Gene Expression And Ctional Identification Of Ligodendrocyte Regulator Lingo1in Zebrafish

The complex nervous systems consist of two major cell types, neurons and glia cells. Myelination is a wrapping process that glial processes attach to the axons and trigger the polarization of the plasma membrane to form multilayer myelin sheath tightly. The myelin is produced by two types of specialized glial cells, oligodendrocytes in the central nervous system (CNS) and Schwann cells in the peripheral nervous system (PNS). Demyelinating diseases include multiple sclerosis, which is a neurodegenerative disease characterized by immune attacks on the CNS, resulting in myelin sheath damage and axonal loss. To explore the causes of CNS demyelination and to find effective treatment approaches, it is necessary to study the demyelination and myelination during the development closely.Leucine-rich repeat (LRR) and immunoglobulin (Ig) domain-containing neurite outgrowth inhibitory protein (Nogo) receptor-interacting protein-1(LINGO-1) is a CNS specific transmembrane protein and has been identified as a negative regulator of oligodendrocytes differentiation. Targeted LINGO-1inhibition promotes neuron survival, axon regeneration, oligodendrocyte differentiation, and remyelination in diverse animal models. Although studies in rodent models have extended our understanding of LINGO-1, its roles in neural development and myelination in zebrafish (Danio rerio) are not yet clear. Here we aim to study the temporal and spatial expression pattern of lingo1and the roles of Lingo1protein during zebrafish development.We first studied the molecular structure of lingo1in zebrafish by bioinformatics tools. Zebrafish have two copies of the lingol gene(lingo1a and lingo1b) that is present as a single copy in mammals. The Lingolb protein has a similar protein structure to the mammals and may play a role in the nervous system. A phylogenetic tree was constructed and showed that zebrafish Lingol was highly conserved within vertebrates. After the specific digoxin-labeling RNA probe was synthesized, we analyzed the temporal and spatial expression pattern of lingol by whole-mount in situ hybridization (WISH). The expression of lingo1b started1(mRNA) and2(protein) days post-fertilization (dpf) in the CNS by real-time quantitative-polymerase chain reaction (Q-PCR) and western blot (WB) analysis, In addition, by virtue of morpholino oligonucleotide (MO) knockdown technology, the MO targeted green fluorescent protein (GFP) mRNA report sequence and antibodies of Lingolb protein were synthesized in vitro for the knockdown specificity and efficiency analysis. The morphology analysis of4dpf lingo1b knockdown zebrafish showed some nervous system developmental abnormalities, including less dark pigment, small eyes, and a curly spinal cord (SC). The transmit electron microscopy (TEM) image and G-ratio calculation of lingo1b knockdown showed a thick myelin and an early-onset myelination of the Mauthner axons (MAs) and other axons, further analysis also implied that knockdown of Lingolb protein may promote the oligodendrocytes differentiation and maturation. We further measured the total and MA circumferences with TEM in4dpf larvae. The significantly decreased MA circumferences were accompanied with the increased myelin sheath thickness. To further confirm the decreasion in MA diameter, we counted the average diameter of MAs using whole-mount immunohistochemisty (WIHC) labeling confocal image. These results implied that the early-onset myelination caused by lingo1b knockdown may inhibit the development of primary motor neurons (pMNs). The outcome of early-onset CNS myelination and abnormal motor neuron development has been shown by the spontaneous movement analysis and larvae optokinetic response (OKR) tests. The accumulation distance (Acc Dist) value of spontaneous movement and the frequencies of eye movements were both decreased after lingolb knockdown that analyzed with Image-Pro Plus (IPP)6software. The lingo1b gene promoter sequences were cloned from zebrafish genomic DNA after bioinformatics predication and inserted into recombinant plasmid with transposase (Tol2) or meganuclease (I-Scel) sites and fluorescent protein report sequence. The fluorescent signal patterns of injected embryos were analyzed with confocal image, and the primary Lingolb specific gene marker of transgenic zebrafish lines Tg (lingo lb:EGFP) were also primarily constructed.Our results, to date, indicated that the lingo1is highly conserved and specific expressed in zebrafish CNS. Furthermore, the MOs knockdown effects of lingo1b suggested that Lingolb may play a role as a negative regulator of myelination and oligodendrocyte differentiation during zebrafish development. The morphology and ethology results also implied that the early-onset myelination caused by lingo1b knockdown may inhibit the development of primary motor neurons. These may provide a new platform for further understanding mechanisms underlying nervous system damage and repair as well as benefit to the large-scale potential compounds screening for the demyelinating disease in the future.