Molecular Mechanisms Of EphA7 And EphB6 In Regulating The Soma Spacing And Self-avoidance

The brain is the body’s most important and complex organ,enabling us feeling,thinking,remembering and acting.Various brain functions are performed by a neural network formed between neurons.The somas and dendrites of neurons receive information,which is transmitted by axons to the next level of neurons in the network.In nervous system development,spacing and dendrite branching are key events for establishing correct neural circuits.Taking the cerebral cortex as an example,the neural stem cells located in the area around the lateral ventricle of the medial cerebral cortex settle down in a specific location through continuous proliferation,differentiation,migration and other processes,forming a highly complex brain cell architecture consisting of six layers of different types of neurons.After migration,the cell bodies kept a certain distance to form regular layered structures.At the same time,the dendrites of the neurons begin to develop into lush dendritic trees.A basic rule is that the dendrite branches of neurons keep a certain distance from each other during growth,which is called self-avoidance.The genes controlling dendrite self-avoidance of sensory neurons have been found in nematodes,but the mechanism of dendrite self-avoidance in mammalian brain is still unknown.Previous studies have observed expression of the transcription factor Satb2 in cortical excitatory neurons.Knockdown of the transcription factor Satb2 in cortical pyramidal neurons causes cell body clustering,and their dendrites failed to expand within the upper layers of the cortex but instead formed fascicles,but its downstream mechanisms have been poorly understood.In gyrencephalic species,the combination of basal progenitor cell amplification and divergent migration contributes to cortical expansion along the radial and tangential axis and finally cortical folding.Failure in these processes can cause severe abnormalities in the folding such as huamn lissencephaly.Therefore,it is of great significance to elucidate Satb2 regulated signaling pathways for understanding the molecular mechanisms of neuronal development and the pathogenesis of developmental brain diseases.Based on this,we screened the differentially expressed genes in the brain of control and Satb2 conditional knockout mice by sequencing and found significant changes in the Eph family of genes,and previous studies have shown that Ephrin-Eph signaling is involved in regulating the balance between cell-cell adhesive and/or repulsive properties of cerebral pyramidal neurons,which is critical for various cellular events.Based on this,we investigated the role of Eph family in somas spacing and dendrite branch formation during cortical development,as well as its regulatory relationship with transcription factor Satb2.First,the differentially expressed genes between the control group and Satb2^Emx1CKO mice were analyzed by RNA-seq sequencing combined with RT-q PCR and in situ hybridization（ISH）.Among them,3 were up-regulated（Eph B1,Eph A6,Eph A7）and3 were down-regulated（Ephrin A5,Eph A4,Eph B6）in cortical layer II/III of Satb2CKO mice.According to the principle of phenotypic consistency,overexpression of Eph A7 by in utero electroporation（IUE）in late-born cortical neurons of wild-type mice resulted in impaired pyramid-somas clustering and dendrite self-avoidance,similar to the phenotype induced by downregulation of Satb2 expression.Importantly,the phenotype caused by Satb2 knockdown can be rescued by reducing cortical Eph A7expression.Ch IP and dual luciferase reporter gene assay showed that Satb2 could directly bind to the nuclear matrix attachment region of Eph A7 gene and negatively regulate Eph A7 expression.These results suggest that the effect of Eph A7 on cortical cell body spatial distribution and dendrite self-avoidance depends on the regulation of transcription factor Satb2.In addition,our results indicate that Eph A7 overexpression affects somas spacing and dendrite self-avoidance in a cellular autonomous manner.Eph A7 overexpression affects neuronal migration and radial glial fiber arrangement,but does not change layer II/III cell fate,neuronal progenitor cell proliferation and cell survival.The second part of this paper is about the role of Eph B6 in cellular spatial distribution and dendrite self-avoidance.In Satb2 CKO,Eph B6 expression was significantly down-regulated in superficial cortical neurons.According to the principle of phenotypic consistency,Eph B6 may be the downstream target gene of Satb2 if the phenotype of Eph B6 is similar to that of Satb2 knocked down in wild-type mice cortex.We found that Eph B6 is dynamically expressed during cortical development by in situ hybridization.After knockdown of Eph B6 in superficial cortical neurons by in utero electroporation experiment,it was found that the distribution of neurons was uniform between control and knockdown of Eph B6,and there was no significant difference in the distribution of cell body,dendrite distribution and neuron migration.These results suggest that the down-regulation of Eph B6 expression is not involved in the cellular and dendritic phenotypes induced by Satb2deficiency.Previous studies have found that Eph B6 is a risk gene for autism spectrum disorder,and the loss of Eph B6 leads to autism-like behavior in mice,some studies have also detected elevated Eph B6 expression in the whole blood of children with autism by using genomic profiling.These reports suggest that Eph B6 plays an important role in the development and progression of brain developmental diseases such as autism,but its role in the development of the nervous system and its relationship with the disease has not been understood.Surprisingly,when Eph B6 was overexpressed by in utero electroporation,the somas of neurons clustered significantly.This phenotype disappeared when both Eph B6 and its ligand Ephrin B2were overexpressed,suggesting that Eph B6/Ephrin B2 is a mutually inhibitory mechanism controlling cortical development.In addition,our study found that overexpression of Ephb6 can lead to impaired neuronal migration and increased proliferation of neural progenitor cells,but does not affect the projection mode and the cell fate of neurons with impaired migration.The cell survival of neurons overexpressing Ephb6 and the fiber structure of radial glial cells are not affected.As for the downstream molecular mechanism,p-Erk may be partially involved in the downstream pathway of Eph B6,but the specific mechanism needs to be further explored.In conclusion,our study found that Eph A7,a member of the Eph family,is critical to the control of cortical neuron somas spacing and dendrite self-avoidance,and this effect depends on the inhibitory regulation of Satb2.These findings provide new insights into the complexity of transcriptional regulation of the morphogenesis of cerebral cortex and the pathogenesis of SATB2-related syndromes.In addition,the Eph family member Eph B6 also plays an important role in the spatial distribution of cortical neurons,but it is not regulated by Satb2.Eph B6 and Ephrin B2 controls soma spacing of cortical neurons in a mutual inhibitory way,and its mechanism may be realized through the cis-interaction between Eph B6 and Ephrin B2 ligand.Thus,our results not only reveal the importance of Eph B6/Ephrin B2 in controlling soma spacing in cortical neurons,but also provide new insights into the potential mechanisms underlying neurodevelopmental disorders.