Spatiotemporal expression of ninjurin1/2 in zebrafish and Construction of ninjurin1 knockout zebrafish using CRISPR/Cas9 system

Abstract

Nerve injury induced proteins (Ninjurin) are cell surface adhesion proteins that are firstly isolated in Schwann cells surrounding the distal segment of injured nerves. There are two types of proteins in Ninjurin family, Ninjurin1 and Ninjurin2, and they are known to promote neurite outgrowth from primary cultured dorsal root ganglion neurons. Ninjurin1 and Ninjurin2 share conserved hydrophobic regions in their transmembrane domains but the adhesion motifs in their N-terminal are different. In mammals, ninjurin1 is mainly expressed in tissues with epithelial origin but ninjurin2 is highly expressed in lymphatic and hematopoietic organs. Most of previous studies revealed the functions of ninjurin in inflammatory responses, however, a role of ninjurin during developmental process is unknown. Therefore, I investigated expression patterns of ninjurin1/2 in developing zebrafish embryos and examined their functions using morpholino-based knockdown strategies. Sequence alignment indicated that both zebrafish ninjurins show approximately 50-70% homologies when compared to human and mouse forms. RT-PCR analysis revealed the temporal mRNA expression patterns of ninjurins at different developmental stages. ninjurin1 transcripts were detected from 1 cell stage with gradual increase from shield period to 3 dpf, while ninjurin2 transcripts were constantly expressed during the development of zebrafish. To verify a spatial mRNA expression, in situ hybridization was performed. ninjurin1 was detected ubiquitously during gastrulation, later it localized to the somites and head region especially cerebellum and tectum. The expression of ninjurin2 was also observed ubiquitously in gastrulation period and restricted to the somites from segmentation period. In addition, ninjurin1/2 morphants showed similar developmental abnormalities such as small head, curved trunk, and short body length. Interestingly, ninjurin1 morphants exhibited more severe defects than ninjurin2 morphants, and the viability of ninjurin1 morphants was dramatically decreased in a dose dependent manner. To further confirm roles of Ninjurins in zebrafish development, I employed Clustered regularly interspaced short palindromic repeats (CRISPR)/ CRISPR-associated proteins (Cas) system which has already been successfully used to construct knockout models in many cell lines and organisms, including human, bacteria, mice, and zebrafish. ninjurin1-targeting sgRNA was constructed and CRISPR/Cas9-mediated mutation of ninjurin1 was confirmed by T7E1 assay. The homogeneous ninjurin1 knockout zebrafish is expected to be generated in F3 generation. Taken together, these data suggests that Ninjurin1 and Ninjurin2 are possibly associated with development of brain and somites in zebrafish embryos. Also these studies can be used for preliminary data of development study.