FOXG1 Drives Transcriptomic Networks To Specify Principal Neuron Subtypes During The Development Of The Archicortex And Retrosplenial Cortex

In mammals,the Medial pallium(MP)of the telencephalon comprises the archicortex,which consists of hippocampal CA1,CA3,the dentate gyrus(DG),and the subiculum(Sub C),and the ‘intermediate’ retrosplenial cortex(RSC),which is located between the neocortex and the archicortex.These components of the MP are anatomically and functionally interconnected underpinning a range of cognitive functions,including learning and memory,spatial navigation,and emotional regulation.In the MP,the orderly functioning of hippocampal CA1,CA3,DG,Sub C and RSC depends on the normal specialization of subtype principal neurons,and the abnormal development of MP subtypes will lead to many neurological and psychiatric diseases.However,the mechanisms underlying the specification of its principal neuron subtypes remain largely unexplored.The transcription factor FOXG1 has been demonstrated to control a variety of developmental processes ranging from dorsal-ventral pattern formation of the telencephalon and cell fate determination to cortical circuit specialization by exerting both repressive and activating function.However,no study has examined the contribution of FOXG1 to postmitotic subtype specificity in MP.In this study,by postmitotic deletion of Foxg1 using Nex-Cre mice,and single-cell RNA sequencing of E17.5 MP in mice,we explored the function of FOXG1 during the specification of MP principal neurons.We found that loss of Foxg1 in postmitotic neurons results in severe morphological abnormalities and dysregulation of the identities of principal neuron subtypes in the MP: RSC-Py Ns(UL)and Sub C-Py Ns were lost,and CA3-Py Ns and DG-GCs expanded to the entire MP.Meanwhile,some of the characteristics of CA1-Py Ns were transformed into the characteristics of CA3-Py Ns.By single-cell RNA sequencing using E17.5 MP,we characterized the molecular profiles of principal neuron subtypes in the developing MP,and identified the differentially expressed genes(DEGs)between the control and Foxg1 conditional knockout mice.The transcriptional networks that FOXG1 regulates the specification of MP principal neuron subtypes were analyzed at three levels: subtype-specific genes,subtype-coexpressed genes,and subtype-nonenriched genes.In supporting of the regulatory function of FOXG1 at the above three levels,we used Ch IP-q PCR,luciferase assays and in utero electroporation(IUE)methods,and found that FOXG1 transcriptionally represses Zbtb20,Prox1,Epha4 to prevent CA3 pyramidal neuron and DG granule cell identities during the specification of RSC-Py Ns(UL)and Sub C-Py Ns.Meanwhile,FOXG1 directly activates Nr4a2 to promote Sub C-Py N identity,and activates Satb2 to promote RSC-Py Ns(UL).We also showed that TBR1,controlled by FOXG1 during CA1-Py N specification,was downregulated.Taken together,our results suggest that FOXG1 specifies MP principal neuron subtypes by controlling subtype-specific,subtype-coexpressed and subtype-nonenriched transcriptional programs.These findings will expand our understanding of mechanism underlying MP development,and help us to elucidate the pathogenesis of MP-related high brain function deficits and related neurodevelopmental disorder.