ABSTRACT
Skeletal muscle is highly regenerative and is mediated by a population of migratory adult muscle stem cells (muSCs). Effective muscle regeneration requires a spatio-temporally regulated response of the muSC population to generate sufficient muscle progenitor cells that then differentiate at the appropriate time. The relationship between muSC migration and cell fate is poorly understood and it is not clear how forces experienced by migrating cells affect cell behaviour. We have used zebrafish to understand the relationship between muSC cell adhesion, behaviour and fate in vivo. Imaging of pax7-expressing muSCs as they respond to focal injuries in trunk muscle reveals that they migrate by protrusive-based means. By carefully characterizing their behaviour in response to injury we find that they employ an adhesion-dependent mode of migration that is regulated by the RhoA kinase ROCK. Impaired ROCK activity results in reduced expression of cell cycle genes and increased differentiation in regenerating muscle. This correlates with changes to focal adhesion dynamics and migration, revealing that ROCK inhibition alters the interaction of muSCs to their local environment. We propose that muSC migration and differentiation are coupled processes that respond to changes in force from the environment mediated by RhoA signalling.
Subject(s)
Adult Stem Cells , Zebrafish , Animals , Cell Differentiation , Signal Transduction , Muscle, SkeletalABSTRACT
Drosophila Ceramide Synthase (CerS) Schlank regulates both ceramide synthesis and fat metabolism. Schlank contains a catalytic lag1p motif and, like many CerS in other species, a homeodomain of unknown function. Here, we show that the Drosophila CerS Schlank is imported into the nucleus and requires two nuclear localization signals (NLSs) within its homeodomain and functional Importin-ß import machinery. Expression of Schlank variants containing the homeodomain without functional lag1p motif rescued the fat metabolism phenotype of schlank mutants whereas a variant with a mutated NLS site did not rescue. Thus, the homeodomain of Schlank is involved in the regulation of lipid metabolism independent of the catalytic lag1p motif.
Subject(s)
Cell Nucleus/metabolism , Drosophila Proteins/metabolism , Drosophila melanogaster/metabolism , Fat Body/metabolism , Lipid Metabolism , Nuclear Localization Signals/metabolism , Sphingosine N-Acyltransferase/metabolism , Active Transport, Cell Nucleus , Amino Acid Motifs , Amino Acid Substitution , Animals , Animals, Genetically Modified , Catalytic Domain , Cell Line , Cell Nucleus/enzymology , Drosophila Proteins/antagonists & inhibitors , Drosophila Proteins/chemistry , Drosophila Proteins/genetics , Drosophila melanogaster/cytology , Drosophila melanogaster/genetics , Fat Body/cytology , Fat Body/enzymology , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Homeodomain Proteins/chemistry , Homeodomain Proteins/genetics , Homeodomain Proteins/metabolism , Mutation , Nuclear Localization Signals/chemistry , Nuclear Localization Signals/genetics , Peptide Fragments/antagonists & inhibitors , Peptide Fragments/chemistry , Peptide Fragments/genetics , Peptide Fragments/metabolism , Protein Interaction Domains and Motifs , RNA Interference , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/metabolism , Sphingosine N-Acyltransferase/antagonists & inhibitors , Sphingosine N-Acyltransferase/chemistry , Sphingosine N-Acyltransferase/genetics , beta Karyopherins/antagonists & inhibitors , beta Karyopherins/genetics , beta Karyopherins/metabolismABSTRACT
BACKGROUND: Sphingolipids represent a major class of lipids which both serve as structural components of membranes and as bioactive molecules involved in lipid signaling. Ceramide synthases (cers) reside in the center of sphingolipid metabolism by producing ceramide through de novo synthesis or degradative pathways. While the six mammalian cers family members have been extensively studied in cell culture and in adult tissues, a systematic analysis of cers expression and function during embryogenesis is still lacking. RESULTS: Using bioinformatic and phylogenetic analysis, we identified nine highly conserved homologs of the vertebrate cers gene family in the zebrafish genome. A systematic expression analysis throughout five developmental stages indicates that, whereas until 48 hours post fertilization most zebrafish cers homologs are expressed in distinct patterns, e.g., in the intermediate cell mass and the pronephric duct, they show a highly overlapping expression during later stages of embryonic development, mostprominently in the developing brain. CONCLUSIONS: In this study, the expression of the cers gene homologs is comprehensively analyzed for the first time during vertebrate embryogenesis. Our data indicate that each embryonic tissue has a unique profile of cers expression during zebrafish embryogenesis suggesting tissue-specific profiles of ceramides and their derivatives.
