Selection Of Callosal Projection Neuron Fate By Zbtb18

Background Callosal projection neuron(CPN),also known as intercortical projection neurons,are a major subtype of cortical projection neurons.The majority of CPNs populate the upper layers of the cerebral cortex(layer ?,? and ?),and their axons project to the contralateral hemisphere and contribute to the intricate neuronal network.In the past a few decades,significant discoveries have been made regarding the origin and development of CPNs.Taking human and the lab mouse,the most studied mammalian models as example,studies show CPNS originate from intermediate progenitors(IPCs)residing in the embryonic subventricular zone(SVZ),after mitosis they migrate past early-born subcerebral projection neurons and reach the upper layers of the cerebral cortex.Once CPNs reach their destination,they send axons to the contralateral hemisphere in response to axon guidance molecules.From the view of vertebrate evolution,CPNs are unique to mammals;and they have expanded in population and also evolved in structure and functionality as the mammalian species evolve.In humans,CPNs far exceed another major type of projection neuron,subcerebral projection neurons(ScPNs)in number and play indispensable role in higher order brain activities.In placental mammals,the axonal projections of CPNs form the corpus callosum,which connects the two cerebral hemisphere as a“short-cut” and thus facilitates the communication between the two hemispheres.The emergence of CPN and the corpus callosum may have contributed tremendously to the evolution of mammalian intelligence,but the molecular control as well as the underlying drive for CPN cell fate,differentiation,maturation and evolution was still not clear.Studies have shown that potent transcription factors are able to control cell fate choice through regulating the activity of cisregulatory elements,and it is possible there is a key transcription factor/transcription factors plays crucial role in CPN fate choice.Objective:1.To understand the molecular control in the genesis,differentiation and maturation of allosal projection neurons;2.To understand the role of transcription factors and cis-regulatory elements and their nteraction in regulating callosal projection neurons and corpus callosum development;3.To understand the evolutionary incidents that promoted the emergence of mammalian allosal projection neuron and corpus callosum;4.To explore the role of the evolution and adaptive mutation of cis-regulatory elements in he emergence and evolution of corpus callosum,neuronal network and intelligence in ammals.Methods:The brain of transgenic mice Fezf2-Gfp and Arpp21-Gfp were analyzed and GFP labeled cortical cells were determined to be subcerebral projection neuron(Fezf2-Gfp)and callosal projection neuron(Arpp21-Gfp),respectively.Neocortices of postnatal day 0 mice were dissected and dissociated and GFP positive cells were selected by Fluoroscence Activated Cell Sorting(FACS).FACS collected cells were used for two purposes:1)RNA-sequencing to acquire the transcriptome information;and 2)Chromatin immuno-precipitation and sequencing(ChIP-seq)with H3K27 ac antibody to acquire genome-wide map of active cis-regulatory elements of these two projection neuron subtypes.Next,integrated analysis were performed to screen for important transcription factors and candidate callosal projection neuron enhancers.Transcription factor ZBTB18 was considered as a key transcription factor because of its enriched expression in callosal projection neurons and its ability in activating a characteristic callosal projection neuron enhancer,Cux2 intronic enhancer 7(Cux2-E7).Further investigation of Zbtb18 knockout mouse brain showed a significant loss of callosal projection neurons.IDU,CIDU,BRDU injection were performed on timed-pregnant mice to examine the genesis and migration of callosal projection neurons as well as subcerebral projection neurons.Breed Zbtb18flox/flox;Neurod6-cre;Cag-Cat-Gfp mice to conditionally knockout Zbtb18 in postmitotic neurons and label all cortical projection neurons and their projections with GFP.Examine GFP labeled cortical projection changes in Zbtb18flox/flox;Neurod6-cre;Cag-Cat-Gfp mice.Rescue CPN migration and projection defects by delivery of targeted molecules into e14.5-e15.5 IPCs by in uteroelectroporation.Analyze the DNA sequence conservation and evolutionary mutations of ZBTB18 sites in CPN enhancers,and validate the activity change of CPN enhancers with reptilian and marsupial mutation.Results:Part1 Identification of cis-regulatory network regulating CPN and CC genesis and development1,Co-immunostaining of GFP and projection neurons markers on Fezf2-Gfp andArpp21-Gfp mice brain sections showed Fezf2-Gfp and Arpp21-Gfp cells were ubcerebral projection neurons(ScPN)and callosal projection neurons(CPN),respectively.2,Fezf2-Gfp and Arpp21-Gfp cells were purified and collected by FACS,and RNA-seq evealed Fezf2-Gfp and Arpp21-Gfp cells are representative populations of ubcerebral projection neurons and callosal projection neurons by gene expression rofile.3,ChIP-seq showed that the majority of H3k27 ac signatures were similar across the nome in Fezf2-Gfp and Arpp21-Gfp cells,but in certain loci,distinct H3k27acsignatures were detected,for example,the peaks adjacent to genomic regions of Cux2, ux2,Satb2,Robo1,Robo2,Fezf2,etcs.An enhancer at intronic region of Cux2, ux2-E7 has significantly enriched H3K27 ac signal in Arpp21-Gfp cell but not Fezf2- fp,thus was considered as a CPN-specific enhancer.4,Transgenic mice assay using Cux2-E7 showed it drove GFP expression specifically inlayer ?-? CPNs.5,The integrated analysis of transcription factor binding sites enrichment within Cux2-7 DNA sequence,and transcription factor expression enrichments in developing PNs indicated transcription factor ZBTB18 as a possible regulator of Cux2-E7 ctivity.Luciferase assay validated ZBTB18 activated Cux2-E7 in vitro.6,The investigation of Zbtb18 knockout mice showed inactivation of Zbtb18 from early orebrain progenitors caused agenesis of the upper layers,and agenesis of the corpuscallosum;Zbtb18-/-neocortex has a 3 –layered structure resembling that of reptiles.Part ? Biological process and molecular mechanism of ZBTB18 regulating mammalian CPN fate decision1.Investigation of embryonic mice brain and human cortical tissue showed ZBTB18 is ighly expressed in the subventricular zone(SVZ),Intermiediate zone(IZ),and cortical late(CP)in both the human and mouse embryonic brain.Results:2.RNA-seq revealed loss of Zbtb18 led to significant down-regulation of CPN genes and p-regulations of ScPN genes.3.Neuron-specific Zbtb18 inactivated mice showed the thickness of neocortical layer ?,? nd ? were significantly reduced,and the corpus callosum was not formed.4.In Zbtb18fl/fl;Neurod6-cre mice the dendrites and dendritic spines of CPN are severlyaffected.5.IDU,CIDU,BRDU birth-date labeling revealed the extra BCL11B+neurons were not produced by early progenitors,instead,they were produced by e14.5-e15.5 IPCs,which gave rise to SATB2+ neurons in wild type mice.Examination of e13.5,e14.5,e16.5 and e18.5 neuron-specific Zbtb18 knockout mice showed starting from e14.5,neuronal progenitors produced more BCL11B+ and less of SATB2+ neurons?6.The phenotype of Zbtb18fl/fl;Neurod6-cre mice cortex is highly similar to btb18fl/fl;Emx1-cre mice,indicating a neuronal autonomous role of ZBTB18.The elayed expression of SATB2 and the ectopic expression of BCL11 B are the prominent henotypes in ZBTB18-deficent cortex.7.Delivering microRNA targeting Bcl11 b mRNA into VZ progenitor cells at e14.5-15.5 by n utero electroporation(IUE)rescued the migration defects of CPNs,but not the rojection defects.8.Analysis of transcription factor binding sites enrichment in putative Bcl11 b enhancers dentified by ChIP-seq showed two candidate enhancers had ZBTB18 binding sites. hIP-qPCR confirmed ZBTB18 binding of these two enhancers and luciferase assay howed one of them,Bcl11b-E3 is significantly repressed by ZBTB18.Part ? ZBTB18 regulates CC formation intrinsically1.Examination of Zbtb18flox/flox;Neurod6-cre;Cag-Cat-Gfp mice brain showed while FP-labeled subcerebral projections were mostly normal,GFP-labeled intracortical rojections were severely affected?2.Delivering expression plasmids of ZBTB18 into VZ progenitor cells at e14.5-15.5 by in tero electroporation(IUE)fully rescued the migration and projection defects of CPNs.3.Analysis of RNA-seq data acquired from Zbtb18 knockout mice neocortex showed a mis-expression of axon guidance molecules and transmembrane receptors,most prominently,the down-regulation of Robo1,Robo2,Dcc,Unc5 d,and the up-regulation of Robo3.4.RNA ISH confirmed the mis-expression of selected axon guidance molecules including CC,ROBO1,ROBO2 and ROBO3.5.Delivering expression plasmids of ROBO1,but not DCC or UNC5 D into VZ rogenitor cells at e14.5-15.5 by in utero electroporation(IUE)partially rescued the igration and projection defects of CPNs.Part ? The evolution of cis-regulatory elements possibly contributed to the emergence and evolution of CPN and CC1.Analysis of the conservation of CPN-active cis-regulatory elements revealed significant utations across vertebtate evolution.Take ROBO molecules as an example,3 ROBO nhancers emerged after placental mammals separated from marsupials.2.Analysis of the conservation and evolutionary mutations of Cux2-E7 and Bcl11b-E3 howed at key position within ZBTB18 binding sites mutations happened from reptiles to arsupials,and from marsupials to placental mammals.3.Introducing reptile and marsupial mutation into mouse ZBTB18 binding sites within ux2-E7 and Bcl11b-E3 enhancers significantly changed their responsiveness to BTB18.Conclusions:1,ZBTB18(RP58/ZFP238)intrinsically determines the cell fate and projection of allosal projection neurons by directly binding and regulating the activity of callosal rojection neuron enhancers,which in turn regulates doewnstream transcription actors and selects appropriate axon guidance receptors.2,ZBTB18 activates Cux2 through enhancer Cux2-E7 and inhibits Bcl11 b through cl1b-E3 in nascent callosal projection neurons is a key mechanism for this process; nd this mechanism is unique to mammals.3,Projection neuron enhancers possibly have undergone evolutionary elaborations to acilitate the rise of mammalian CPNs and the corpus callosum formation.