Characterization and expression analysis of mcoln1.1 and mcoln1.2, the putative zebrafish co-orthologs of the gene responsible for human mucolipidosis type IV

Abstract

Mucolipidosis type IV (MLIV) is an autosomal recessive lysosomal storage disorder caused by mutations in the MCOLN1 gene coding for mucolipin-1 (TRPML1). TRPML1 belongs to a transient receptor potential channels (TRP) subfamily, which in mammals includes two other members: mucolipin-2 (TRPML2) and mucolipin-3 (TRPML3). Bioinformatic analysis of the Danio rerio (zebrafish) genome and trascriptome revealed the presence of five different genes related to human mucolipins: mcoln1.1, mcoln1.2, mcoln2, mcoln3.1 and mcoln3.2. We focused our efforts on the characterization of the two putative zebrafish MCOLN1 co-orthologs. Transient-expression experiments in human HeLa cells demonstrated that fish Mcoln1.1 and Mcoln1.2, similarly to TRPML1, localize to late endosomal/lysosomal compartments. Real-Time PCR (RT-PCR) experiments showed that both genes are maternally expressed and transcribed at different levels during embryogenesis. RT-PCR analysis in different zebrafish tissues displayed ubiquitary expression for mcoln1.1 and a more tissue-specific pattern for mcoln1.2. Spatial and temporal expression studies using whole-mount in situ hybridization confirmed that both genes are maternally expressed and ubiquitously transcribed during gastrulation and early somitogenesis. Notably, in the next developmental stages they are more expressed in neural regions and in retina layers, tissues affected in MLIV. Interestingly, mcoln1.1 is detected, from 10 somite-stage until to 36 hpf, in the yolk syncytial layer (YSL) and in the intermediate cell mass (ICM), the earliest site of hematopoiesis. Overall, the redundancy of mucolipins together with their expression profile support the biological relevance of this class of proteins in zebrafish. The data herein presented indicate that Danio rerio could be a suitable vertebrate model for the study of some aspects of MLIV pathogenesis.