Role Of Canotical Wnt Signaling In Dendritic Development Of Upper Layer Pyramidal Neurons In Rat Restroplenial Cortex

Dendritogenesis and synaptogenesis are key events of neuronal circuit formation that are finely regulated in time by a multitude of signaling pathways.Abnormal dendritic development of cortical neurons can lead to severe neurodevelopmental disorders.Retrosplenial cortrex(RSC)as one of the important corticol region,has emerged as a key member of a core network of brain regions that underpins a range of cogtive functions.RSC has been reported to be compromised in several neurological and psychiatric disorders and direct lesion of this cortical region is known to result in a significant loss of quality of life for patients.Therefore,finding a treatment to such conditions is now considered of utmost importance to both clinicians and pharmaceutical industry.However,mechanisms that regulate neuron development in RSC remain largely unknown and revealing these mechanisms will be pivotal to develop potential treatment to its pathological conditions.In the recent years,a remarkable candidate has been gaining growing interest in this field,the Wnt signaling pathway.Wnt signaling pathways are implicated in major steps of neocortical development.In vitro studies indicate that Wnt signaling pathways are involved in dendritic growth and arborization,nevertheless,the role in dendritic development in vivo,in the context of cortical development,has not yet been explored.This is the first study reporting molecular mechanism of canonical Wnt signaling in the regulation of dendriticgenesis in RSC pyramidal neurons in vivo.Wnt signaling activity was fisrt examined in layer(L)2 RSC pyramidal neurons.A wellcharacterized canonical Wnt activity reporter was introduced,pCLX-M38TOP-dGFP,in L2 neurons through in utero electroporation at embryonic day(E)18,together with internalcontrol plasmid.At Postnal day(P)0.P4,P10,P21 and P45,a significantly higher percentage of TOPdGFP positive neurons is present in L2.Hence,canonical Wnt signaling is active in L2 pyramidal cells during the postnatal period.Genetic loss-of-function(LOF)and gain-offunction and(GOF)strategies were used to perturb intrinsic canonical Wnt signaling via autoinducible expression lentivector plasmid system(pCWXPGR-pTF-dnTCF4,pCWXPGR-pTF?DIX and pCWXPGR-pTF-?-cat)and in utero electroporation.These plasmids were introduced in L2 pyramidal neurons of RSC by in utero electroporation at E18 and induced gene of interest(GOI)expression by doxycycline(DOX)administration from E21 to P21.To obtain single-cell resolution of dendritic arborization among bundles,we took advantage of iontophoretic post hoc single cell injections.Neurolucida software was used for dendritic reconstruction of injected singel cell.Resutls revealed that neurons with Wnt downregulation displayed reduced dendritic arborization compared with control cells at P21,while Wnt upregulation resulted in increased dendritic complexity.Canonical Wnt LOF effect on dendritic development in L2/3 pyramidal neurons of the somatosensory cortex was also tested and results similar to the ones in RSC results were found.Thus,the involvement of canonical Wnt signaling in dendritic branching seems to be a general phenomenon in the neocortex.The hypothesis of the existence of a key period during which Wnt signaling is required for a correct dendritic development was then investigated.Expression of Wnt downregulation plasmid(pCWXPGR-pTF-dnTCF4)was dox-activated from E21 to P15 and analyzed dendritic morphology at P90.Significant dendritic defects were observed at P90,indicating that the phenotype caused by a transient decrease of canonical Wnt signaling is permanent and irreversible.Then dnTCF4 expressing was induced during different time windows,and the special time window was identified:from E21 to P7.To address how dendritic branching relates to neuronal activity induced mechanisms,neuronal activity was remotely enhanced by esigner drugs(DREADDs)-based chemogenetic approach(pCWXPG-hM3Dq with clozapine-N-oxide(CNO))and neuronal excitability was reduced by overexpressing the inward-rectifying potassium channel Kir2.1(pCWXPGR-pTF-Kir2.1)from E21 to P7,and found no defects in dendritic morphology.Enhanching neural activity in Wnt downregulation cells was not able to rescue dendritic defect either.Futhermore,the link between canonical Wnt signaling and neurotrophin-3(NT3)in dendritogenesis regulation was investigated.First,strong NT3 mRNA expression was found from P0 to P9 in RSC.To test if NT3 plays a role in dendritic development,a short hairpin RNA(shRNA)-specific for NT3(shNT3)was created and its expression from E21 to P7 resulted in neurons with shorter and less branched dendrites compared with control cells at P7.L2 RSC neurons were then electroporated with either a control plasmid(GFP)or pCWXPGRpTF-dnTCF4 plasmid and qPCR on dissected,fluorescence-activated cell sorting(FACS)sorted P7 cells was performed.dnTCF4 induction from E21 to P7 produced significant reduction in NT3 mRNA expression relative to control cells.Combined with bioinformatic analysis,NT3 was suggested as being downstream of Wnt transcriptional activity.To further confirm it,a new experiment using NT3 overexpression to rescue Wnt LOF-induced dendritic defects was conducted.Indeed,NT3 overexpression during Wnt LOF completely restored dendritic arborization at P7.These data strongly support the hypothesis that canonical Wnt signaling acts on early dendritic development of L2 RSC neurons through a transcriptional mechanism regulating NT3 expression.Finally,a different role was hypothesized for canonical Wnt signaling in the late phase of dendritogenesis.Spine development was analyzed from cells that expressed dnTCF4 from P21 to P30.This showed a substantial reduction in spine density compared with control cells at P30.Moreover,statistical analysis of spine morphology indicated that dnTCF4 cells have an increased proportion mature spines,indicating canonical Wnt LOF impairs spine development.hM3Dq was overexpressed and activated with CNO from P21 to P30.hM3Dq-overexpressing cells showed increased spine density compared with controls at P30.Moreover,overexpression of hM3Dq in dnTCF4 neurons was able to rescue Wnt LOF-induced spine deficit.These results suggested that spine formation in the late phase is affected by neuronal activity.NT3 mRNA was then detected in L2 of the RSC at P21 and P25.NT3 and dnTCF4 were overexpressed from P21 to P30 leading to both spine density and maturation being completely rescued.These data suggest that during the late developmental phase,Wnt signaling acts on spine formation by regulating the expression of NT3.Together,these results reveal a novel role for canonical Wnt transcriptional activity in dendritic development of L2 pyramidal neurons in vivo.The present study may provide a new framework for understanding how deregulation of Wnt signaling could affect cortical development,thereby potentially underlying neuropsychiatric disorders previously linked to abnormal dendrites development.