45Vz2r7qTgPDsYrr4RCzIpXUybTecShdbIqFJC8tFHVIudjDAHqNvTC7T9FKFKc0qmHPGZrcmd834PDD6qFbilVctO3jWVE1sBkQSBIh5EWCX3Mzn-JElih1Wl772R4l8hU3PJEMyfzoZc0I1w-BU14wlzHz_3IA3cv7d49fULmQK6A0SmOgYEnYTksoCh9GkJsDuMwynzDp38L1Y0A83cH-Co0TheHZR1QdT0N79UMlrpvw1pgzD-MPprZSoQKbpWc4qWIzkrr-AiaLGjQbfNx3pIh8ZleePVyJb9RA(mp4, (44.67 MB) Download video Neuronal subtype-specific transcription factors (TFs) instruct key features of neuronal function and connectivity. Activity-dependent mechanisms also contribute to wiring and circuit assembly, but whether and how they relate to TF-directed neuronal differentiation is poorly investigated. Here we demonstrate that the TF Cux1 controls the formation of the layer II/III corpus callosum (CC) projections through the developmental transcriptional regulation of Kv1 voltage-dependent potassium channels and the resulting postnatal switch to a Kv1-dependent firing mode. Loss of Cux1 function led to a decrease in the expression of Kv1 transcripts, aberrant firing responses, and selective loss of CC contralateral innervation. Firing and innervation were rescued by re-expression of Kv1 or postnatal reactivation of Cux1. Knocking down Kv1 mimicked Cux1-mediated CC axonal loss. These findings reveal that activity-dependent processes are central bona fide components of neuronal TF-differentiation programs and establish the importance of intrinsic firing modes in circuit assembly within the neocortex. The corpus callosum (CC) connects the hemispheres of the cerebral cortex and allows the high associative functions of the mammalian brain. Partial or total CC agenesis is a feature of several developmental disorders (Fame et al., 2011Fame R.M. MacDonald J.L. Macklis J.D. Development, specification, and diversity of callosal projection neurons.Trends Neurosci. 2011; 34: 41-50Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar). The CC is formed by myelinated axons that project from neurons in layers II/III (∼80% in mouse) and V (∼20%) and a very minor layer VI population (Fame et al., 2011Fame R.M. MacDonald J.L. Macklis J.D. Development, specification, and diversity of callosal projection neurons.Trends Neurosci. 2011; 34: 41-50Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar). Cortical pyramidal layer II/III neurons are molecularly defined by the expression of the homeodomain transcription factor (TF) Cux1 (Britanova et al., 2008Britanova O. de Juan Romero C. Cheung A. Kwan K.Y. Schwark M. Gyorgy A. Vogel T. Akopov S. Mitkovski M. Agoston D. et al.Satb2 is a postmitotic determinant for upper-layer neuron specification in the neocortex.Neuron. 2008; 57: 378-392Abstract Full Text Full Text PDF PubMed Scopus (479) Google Scholar, Nieto et al., 2004Nieto M. Monuki E.S. Tang H. Imitola J. Haubst N. Khoury S.J. Cunningham J. Gotz M. Walsh C.A. Expression of Cux-1 and Cux-2 in the subventricular zone and upper layers II-IV of the cerebral cortex.J. Comp. Neurol. 2004; 479: 168-180Crossref PubMed Scopus (378) Google Scholar). They include callosally and noncallosally projecting subpopulations in undetermined proportions (Lefort et al., 2009Lefort S. Tomm C. Floyd Sarria J.C. Petersen C.C. The excitatory neuronal network of the C2 barrel column in mouse primary somatosensory cortex.Neuron. 2009; 61: 301-316Abstract Full Text Full Text PDF PubMed Scopus (573) Google Scholar, Petreanu et al., 2007Petreanu L. Huber D. Sobczyk A. Svoboda K. Channelrhodopsin-2-assisted circuit mapping of long-range callosal projections.Nat. Neurosci. 2007; 10: 663-668Crossref PubMed Scopus (665) Google Scholar). Callosal neurons branch their axons at layers II/III and V, both contralaterally (interhemispheric) and ipsilaterally (intrahemispheric) (Fame et al., 2011Fame R.M. MacDonald J.L. Macklis J.D. Development, specification, and diversity of callosal projection neurons.Trends Neurosci. 2011; 34: 41-50Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar, Lefort et al., 2009Lefort S. Tomm C. Floyd Sarria J.C. Petersen C.C. The excitatory neuronal network of the C2 barrel column in mouse primary somatosensory cortex.Neuron. 2009; 61: 301-316Abstract Full Text Full Text PDF PubMed Scopus (573) Google Scholar, Petreanu et al., 2007Petreanu L. Huber D. Sobczyk A. Svoboda K. Channelrhodopsin-2-assisted circuit mapping of long-range callosal projections.Nat. Neurosci. 2007; 10: 663-668Crossref PubMed Scopus (665) Google Scholar). Noncallosal layer II/III subpopulations project only ipsilaterally to layers II/III and V (Mitchell and Macklis, 2005Mitchell B.D. Macklis J.D. Large-scale maintenance of dual projections by callosal and frontal cortical projection neurons in adult mice.J. Comp. Neurol. 2005; 482: 17-32Crossref PubMed Scopus (75) Google Scholar). Activity-dependent mechanisms are fundamental processes contributing to neuronal wiring and to the formation of functional circuits in the brain, but they are poorly understood (Ackman and Crair, 2014Ackman J.B. Crair M.C. Role of emergent neural activity in visual map development.Curr. Opin. Neurobiol. 2014; 24: 166-175Crossref PubMed Scopus (122) Google Scholar, Kano and Hashimoto, 2009Kano M. Hashimoto K. Synapse elimination in the central nervous system.Curr. Opin. Neurobiol. 2009; 19: 154-161Crossref PubMed Scopus (149) Google Scholar, Kirkby et al., 2013Kirkby L.A. Sack G.S. Firl A. Feller M.B. A role for correlated spontaneous activity in the assembly of neural circuits.Neuron. 2013; 80: 1129-1144Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar, Stanley, 2013Stanley G.B. Reading and writing the neural code.Nat. Neurosci. 2013; 16: 259-263Crossref PubMed Scopus (73) Google Scholar). In CC formation, studies have shown that diminishing layer II/III neurons’ excitability by overexpressing the inward-rectifier potassium-ion channel Kir2.1 reduces axonal branching and changes the columnar patterns of callosal innervation in the contralateral hemisphere (Mizuno et al., 2010Mizuno H. Hirano T. Tagawa Y. Pre-synaptic and post-synaptic neuronal activity supports the axon development of callosal projection neurons during different post-natal periods in the mouse cerebral cortex.Eur. J. Neurosci. 2010; 31: 410-424Crossref PubMed Scopus (49) Google Scholar, Suárez et al., 2014Suárez R. Fenlon L.R. Marek R. Avitan L. Sah P. Goodhill G.J. Richards L.J. Balanced interhemispheric cortical activity is required for correct targeting of the corpus callosum.Neuron. 2014; 82: 1289-1298Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, Wang et al., 2007Wang C.L. Zhang L. Zhou Y. Zhou J. Yang X.J. Duan S.M. Xiong Z.Q. Ding Y.Q. Activity-dependent development of callosal projections in the somatosensory cortex.J. Neurosci. 2007; 27: 11334-11342Crossref PubMed Scopus (150) Google Scholar). More recently, it was shown that altered innervation is not due to decreased firing rates per se, but because reducing excitability disrupts the balanced cortical activity of the hemispheres (Suárez et al., 2014Suárez R. Fenlon L.R. Marek R. Avitan L. Sah P. Goodhill G.J. Richards L.J. Balanced interhemispheric cortical activity is required for correct targeting of the corpus callosum.Neuron. 2014; 82: 1289-1298Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). These studies pointed to the intrinsic excitability of neurons as possibly determining CC development by regulating the neurons’ communication and/or forms of cortical activity. Yet much remains to be learned about how activity instructs callosal connectivity. Cux1 and other layer-specific TFs have been shown to govern several aspects of neuronal identity and connectivity (Cubelos et al., 2010Cubelos B. Sebastián-Serrano A. Beccari L. Calcagnotto M.E. Cisneros E. Kim S. Dopazo A. Alvarez-Dolado M. Redondo J.M. Bovolenta P. et al.Cux1 and Cux2 regulate dendritic branching, spine morphology, and synapses of the upper layer neurons of the cortex.Neuron. 2010; 66: 523-535Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar, Fame et al., 2011Fame R.M. MacDonald J.L. Macklis J.D. Development, specification, and diversity of callosal projection neurons.Trends Neurosci. 2011; 34: 41-50Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar, Lodato et al., 2015Lodato S. Shetty A.S. Arlotta P. Cerebral cortex assembly: generating and reprogramming projection neuron diversity.Trends Neurosci. 2015; 38: 117-125Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). However, the possible links between TF determining the molecular identity of neuronal populations and activity-dependent wiring are only beginning to be investigated (García-Frigola and Herrera, 2010García-Frigola C. Herrera E. Zic2 regulates the expression of Sert to modulate eye-specific refinement at the visual targets.EMBO J. 2010; 29: 3170-3183Crossref PubMed Scopus (47) Google Scholar). A recent report demonstrated the coupling of Er81 expression and intrinsic excitability through the regulation of Kv1 channels in a subpopulation of interneurons. Importantly, it was shown that Er81 expression levels are dynamically modulated in an activity-dependent manner along the life of the neuron, providing a mechanistic explanation for the activity-dependent regulation of the excitability of these interneurons (Dehorter et al., 2015Dehorter N. Ciceri G. Bartolini G. Lim L. del Pino I. Marín O. Tuning of fast-spiking interneuron properties by an activity-dependent transcriptional switch.Science. 2015; 349: 1216-1220Crossref PubMed Scopus (95) Google Scholar). In contrast, it is not known whether neuronal subtype-specific transcriptional programs instruct the specific firing responses of pyramidal neurons. Some reports have linked the identity of mature adult pyramidal neurons to their distinct firing responses (De la Rossa et al., 2013De la Rossa A. Bellone C. Golding B. Vitali I. Moss J. Toni N. Lüscher C. Jabaudon D. In vivo reprogramming of circuit connectivity in postmitotic neocortical neurons.Nat. Neurosci. 2013; 16: 193-200Crossref PubMed Scopus (139) Google Scholar, Otsuka and Kawaguchi, 2011Otsuka T. Kawaguchi Y. Cell diversity and connection specificity between callosal projection neurons in the frontal cortex.J. Neurosci. 2011; 31: 3862-3870Crossref PubMed Scopus (58) Google Scholar) but did not address to what extent this association is due to their intrinsic differentiation. Furthermore, it is unknown if and how TF-directed regulation of excitability might be related to selective wiring. The acquisition of specific firing modes is a gradual process of differentiation that involves the dynamic expression of a wide repertoire of ion channels. Maturation of firing responses during the development of layer II/III neurons correlates with the onset of the critical period of maximal plasticity (Maravall et al., 2004Maravall M. Stern E.A. Svoboda K. Development of intrinsic properties and excitability of layer 2/3 pyramidal neurons during a critical period for sensory maps in rat barrel cortex.J. Neurophysiol. 2004; 92: 144-156Crossref PubMed Scopus (66) Google Scholar). From postnatal day (P)10 to approximately P17 in rodent somatosensory (SS) cortex, spiking behavior of layer II/III neurons shifts from phasic (more spike-frequency adaptation) to regular (less spiking adaptation) over time, such that the number of action potentials (APs) elicited by a given stimulus increases (Locke and Nerbonne, 1997Locke R.E. Nerbonne J.M. Role of voltage-gated K+ currents in mediating the regular-spiking phenotype of callosal-projecting rat visual cortical neurons.J. Neurophysiol. 1997; 78: 2321-2335PubMed Google Scholar, Maravall et al., 2004Maravall M. Stern E.A. Svoboda K. Development of intrinsic properties and excitability of layer 2/3 pyramidal neurons during a critical period for sensory maps in rat barrel cortex.J. Neurophysiol. 2004; 92: 144-156Crossref PubMed Scopus (66) Google Scholar). Concurrent with these electrical changes, the expression of voltage-gated potassium channels (Kv1) increases in cortical pyramidal neurons from P8 onward (Guan et al., 2011Guan D. Horton L.R. Armstrong W.E. Foehring R.C. Postnatal development of A-type and Kv1- and Kv2-mediated potassium channel currents in neocortical pyramidal neurons.J. Neurophysiol. 2011; 105: 2976-2988Crossref PubMed Scopus (35) Google Scholar). Kv1 channels open at voltages close to AP threshold, limit excitability, and contribute to the emergence of distinct firing modes (Goldberg et al., 2008Goldberg E.M. Clark B.D. Zagha E. Nahmani M. Erisir A. Rudy B. K+ channels at the axon initial segment dampen near-threshold excitability of neocortical fast-spiking GABAergic interneurons.Neuron. 2008; 58: 387-400Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar, Kole and Stuart, 2012Kole M.H. Stuart G.J. Signal processing in the axon initial segment.Neuron. 2012; 73: 235-247Abstract Full Text Full Text PDF PubMed Scopus (286) Google Scholar, Locke and Nerbonne, 1997Locke R.E. Nerbonne J.M. Role of voltage-gated K+ currents in mediating the regular-spiking phenotype of callosal-projecting rat visual cortical neurons.J. Neurophysiol. 1997; 78: 2321-2335PubMed Google Scholar, Shu et al., 2007Shu Y. Yu Y. Yang J. McCormick D.A. Selective control of cortical axonal spikes by a slowly inactivating K+ current.Proc. Natl. Acad. Sci. USA. 2007; 104: 11453-11458Crossref PubMed Scopus (144) Google Scholar). These channels are key to the strong adapting behavior of P10–P12 layer II/III neurons: pharmacological blockade of Kv1 currents in these neurons decreased spiking adapting capacity and increased firing rates such that they resembled the firing responses of more mature P16 neurons (Locke and Nerbonne, 1997Locke R.E. Nerbonne J.M. Role of voltage-gated K+ currents in mediating the regular-spiking phenotype of callosal-projecting rat visual cortical neurons.J. Neurophysiol. 1997; 78: 2321-2335PubMed Google Scholar). Here we investigate the role of the transcription factor Cux1 in callosal axon development. We find that downmodulation of Cux1 expression results in complete impairment of callosal axonal innervation in the contralateral cortical plate and show that this is due to the inability to switch on Kv1-dependent firing responses. Our data indicate that transcriptional regulation of Kv1.1 and Kv1.3 genes by Cux1 contributes to the normal electrical differentiation and the developmental upregulation of Kv1 channels in layer II/III neurons. We also show that restoring Kv1 currents in Cux1-deficient neurons allows the acquisition of a strong-adapting firing mode and rescues contralateral CC innervation, while knocking down Kv1 channels eliminates contralateral innervation. Restoring Cux1 expression after P8 is sufficient to rescue electrical and axonal defects of shRNACux1 layer II/III neurons. Our data thus demonstrate that Cux1-mediated regulation of Kv1-dependent firing is a developmental mechanism that determines CC contralateral innervation of callosal layer II/III neurons. The results demonstrate the importance of electrical differentiation for the establishment of neuronal networks, which has implications for the treatment of neuronal disorders. To determine the role of Cux1 in connectivity, we knocked down gene expression in layer II/III neurons of the SS cortex using a previously reported shRNA (shRNACux1) in conjunction with in utero electroporation (IUE) of wild-type (WT) embryonic day (E)15 mouse cortices (Cubelos et al., 2010Cubelos B. Sebastián-Serrano A. Beccari L. Calcagnotto M.E. Cisneros E. Kim S. Dopazo A. Alvarez-Dolado M. Redondo J.M. Bovolenta P. et al.Cux1 and Cux2 regulate dendritic branching, spine morphology, and synapses of the upper layer neurons of the cortex.Neuron. 2010; 66: 523-535Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar). This knockdown strategy circumvents the prenatal lethality of Cux1 knockout mice (Cubelos et al., 2010Cubelos B. Sebastián-Serrano A. Beccari L. Calcagnotto M.E. Cisneros E. Kim S. Dopazo A. Alvarez-Dolado M. Redondo J.M. Bovolenta P. et al.Cux1 and Cux2 regulate dendritic branching, spine morphology, and synapses of the upper layer neurons of the cortex.Neuron. 2010; 66: 523-535Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar) and allows the visualization of ipsilateral and contralateral axons of the targeted neurons via coelectroporation of a plasmid encoding the green fluorescent protein (CAG-GFP) (Cubelos et al., 2010Cubelos B. Sebastián-Serrano A. Beccari L. Calcagnotto M.E. Cisneros E. Kim S. Dopazo A. Alvarez-Dolado M. Redondo J.M. Bovolenta P. et al.Cux1 and Cux2 regulate dendritic branching, spine morphology, and synapses of the upper layer neurons of the cortex.Neuron. 2010; 66: 523-535Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar; Figures 1A, 1B, and S1A, available online). Analysis of callosal contralateral axons appeared relevant because axonal retrotracing labeling of contralateral projections demonstrated that all labeled callosal layer II/III neurons expressed Cux1 (Figure S1B), as expected by the widespread expression of Cux1 in layer II/III (Britanova et al., 2008Britanova O. de Juan Romero C. Cheung A. Kwan K.Y. Schwark M. Gyorgy A. Vogel T. Akopov S. Mitkovski M. Agoston D. et al.Satb2 is a postmitotic determinant for upper-layer neuron specification in the neocortex.Neuron. 2008; 57: 378-392Abstract Full Text Full Text PDF PubMed Scopus (479) Google Scholar, Cubelos et al., 2010Cubelos B. Sebastián-Serrano A. Beccari L. Calcagnotto M.E. Cisneros E. Kim S. Dopazo A. Alvarez-Dolado M. Redondo J.M. Bovolenta P. et al.Cux1 and Cux2 regulate dendritic branching, spine morphology, and synapses of the upper layer neurons of the cortex.Neuron. 2010; 66: 523-535Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar). We first analyzed GFP-positive layer II/III axonal projections in control shRNA-electroporated mice at P16, an age at which the branching pattern reflects that of the mature circuit (Mizuno et al., 2010Mizuno H. Hirano T. Tagawa Y. Pre-synaptic and post-synaptic neuronal activity supports the axon development of callosal projection neurons during different post-natal periods in the mouse cerebral cortex.Eur. J. Neurosci. 2010; 31: 410-424Crossref PubMed Scopus (49) Google Scholar, Suárez et al., 2014Suárez R. Fenlon L.R. Marek R. Avitan L. Sah P. Goodhill G.J. Richards L.J. Balanced interhemispheric cortical activity is required for correct targeting of the corpus callosum.Neuron. 2014; 82: 1289-1298Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). As shown in Figure 1C, in the electroporated ipsilateral side, GFP-positive dendritic and axonal processes intermingled with the somas of layer II/III targeted neurons, and axonal branches developed in layer V; in the contralateral hemisphere, callosal axons formed columns with profuse branching in layers II/III and V in the SS areas and a diffuse layer pattern in the insular cortex (Figures 1A and 1D). Upon shRNA-mediated Cux1 knockdown, we did not detect any major defect in ipsilateral axonal projections in layers II/III and V (Figures 1E and 1G). In contrast, loss of Cux1 function eliminated most of the GFP-positive axons in the contralateral hemisphere, both in the SS and the insular cortex, and greatly reduced those within the contralateral white matter (WM) (Figures 1F–1I). These defects were not due to delayed innervation, as they persisted at P28, nor could they be explained by alternative GFP axonal tracts toward ventral, frontal, or rostral areas (data not shown). They were also not due to neuronal death because, as we previously reported (Cubelos et al., 2010Cubelos B. Sebastián-Serrano A. Beccari L. Calcagnotto M.E. Cisneros E. Kim S. Dopazo A. Alvarez-Dolado M. Redondo J.M. Bovolenta P. et al.Cux1 and Cux2 regulate dendritic branching, spine morphology, and synapses of the upper layer neurons of the cortex.Neuron. 2010; 66: 523-535Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar), shRNACux1-electroporated neurons did not show signs of degeneration or increased expression of the apoptotic marker Cleaved Caspase 3 (Figure S1C). The specificity of shRNACux1-mediated defects in CC innervation was demonstrated by reversion of the innervation phenotype via coelectroporation of a shRNACux1-resistant Cux1 construct (Supplemental Experimental Procedures and Figures S1D and S1E). As most layer II/III neurons also express the Cux1 homolog Cux2 (Cubelos et al., 2010Cubelos B. Sebastián-Serrano A. Beccari L. Calcagnotto M.E. Cisneros E. Kim S. Dopazo A. Alvarez-Dolado M. Redondo J.M. Bovolenta P. et al.Cux1 and Cux2 regulate dendritic branching, spine morphology, and synapses of the upper layer neurons of the cortex.Neuron. 2010; 66: 523-535Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar), we also analyzed the effects of loss of function of Cux2 on contralateral innervation using Cux2 knockout mice or shRNACux2 in WT mice. These experiments did not reveal impaired CC contralateral connections (Figures S1D and S1E), which indicates that control of contralateral CC innervation is a Cux1-specific function. Overall, the results show that Cux1 is essential for the establishment of CC contralateral axonal innervation. We next analyzed earlier stages of CC axonal development to investigate the process leading to the absence of P16 contralateral CC axons induced by loss of Cux1 function. At P2–P4, soon after axons cross the midline, no major differences were observed between control and Cux1-deficient axons (Figures S2Aand S2B). At P8, when axons branch in the WM and begin to invade the contralateral cortical plate, the only apparent difference was a slight increase in GFP signal in the WM of shRNACux1-electroporated brains as compared to controls (Figures 2A, 2B, and 2H ). In contrast, by P10, the phenotype resulting from Cux1 knockdown was striking and equivalent to that of P16 brains: while in control mice GFP-positive axons profusely invaded the contralateral territories in a still-unrefined columnar pattern (Figures 2C and 2E), only a few axons appeared in the cortical plate in shRNACux1-electroporated brains (Figures 2D, 2F, and 2G). There was also a significant decrease of GFP-positive axons in the WM at both P10 and P12 (Figures 2H and S2A), indicating a reduced number of WM branches compared to controls. At P28, the reduction in GFP signal in the contralateral WM of shRNACux1-targeted brains was as marked as, but not higher than, that observed at P16, indicating that not all WM axons are eventually eliminated (not shown). These data indicate that axons of Cux1-deficient neurons initiate invasion of the cortical plate normally but cannot sustain further development after P8. The reduction of GFP-positive axons in the cortical plate and WM observed between P8 and P10 suggests that Cux1-deficient axons retract. The onset of axonal loss in Cux1-deficient conditions coincides with the earliest establishment of synapses, and it has been reported that CC axons that are unable to make synapses are eliminated (Wang et al., 2007Wang C.L. Zhang L. Zhou Y. Zhou J. Yang X.J. Duan S.M. Xiong Z.Q. Ding Y.Q. Activity-dependent development of callosal projections in the somatosensory cortex.J. Neurosci. 2007; 27: 11334-11342Crossref PubMed Scopus (150) Google Scholar). However, coelectroporation experiments with a construct encoding fluorescently tagged synaptophysin showed indistinguishable patterns of presynaptic clusters in the axons of control and Cux1-deficient neurons at P8 in both ipsilateral and contralateral territories (Figures S3A–S3C), indicating that the loss of shRNACux1 CC axons is not due to a general inability to make synapses. We next investigated whether a general deficit in branching mechanisms could explain the loss of CC innervation in Cux1-deficient neurons. To be able to reconstruct the branches of individual neurons, we activated GFP expression in a sparse population of control or shRNACux1 cells by coelectroporating a GFP-floxed construct (pCALNL-GFP) and very low concentrations of a CRE plasmid (see Experimental Procedures). We then reconstructed individual axons at the contralateral cortical plate and analyzed the number of branches. This analysis showed no differences between P8 shRNACux1 and control branches (Figures S3D and S3E). This indicated that Cux1-deficient callosal neurons can branch normally in contralateral territories until at least P8. At P10 there were very few axons in the cortical plate of shRNACux1-electroporated brains, but these few axons did not show defective branching compared to controls (Figures S3D and S3E). Thus, in Cux1-deficient contralateral axons, we did not observe any evidence to suggest a general branching deficit. We further investigated potential branching deficits by analyzing the ipsilateral axons of Cux1-deficient layer II/III neurons projecting callosally. This also assessed whether axonal loss is specific to the contralateral axonal territory. As in P16 (Figure 1E), in IUE experiments targeting the majority of layer II/III neurons, no obvious differences were detected between ipsilateral axons of control and shRNACux1-targeted neurons at P4 (not shown) or P8 (Figure S2C). To distinguish possible differences with neurons projecting only ipsilaterally, the subpopulation of control and shRNACux1-targeted neurons with axons crossing to the contralateral hemisphere were retrograde labeled at P5 by fluorescently labeled cholera toxin subunit B (CTB) injections in the CC at the midline. Ipsilateral branches of individual CTB+ neurons were analyzed at P11, when the major loss of contralateral axons has already occurred in shRNACux1 brains, using the CRE-floxed-GFP dilution strategy. Reconstructions and quantifications of the number of branches in layers II/III and V showed that ipsilateral arbors from all Cux1-deficient CTB+ callosal neurons were indistinguishable from controls (Figure 3 and Movie S1). Importantly, this demonstrates that Cux1-deficient axons branch and develop normally in ipsilateral territories. Altogether, our data demonstrate that loss of Cux1 does not generally alter the capacity of callosal neurons to establish synapses or axonal branches but specifically disrupts the development of contralateral callosal connectivity. Activity has a role in regulating CC axonal development (Mizuno et al., 2010Mizuno H. Hirano T. Tagawa Y. Pre-synaptic and post-synaptic neuronal activity supports the axon development of callosal projection neurons during different post-natal periods in the mouse cerebral cortex.Eur. J. Neurosci. 2010; 31: 410-424Crossref PubMed Scopus (49) Google Scholar, Suárez et al., 2014Suárez R. Fenlon L.R. Marek R. Avitan L. Sah P. Goodhill G.J. Richards L.J. Balanced interhemispheric cortical activity is required for correct targeting of the corpus callosum.Neuron. 2014; 82: 1289-1298Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, Wang et al., 2007Wang C.L. Zhang L. Zhou Y. Zhou J. Yang X.J. Duan S.M. Xiong Z.Q. Ding Y.Q. Activity-dependent development of callosal projections in the somatosensory cortex.J. Neurosci. 2007; 27: 11334-11342Crossref PubMed Scopus (150) Google Scholar). In order to ascertain a possible role for neuronal activity in Cux1-mediated CC contralateral patterning, we perturbed electrical activity by utilizing the inwardly rectifying K+ channel Kir2.1. Overexpression of Kir2.1 in WT brains eliminated many of the branches of the primary SS areas (60% innervation compared to controls) and altered the normal CC contralateral layer-specific branching pattern (Figures 4A, 4C, and 4E ), coinciding with previously reported data (Mizuno et al., 2010Mizuno H. Hirano T. Tagawa Y. Pre-synaptic and post-synaptic neuronal activity supports the axon development of callosal projection neurons during different post-natal periods in the mouse cerebral cortex.Eur. J. Neurosci. 2010; 31: 410-424Crossref PubMed Scopus (49) Google Scholar, Suárez et al., 2014Suárez R. Fenlon L.R. Marek R. Avitan L. Sah P. Goodhill G.J. Richards L.J. Balanced interhemispheric cortical acti" @default.
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• W2264766091 date "2016-02-01" @default.
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• W2264766091 title "Cux1 Enables Interhemispheric Connections of Layer II/III Neurons by Regulating Kv1-Dependent Firing" @default.
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• W2264766091 cites W1970876750 @default.
• W2264766091 cites W1974893701 @default.
• W2264766091 cites W1977035226 @default.
• W2264766091 cites W1983281184 @default.
• W2264766091 cites W1983731822 @default.
• W2264766091 cites W2001463755 @default.
• W2264766091 cites W2008693831 @default.
• W2264766091 cites W2012404665 @default.
• W2264766091 cites W2015816905 @default.
• W2264766091 cites W2016716381 @default.
• W2264766091 cites W2017354510 @default.
• W2264766091 cites W2028529429 @default.
• W2264766091 cites W2031030159 @default.
• W2264766091 cites W2032321427 @default.
• W2264766091 cites W2042186471 @default.
• W2264766091 cites W2044171033 @default.
• W2264766091 cites W2046165140 @default.
• W2264766091 cites W2049548746 @default.
• W2264766091 cites W2052156323 @default.
• W2264766091 cites W2055905369 @default.
• W2264766091 cites W2080586470 @default.
• W2264766091 cites W2082023836 @default.
• W2264766091 cites W2086496688 @default.
• W2264766091 cites W2087226217 @default.
• W2264766091 cites W2089518215 @default.
• W2264766091 cites W2103655875 @default.
• W2264766091 cites W2105882251 @default.
• W2264766091 cites W2113054914 @default.
• W2264766091 cites W2123973146 @default.
• W2264766091 cites W2129693714 @default.
• W2264766091 cites W2134036551 @default.
• W2264766091 cites W2135369850 @default.
• W2264766091 cites W2137393514 @default.
• W2264766091 cites W2137614532 @default.
• W2264766091 cites W2145935221 @default.
• W2264766091 cites W2148212562 @default.
• W2264766091 cites W2148998821 @default.
• W2264766091 cites W2149375030 @default.
• W2264766091 cites W2153286410 @default.
• W2264766091 cites W2159662195 @default.
• W2264766091 cites W2163438163 @default.
• W2264766091 cites W2168228198 @default.
• W2264766091 cites W2414752808 @default.
• W2264766091 cites W4293200984 @default.
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