RESUMO
MicroRNAs (miRNAs) are key regulators of developmental processes, such as cell fate determination and differentiation. Previous studies showed Dicer knockdown in honeybee embryos disrupt the processing of functional mature miRNAs and impairs embryo patterning. Here we investigated the expression profiles of miRNAs in honeybee embryogenesis and the role of the highly conserved miR-34-5p in the regulation of genes involved in insect segmentation. A total of 221 miRNAs were expressed in honey bee embryogenesis among which 97 mature miRNA sequences have not been observed before. Interestingly, we observed a switch in dominance between the 5-prime and 3-prime arm of some miRNAs in different embryonic stages; however, most miRNAs present one dominant arm across all stages of embryogenesis. Our genome-wide analysis of putative miRNA-target networks and functional pathways indicates miR-34-5p is one of the most conserved and connected miRNAs associated with the regulation of genes involved in embryonic patterning and development. In addition, we experimentally validated that miR-34-5p directly interacts to regulatory elements in the 3'-untranslated regions of pair-rule (even-skipped, hairy, fushi-tarazu transcription factor 1) and cytoskeleton (actin5C) genes. Our study suggests that miR-34-5p may regulate the expression of pair-rule and cytoskeleton genes during early development and control insect segmentation.
Assuntos
Citoesqueleto/genética , Fatores de Transcrição Fushi Tarazu/genética , Proteínas de Homeodomínio/genética , Proteínas de Insetos/genética , MicroRNAs/metabolismo , Regiões 3' não Traduzidas , Actinas/química , Actinas/genética , Actinas/metabolismo , Animais , Sequência de Bases , Abelhas/genética , Sítios de Ligação , Desenvolvimento Embrionário/genética , Fatores de Transcrição Fushi Tarazu/química , Fatores de Transcrição Fushi Tarazu/metabolismo , Genoma , Proteínas de Homeodomínio/química , Proteínas de Homeodomínio/metabolismo , Proteínas de Insetos/química , Proteínas de Insetos/metabolismo , MicroRNAs/química , MicroRNAs/genética , Alinhamento de Sequência , TranscriptomaRESUMO
In honeybees, the haplodiploid sex determination system promotes a unique embryogenesis process wherein females develop from fertilized eggs and males develop from unfertilized eggs. However, the developmental strategies of honeybees during early embryogenesis are virtually unknown. Similar to most animals, the honeybee oocytes are supplied with proteins and regulatory elements that support early embryogenesis. As the embryo develops, the zygotic genome is activated and zygotic products gradually replace the preloaded maternal material. The analysis of small RNA and mRNA libraries of mature oocytes and embryos originated from fertilized and unfertilized eggs has allowed us to explore the gene expression dynamics in the first steps of development and during the maternal-to-zygotic transition (MZT). We localized a short sequence motif identified as TAGteam motif and hypothesized to play a similar role in honeybees as in fruit flies, which includes the timing of early zygotic expression (MZT), a function sustained by the presence of the zelda ortholog, which is the main regulator of genome activation. Predicted microRNA (miRNA)-target interactions indicated that there were specific regulators of haploid and diploid embryonic development and an overlap of maternal and zygotic gene expression during the early steps of embryogenesis. Although a number of functions are highly conserved during the early steps of honeybee embryogenesis, the results showed that zygotic genome activation occurs earlier in honeybees than in Drosophila based on the presence of three primary miRNAs (pri-miRNAs) (ame-mir-375, ame-mir-34 and ame-mir-263b) during the cleavage stage in haploid and diploid embryonic development.
Assuntos
Abelhas/genética , Diploide , Regulação da Expressão Gênica no Desenvolvimento , Haploidia , Proteínas de Insetos/genética , Animais , Embrião não Mamífero , Feminino , Perfilação da Expressão Gênica , MasculinoRESUMO
BACKGROUND: Hexamerins are hemocyanin-derived proteins that have lost the ability to bind copper ions and transport oxygen; instead, they became storage proteins. The current study aimed to broaden our knowledge on the hexamerin genes found in the honey bee genome by exploring their structural characteristics, expression profiles, evolution, and functions in the life cycle of workers, drones and queens. RESULTS: The hexamerin genes of the honey bee (hex 70a, hex 70b, hex 70c and hex 110) diverge considerably in structure, so that the overall amino acid identity shared among their deduced protein subunits varies from 30 to 42%. Bioinformatics search for motifs in the respective upstream control regions (UCRs) revealed six overrepresented motifs including a potential binding site for Ultraspiracle (Usp), a target of juvenile hormone (JH). The expression of these genes was induced by topical application of JH on worker larvae. The four genes are highly transcribed by the larval fat body, although with significant differences in transcript levels, but only hex 110 and hex 70a are re-induced in the adult fat body in a caste- and sex-specific fashion, workers showing the highest expression. Transcripts for hex 110, hex 70a and hex70b were detected in developing ovaries and testes, and hex 110 was highly transcribed in the ovaries of egg-laying queens. A phylogenetic analysis revealed that HEX 110 is located at the most basal position among the holometabola hexamerins, and like HEX 70a and HEX 70c, it shares potential orthology relationship with hexamerins from other hymenopteran species. CONCLUSIONS: Striking differences were found in the structure and developmental expression of the four hexamerin genes in the honey bee. The presence of a potential binding site for Usp in the respective 5' UCRs, and the results of experiments on JH level manipulation in vivo support the hypothesis of regulation by JH. Transcript levels and patterns in the fat body and gonads suggest that, in addition to their primary role in supplying amino acids for metamorphosis, hexamerins serve as storage proteins for gonad development, egg production, and to support foraging activity. A phylogenetic analysis including the four deduced hexamerins and related proteins revealed a complex pattern of evolution, with independent radiation in insect orders.
Assuntos
Abelhas/genética , Proteínas de Insetos/genética , Animais , Abelhas/crescimento & desenvolvimento , Abelhas/fisiologia , Expressão Gênica , Proteínas de Insetos/química , Hormônios Juvenis/metabolismo , Larva/genética , ReproduçãoRESUMO
Though introduced recently, complex networks research has grown steadily because of its potential to represent, characterize and model a wide range of intricate natural systems and phenomena. Because of the intrinsic complexity and systemic organization of life, complex networks provide a specially promising framework for systems biology investigation. The current article is an up-to-date review of the major developments related to the application of complex networks in biology, with special attention focused on the more recent literature. The main concepts and models of complex networks are presented and illustrated in an accessible fashion. Three main types of networks are covered: transcriptional regulatory networks, protein-protein interaction networks and metabolic networks. The key role of complex networks for systems biology is extensively illustrated by several of the papers reviewed.
RESUMO
BACKGROUND: In honeybees, differential feeding of female larvae promotes the occurrence of two different phenotypes, a queen and a worker, from identical genotypes, through incremental alterations, which affect general growth, and character state alterations that result in the presence or absence of specific structures. Although previous studies revealed a link between incremental alterations and differential expression of physiometabolic genes, the molecular changes accompanying character state alterations remain unknown. RESULTS: By using cDNA microarray analyses of >6,000 Apis mellifera ESTs, we found 240 differentially expressed genes (DEGs) between developing queens and workers. Many genes recorded as up-regulated in prospective workers appear to be unique to A. mellifera, suggesting that the workers' developmental pathway involves the participation of novel genes. Workers up-regulate more developmental genes than queens, whereas queens up-regulate a greater proportion of physiometabolic genes, including genes coding for metabolic enzymes and genes whose products are known to regulate the rate of mass-transforming processes and the general growth of the organism (e.g., tor). Many DEGs are likely to be involved in processes favoring the development of caste-biased structures, like brain, legs and ovaries, as well as genes that code for cytoskeleton constituents. Treatment of developing worker larvae with juvenile hormone (JH) revealed 52 JH responsive genes, specifically during the critical period of caste development. Using Gibbs sampling and Expectation Maximization algorithms, we discovered eight overrepresented cis-elements from four gene groups. Graph theory and complex networks concepts were adopted to attain powerful graphical representations of the interrelation between cis-elements and genes and objectively quantify the degree of relationship between these entities. CONCLUSION: We suggest that clusters of functionally related DEGs are co-regulated during caste development in honeybees. This network of interactions is activated by nutrition-driven stimuli in early larval stages. Our data are consistent with the hypothesis that JH is a key component of the developmental determination of queen-like characters. Finally, we propose a conceptual model of caste differentiation in A. mellifera based on gene-regulatory networks.
Assuntos
Abelhas , Comportamento Animal/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Genes de Insetos , Hierarquia Social , Animais , Abelhas/anatomia & histologia , Abelhas/genética , Abelhas/fisiologia , Etiquetas de Sequências Expressas , Feminino , Perfilação da Expressão Gênica , Redes Reguladoras de Genes , Hormônios Juvenis/metabolismo , Larva/anatomia & histologia , Larva/fisiologia , Análise de Sequência com Séries de OligonucleotídeosRESUMO
A comparison of the most conserved sex-determining genes between the fruit fly, Drosophila melanogaster, and the honey bee, Apis mellifera, was performed with bioinformatics tools developed for computational molecular biology. An initial set of protein sequences already described in the fruit fly as participants of the sex-determining cascade was retrieved from the Gene Ontology database (http://www.geneontology.org/) and aligned against a database of protein sequences predicted from the honey bee genome. The doublesex (dsx) gene is considered one of the most conserved sex-determining genes among metazoans, and a male-specific partial cDNA of putative A. mellifera dsx gene (Amdsx) was identified experimentally. The theoretical predictions were developed in the context of sequence similarity. Experimental evidence indicates that dsx is present in embryos and larvae, and that it encodes a transcription factor widely conserved in metazoans, containing a DM DNA-binding domain implicated in the regulation of the expression of genes involved in sexual phenotype formation.