Peroxin3, a newly identified regulator of melanocyte development and melanosome biogenesis in zebrafish <em>Danio rerio</em>

Abstract

Previous efforts aimed to understand the consequences of a dysregulated control of peroxisome biogenesis and of the dynamics of peroxisomal matrix proteins. Nevertheless, previous publications did not fill the existing gap in understanding the molecular events underlying the progression of Peroxisomal Biogenesis Disorders at systemic level. In particular, a vertebrate model deficient for one of the early peroxins (Pex3, Pex16 and Pex19) was never generated (Baes & van Veldhoven 2006). <br /> In this work, the generation and characterization of a zebrafish model for Peroxisomal Biogenesis Disorders (PBDs) is described. The unique homolog of human <i>PEX3</i> was identified and its expression pattern was determined querying different online databases and with experimental techniques (<i>in situ</i> hybridization, qRT-PCR, immunofluorescence), during embryogenesis and at adult stage. This helped to identify new domains in which pex3 is expressed and that were not previously described in mammals: specific regions in the brain, developing sensory organs (optic cup, otic vesicles and olfactory epithelium), gill filaments and clusters of cells in the epidermis, identified as melanocytes. Different genome editing methods (TALENs, CRISPR/Cas9) were successfully applied to generate a loss-of-function model (<i>pex3^CRISPR/ZMP</i>).<br /> Different from what observed in <i>C. elegans</i> (Petriv et al. 2002) or <i>D. melanogaster</i> (Nakayama et al. 2011), zebrafish <i>pex3^CRISPR/ZMP</i> mutants do not show any premature lethality and they survive to adulthood, even if they lack functional peroxisomes. Nevertheless, their peroxisomal and mitochondrial metabolism are impaired, lowering the energy status, increasing the oxidative stress and reducing the mitochondria abundance. This phenotype can be rescued through the activation of pparγ nuclear receptor, that induces the transcription of genes involved in lipid and xenobiotic metabolism, oxidative stress reduction and cell cycle regulation. <br /> Surprisingly, zebrafish<i>pex3^CRISPR/ZMP</i> embryos display additional major defects in the neural crest derived tissues, like pigment cell and cartilage-bone structures in the cranial region. In <i>pex3^CRISPR/ZMP</i> mutants, embryos lack most melanophores, whereas melanophores are poorly pigmented at adult stage. In these animals, there is a prolonged foxd3-induced repression of the transcription of genes involved in the melanin biosynthesis. The phenotype develops independent of peroxisome and the discriminant factor is the identified C-terminal melanosomal targeting signal (MTS) driving pex3 on the melanosomes. In absence of this MTS, pex3 does not localize to melanosomes, whereas peroxisomes do not suffer any dysfunction. Therefore, melanosomes do not mature properly, inducing a delay in the proliferation and migration of zebrafish embryo melanophores. This has an impact also later, during metamorphosis, for establishing of the correct pigmentation stripes pattern. Thus, pex3 presence on melanosome during embryogenesis is necessary for correct proliferation, maturation and migration events, in melanophore cell lineage. <br /> The results of this study offer a model in which pex3 has the same molecular function on melanosomes and on peroxisomes. pex3 mediates the import of membrane and matrix proteins on these two organelles, in order to ensure their function. To achieve this role of importomer complex component on different organelles, pex3 might interact and cooperate with different pools of proteins. When pex3 is missing from the melanosome - either because it is mutated or not targeted due to the lack of the MTS – melanosome function is impaired and melanophore lineage cannot generate the correct striped pattern, typical of zebrafish. Preliminary data hint to a conserved similar function also in mammals.<br /> Moreover, this study offers a valuable animal model for the study of pigmentation disorders. It opens new possibilities for the pharmaceutical research and for the discovery of new drug treatments.