Functional Analyses of Four Cryptochromes From Aquatic Organisms After Heterologous Expression in Drosophila melanogaster Circadian Clock Cells.

Cryptochromes (Crys) represent a multi-facetted class of proteins closely associated with circadian clocks. They have been shown to function as photoreceptors but also to fulfill light-independent roles as transcriptional repressors within the negative feedback loop of the circadian clock. In addition, there is evidence for Crys being involved in light-dependent magneto-sensing, and regulation of neuronal activity in insects, adding to the functional diversity of this cryptic protein class. In mammals, Crys are essential components of the circadian clock, but their role in other vertebrates is less clear. In invertebrates, Crys can function as circadian photoreceptors, or as components of the circadian clock, while in some species, both light-receptive and clock factor roles coexist. In the current study, we investigate the function of Cry proteins in zebrafish (Danio rerio), a freshwater teleost expressing 6 cry genes. Zebrafish peripheral circadian clocks are intrinsically light-sensitive, suggesting the involvement of Cry in light-resetting. Echinoderms (Strongylocentrotus purpuratus) represent the only class of deuterostomes that possess an orthologue (SpuCry) of the light-sensitive Drosophila melanogaster Cry, which is an important component of the light-resetting pathway, but also works as transcriptional repressor in peripheral clocks of fruit flies. We therefore investigated the potential of different zebrafish cry genes and SpuCry to replace the light-resetting and repressor functions of Drosophila Cry by expressing them in fruit flies lacking endogenous cry function. Using various behavioral and molecular approaches, we show that most Cry proteins analyzed are able to fulfill circadian repressor functions in flies, except for one of the zebrafish Crys, encoded by cry4a. Cry4a also shows a tendency to support light-dependent Cry functions, indicating that it might act in the light-input pathway of zebrafish.

Evolution of lineage-specific functions in ancient cis-regulatory modules.

Morphological evolution is driven both by coding sequence variation and by changes in regulatory sequences. However, how cis-regulatory modules (CRMs) evolve to generate entirely novel expression domains is largely unknown. Here, we reconstruct the evolutionary history of a lens enhancer located within a CRM that not only predates the lens, a vertebrate innovation, but bilaterian animals in general. Alignments of orthologous sequences from different deuterostomes sub-divide the CRM into a deeply conserved core and a more divergent flanking region. We demonstrate that all deuterostome flanking regions, including invertebrate sequences, activate gene expression in the zebrafish lens through the same ancient cluster of activator sites. However, levels of gene expression vary between species due to the presence of repressor motifs in flanking region and core. These repressor motifs are responsible for the relatively weak enhancer activity of tetrapod flanking regions. Ray-finned fish, however, have gained two additional lineage-specific activator motifs which in combination with the ancient cluster of activators and the core constitute a potent lens enhancer. The exploitation and modification of existing regulatory potential in flanking regions but not in the highly conserved core might represent a more general model for the emergence of novel regulatory functions in complex CRMs.

A cnidarian homologue of an insect gustatory receptor functions in developmental body patterning.

Insect gustatory and odorant receptors (GRs and ORs) form a superfamily of novel transmembrane proteins, which are expressed in chemosensory neurons that detect environmental stimuli. Here we identify homologues of GRs (Gustatory receptor-like (Grl) genes) in genomes across Protostomia, Deuterostomia and non-Bilateria. Surprisingly, two Grls in the cnidarian Nematostella vectensis, NvecGrl1 and NvecGrl2, are expressed early in development, in the blastula and gastrula, but not at later stages when a putative chemosensory organ forms. NvecGrl1 transcripts are detected around the aboral pole, considered the equivalent to the head-forming region of Bilateria. Morpholino-mediated knockdown of NvecGrl1 causes developmental patterning defects of this region, leading to animals lacking the apical sensory organ. A deuterostome Grl from the sea urchin Strongylocentrotus purpuratus displays similar patterns of developmental expression. These results reveal an early evolutionary origin of the insect chemosensory receptor family and raise the possibility that their ancestral role was in embryonic development.

The Cryptochrome/Photolyase Family in aquatic organisms.

The Cryptochrome/Photolyase Family (CPF) represents an ancient group of widely distributed UV-A/blue-light sensitive proteins sharing common structures and chromophores. During the course of evolution, different CPFs acquired distinct functions in DNA repair, light perception and circadian clock regulation. Previous phylogenetic analyses of the CPF have allowed reconstruction of the evolution and distribution of the different CPF super-classes in the tree of life. However, so far only limited information is available from the CPF orthologs in aquatic organisms that evolved in environments harboring great diversity of life forms and showing peculiar light distribution and rhythms. To gain new insights into the evolutionary and functional relationships within the CPF family, we performed a detailed study of CPF members from marine (diatoms, sea urchin and annelid) and freshwater organisms (teleost) that populate diverse habitats and exhibit different life strategies. In particular, we first extended the CPF family phylogeny by including genes from aquatic organisms representative of several branches of the tree of life. Our analysis identifies four major super-classes of CPF proteins and importantly singles out the presence of a plant-like CRY in diatoms and in metazoans. Moreover, we show a dynamic evolution of Cpf genes in eukaryotes with various events of gene duplication coupled to functional diversification and gene loss, which have shaped the complex array of Cpf genes in extant aquatic organisms. Second, we uncover clear rhythmic diurnal expression patterns and light-dependent regulation for the majority of the analyzed Cpf genes in our reference species. Our analyses reconstruct the molecular evolution of the CPF family in eukaryotes and provide a solid foundation for a systematic characterization of novel light activated proteins in aquatic environments.