Abstract
Previously, fidgetin (fign) and its family members fidgetin-like 1 (fignl1) and fidgetin-like 2 (fignl2) were found to be highly expressed during zebrafish brain development, suggesting their functions in the nervous system. In this study, we report the effects of loss-of-function of these genes on development. We designed and identified single-guide RNAs targeted to generate fign, fignl1, and fignl2 mutants and then observed the overall morphological and behavioral changes. Our findings showed that while fign and fignl1 null mutants displayed no significant defects, fignl2 null zebrafish mutants displayed pericardial edema, reduced heart rate, and smaller eyes; fignl2 null mutants responded to the light-darkness shift with a lower swimming velocity. fignl2 mRNAs were identified in vascular endothelial cells by in situ hybridization and re-analysis of an online dataset of single-cell RNAseq results. Finally, we used morpholino oligonucleotides to confirm that fignl2 knockdown resulted in severe heart edema, which was caused by abnormal vascular branching. The zebrafish fignl2 morphants also showed longer axonal length and more branches of caudal primary neurons. Taken together, we summarize that Fignl2 functions on cellular branches in endothelial cells and neurons. This study reported for the first time that the microtubule-severing protein Fignl2 contributes to cell branching during development.
Highlights
Morphology and motility of a cell are determined by the regulation of the cytoskeleton, especially microtubules
Embryos with fignl2 mutations were found to have pericardial edema and smaller eyes, and the phenotypes were dependent on gene dose because homozygous mutants showed more severe edema and heart congestion (Figure 1D), while Fign and Fidgetin-like 1 (Fignl1) mutants showed no significant phenotypes in the pericardial area and the heart (Figures 1B,C)
We previously investigated and compared the expression patterns of fign, fignl1, and fignl2 during zebrafish development and found that Fidgetinlike 2 (Fignl2) is expressed at higher levels in the nervous system and other tissues than fign and fignl1 genes, which is consistent with the result of re-analyzed online single-cell RNAseq data (GEO GSE112294) (Wagner et al, 2018)
Summary
Morphology and motility of a cell are determined by the regulation of the cytoskeleton, especially microtubules. Microtubule dynamics are regulated by microtubule-associated proteins, of which microtubule severing proteins (MSPs) such as spastin, katanin, and fidgetin are key regulatory factors of microtubule dynamics (Karabay et al, 2004; Butler et al, 2010; Leo et al, 2015; Menon and Gupton, 2016) These MSPs are members of the AAA (ATPase family associated with various cellular activities) family, which is capable of severing microtubules into short fragments by forming a hexamer that consumes ATP (Sharp and Ross, 2012; McNally and Roll-Mecak, 2018). Drosophila Fign facilitates microtubule disassembly in dendrites but not in axons after neuron injury (Tao et al, 2016) These inconsistent functional features of Fign family members in different species or cell types have aroused our interest. We attempted to comparatively study the functions of Fign family members in one animal model
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