The Long Non-coding RNA-LncBAR Temporally Controls Neocortical And Adult Neurogenesis

Higher cognitive and sensory functions in mammals depend on the cerebral cortex（also known as the neocortex）,which is a six-layered structure largely composed of projection neurons.Projection neurons are derived sequentially from neural progenitor cells（NPC）in the ventricular and subventricular zones（V-SVZ）of embryonic brains in an“inside-out”pattern:early-born projection neurons are located in deep layers,and those generated later are located in upper layers.The main types of neural progenitors include radial glia（RG）and intermediate progenitors（IP）.Indirect neurogenesis is the dominant pattern of neocortex neurogenesis:radial glia divides asymmetrically to produce intermediate progenitors,which then differentiate into projection neurons after several proliferative divisions（transit-amplification）.During development and evolution,the number and proliferative capacity of intermediate progenitor cells directly regulate the development and expansion of the cerebral cortex.Neurogenesis persists in adult mammalian brains.In the adult mammalian brain,active neurogenesis takes place in the subventricular zone of lateral ventricles and the subgranular zone（SGZ）of hippocampal dentate gyrus.Neurogenesis in SVZ and SGZ complements the production of olfactory bulb interneurons and hippocampal neurons respectively.During neurogenesis,the temporal plasticity of neural progenitors and fate determination of projection neurons are largely controlled by actions of specific transcription factors.Moreover,the epigenetic regulation is key to selective expression of transcription factors in various developmental stages and cell types.Epigenetic regulatory mechanisms include chromatin conformation,histone modification,DNA/RNA modification and long non-coding RNAs.This study focused on the regulatory function of long non-coding RNA（lnc RNA）in neurogenesis.Although a large number of lnc RNAs have been identified in the nervous system,the functions of most lnc RNAs were not clear and many lnc RNA knockout mice did not show physiological changes or behavioral abnormalities.In our lab’s previous work,we analyzed transcriptome data from the developing cerebral cortices of mice,and constructed co-expression modules of coding and non-coding genes.We identified a large number of lnc RNAs expressed in the developing cerebral cortex.Then in situ hybridization was applied to unveil expression profiles of several lnc RNA at various developmental stages.A batch of lnc RNAs with regional specificity and/or relatively high expression levels were selected for further functional studies.In utero electroporation assay was conducted to screen lnc RNAs with regulatory functions in neurogenesis.A lnc RNA-LncBAR（BAF complex associated non-coding RNA）that regulates neurogenesis was identified.LncBAR is highly expressed in the ventricular-subventricular region in the early stage of cerebral cortex development,while in late stage,it is highly expressed in the cortical plate.The whole-body knockout mice of LncBAR(LncBAR^KO)were constructed by using CRISPR/Cas9 technique.The adult LncBAR knockout neocortex contains more deep-layer but fewer upper-layer projection neurons.During embryonic development,the loss of LncBAR leads to an overproduction of intermediate progenitors,but the proliferative capacity of intermediate progenitors differed at developmental stages:in early neurogenesis,intermediate progenitors in LncBAR^KO cortices proliferated actively;However,in the mid-later stage of neurogenesis,the LncBAR^KO intermediate progenitors had prolonged cell cycle duration and hampered proliferation.Therefore,LncBAR depletion results in an increase of deep-layer projection neurons in early cortical development and a decrease of upper-layer projection neurons in mid-late cortical development.Furthermore,the olfactory bulbs（OB）of adult LncBAR^KO brains were significantly smaller than control brains,with numbers of Calretinin-and TBR2-positive interneurons in olfactory bulb decreased.In adult LncBAR^KO brains,neuroblasts migrate normally in rostral migratory stream（RMS）,but the number of adult neural stem cells（a NSC）in the V-SVZ of lateral ventricles decreases,leading to a compromised adult neurogenesis.The adult neural stem cells in the V-SVZ of the lateral ventricles are descendants of embryonic neural progenitors,loss of LncBAR leads to shrunken a NSC pool due to diminished NPC pool in embryonic stages.We further studied the regulatory mechanism of LncBAR,and found that LncBAR bound to multiple subunits of the BAF（SWI/SNF）chromatin remodeling complex.RNA immunoprecipitation assay demonstrated that the BAF complex ATPase subunit-BRG1could associate with LncBAR.However,LncBAR deletion did not affect the expression and assembly of BAF core subunit in cortex.When combining RNA-seq and BRG1 Ch IP-seq data of embryonic neural progenitor cells,LncBAR/BAF complex was found to maintain the expression of transcription factor gene Zbtb20.Studies have shown that ZBTB20 plays essential roles in cortical development and adult neurogenesis.The depletion of LncBAR enhances the association of BRG1 with multiple sites in Zbtb20genomic regions,leading to inhibited transcription of Zbtb20.ZBTB20 overexpression in LncBAR-knockout neural precursors reverses compromised cell cycle progressions of intermediate progenitors.Finally,LncBAR overexpression also promotes divisions of intermediate progenitors.In conclusion,this study revealed a lnc RNA-mediated epigenetic regulatory mechanism.The LncBAR/BAF complex-Zbtb20 circuit regulates the number and cell cycle progression of intermediate progenitors,thereby controlling cortical neurogenesis and fate decision of projection neurons.In addition,LncBAR regulates the size of adult V-SVZ neural stem cell pool by maintaining the number of embryonic neural progenitor cells.