The Role Of The Protocadherin Gene Celsr3 In Brain Development And Its Expression Regulation Mechanism

Celsr3 gene encodes a seven transmembrane receptor with several cadherin ectodomains. It’s specifically and widely expressed in the central and peripheral nervous system. Celsr3 gene knockout mice die at birth, suggesting the important functions of this gene in brain development. Celsr3 has been found to regulate the migration of cortical interneurons, as well as the development of axons and dendrites. However, comparing to the expression pattern, the functions of Celsr3 and Celsr family in brain development are largely unknown.Globus pallidus(GP), a supallium nucleus in the ventral forebrain, is a central component of basal ganglia who functions in motor regulation, especially voluntary movement. The malfunctions of globus pallidus participated in the development of many neuropsychiatric symptoms, such as the neurodegenerative disease Parkinsonism. However, little is known about the development regulation of globus pallidus. We found that the protocadherin gene Celsr3 is wildly expressed in the developing and adult globus pallidus, which indicated its function in the development of globus pallidus. In Celsr3 knockout mice, with immunohischemistry and in situ hybridization, we found the morphology of globus pallidus is abnormal, with cell density increase in the dorsal but decrease in the ventral globus pallidus. Ventral GP is occupied by calbindin positive cells and their axons. These calbindin positive cells do not express globus pallidus marker, but instead express cholinergic neuron marker ChAT, and Islet1, we further demonstrated that they are ectopic from the nucleus basalis of Meynert. In addition, Celsr3 participates in the development of the axon bundles between globus pallidus and other brain nuclei. The axonal projections between globus pallidus and striatum, subthalamic nucleus, substantia nigra and dorsal raphe are compromised in Celsr3 knock out mice, which is due to the projection failure of these axons mostly in the telencephalon-diencephalon boundary. Furthermore, the Nkx2-1 positive IC cells(internal capsule cells) are absent in the Celsr3 mutant globus pallidus of early embryonic stage, which may be the reason of the projection defects of thalamocortical and corticothalamic tracts. Through rescue and shRNA knockdown, we demonstrated that the function of Celsr3 in globus pallidus development is both cell autonomous and non-cell autonomous. Finally, to confer whether Celsr3 regulate GP and axonal development through regulating actin dynamics, we made the Celsr3 WIRS point mutation mice using Crispr/Cas9 system and will do further analysis. These data suggest the important function of Celsr3 in globus pallidus development and shed light onto the mechanisms of Celsr3 in regulating axonal development.We further investigate the expression regulation of Celsr genes. Consistent with its neural specific expression and function, we found the repressor neuron restrictive silencer factor(NRSF, also known as REST) specifically binds to exon 1 of the Celsr3 gene, and regulate the neural-specific expression of Celsr3, but not Celsr1 or Celsr2 gene. ChIP-seq data showed that the insulator CCCTC-binding factor(CTCF) binds to the promoter region of Celsr1, Celsr2 and Celsr3 genes, and recruit cohesin to these regions. Knockdown of CTCF or cohesin increase the expression of Celsr1 and Celsr3 genes. The regulation function of CTCF in Celsr3 expression may be related to the looping interaction between promoters of Celsr3 and other genes.