Abstract
BackgroundKeratins make up the largest subgroup of intermediate filaments, and, in chordates, represent the most abundant proteins in epithelial cells. They have been associated with a wide range of functions in the cell, but little information is still available about their expression profile and regulation during flatfish metamorphosis. Senegalese sole (Solea senegalensis) is a commercially important flatfish in which no keratin gene has been described yet.ResultsThe development of large-scale genomics of Senegalese sole has facilitated the identification of two different type I keratin genes referred to as sseKer1 and sseKer2. Main characteristics and sequence identities with other fish and mammal keratins are described. Phylogenetic analyses grouped sseKer1 and sseKer2 in a significant clade with other teleost epidermal type I keratins, and have allowed for the identification of sseKer2 as a novel keratin. The expression profile of both genes was studied during larval development and in tissues using a real-time approach. sseKer1 and sseKer2 mRNA levels were significantly higher in skin than in other tissues examined. During metamorphosis, sseKer1 transcripts increased significantly at first stages, and reduced thereafter. In contrast, sseKer2 mRNA levels did not change during early metamorphosis although a significant drop at metamorphosis climax and late metamorphosis was also detected. To study the possible regulation of sseKer gene expressions by thyroid hormones (THs), larvae were exposed to the goitrogen thiourea (TU). TU-treated larvae exhibited higher sseKer1 and sseKer2 mRNA levels than untreated control at both 11 and 15 days after treatment. Moreover, addition of exogenous T4 hormone to TU-treated larvae restored or even reduced the steady-state levels with respect to the untreated control, demonstrating that expression of both genes is negatively regulated by THs.ConclusionWe have identified two keratin genes, referred to as sseKer1 and sseKer2, in Senegalese sole. Phylogenetic analyses revealed sseKer2 as a novel keratin. Although they exhibit different expression patterns during larval development, both of them are negatively regulated by THs. The co-regulation by THs could explain the reduction of both keratin transcripts after the metamorphosis climax, suggesting their role in the tissue remodelling processes that occur during metamorphosis.
Highlights
Keratins make up the largest subgroup of intermediate filaments, and, in chordates, represent the most abundant proteins in epithelial cells
Molecular characterization and phylogeny of Senegalese sole keratins Two sseKer genes were identified after Expressed sequence tags (ESTs) analysis of a normalized cDNA library constructed from different larval stages, undifferentiated gonads, and six adult tissues
The PCR conditions were the same as used in real-time assays. This strategy allowed us to rule out amplification of additional Senegalese sole keratin genes
Summary
Keratins make up the largest subgroup of intermediate filaments, and, in chordates, represent the most abundant proteins in epithelial cells. They have been associated with a wide range of functions in the cell, but little information is still available about their expression profile and regulation during flatfish metamorphosis. Keratins are the major structural proteins in epithelial cells, and are encoded by a large multigene family that accounts for about 75% of all IF genes in chordates [5]. The heptad periodicity within the rod domain is interrupted in several places, resulting in four consecutive segments (or coils) of different size but identical number of amino acids (aa) among keratins (1A, 1B, 2A, and 2B), separated by three spacers or "linkers" (L1, L12, and L2) of highly variable length and primary structure [1,9,16]
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