RESUMEN
Clonal marking techniques based on the Cre/lox and Flp/FRT systems are widely used in multicellular model organisms to mark individual cells and their progeny, in order to study their morphology, growth properties and developmental fates. The same tools can be adapted to introduce specific genetic changes in a subset of cells within the body, i.e. to perform mosaic genetic analysis. Marking and manipulating distinct cell clones requires control over the frequency of clone induction, which is sometimes difficult to achieve. Here, we present Valcyrie, a new method that replaces the conventional Cre or Flp recombinase-mediated excision of a marker cassette by CRISPR-mediated excision. A major advantage of this approach is that CRISPR efficiency can be tuned in a predictable fashion by manipulating the degree of sequence complementarity between the CRISPR guide RNA and its targets. We establish the method in the beetle Tribolium castaneum We demonstrate that clone marking frequency can be tuned to generate embryos that carry single marked clones. The Valcyrie approach can be applied to a wide range of experimental settings, for example to modulate clone frequency with existing tools in established model organisms and to introduce clonal analysis in emerging experimental models.
Asunto(s)
Células Clonales/metabolismo , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Animales , Animales Modificados Genéticamente , Secuencia de Bases , Embrión no Mamífero/metabolismo , Integrasas/metabolismo , Factores de Tiempo , Tribolium/embriología , Tribolium/genéticaRESUMEN
Insects and other arthropods utilise external sensory structures for mechanosensory, olfactory, and gustatory reception. These sense organs have characteristic shapes related to their function, and in many cases are distributed in a fixed pattern so that they are identifiable individually. In Drosophila melanogaster, the identity of sense organs is regulated by specific combinations of transcription factors. In other arthropods, however, sense organ subtypes cannot be linked to the same code of gene expression. This raises the questions of how sense organ diversity has evolved and whether the principles underlying subtype identity in D. melanogaster are representative of other insects. Here, we provide evidence that such principles cannot be generalised, and suggest that sensory organ diversification followed the recruitment of sensory genes to distinct sensory organ specification mechanism. RESULTS: We analysed sense organ development in a nondipteran insect, the flour beetle Tribolium castaneum, by gene expression and RNA interference studies. We show that in contrast to D. melanogaster, T. castaneum sense organs cannot be categorised based on the expression or their requirement for individual or combinations of conserved sense organ transcription factors such as cut and pox neuro, or members of the Achaete-Scute (Tc ASH, Tc asense), Atonal (Tc atonal, Tc cato, Tc amos), and neurogenin families (Tc tap). Rather, our observations support an evolutionary scenario whereby these sensory genes are required for the specification of sense organ precursors and the development and differentiation of sensory cell types in diverse external sensilla which do not fall into specific morphological and functional classes. CONCLUSIONS: Based on our findings and past research, we present an evolutionary scenario suggesting that sense organ subtype identity has evolved by recruitment of a flexible sensory gene network to the different sense organ specification processes. A dominant role of these genes in subtype identity has evolved as a secondary effect of the function of these genes in individual or subsets of sense organs, probably modulated by positional cues.
Asunto(s)
Expresión Génica , Interferencia de ARN , Órganos de los Sentidos/crecimiento & desarrollo , Tribolium/crecimiento & desarrollo , Animales , Larva/genética , Larva/crecimiento & desarrollo , Tribolium/genéticaRESUMEN
BACKGROUND: The red flour beetle Tribolium castaneum has emerged as an important model organism for the study of gene function in development and physiology, for ecological and evolutionary genomics, for pest control and a plethora of other topics. RNA interference (RNAi), transgenesis and genome editing are well established and the resources for genome-wide RNAi screening have become available in this model. All these techniques depend on a high quality genome assembly and precise gene models. However, the first version of the genome assembly was generated by Sanger sequencing, and with a small set of RNA sequence data limiting annotation quality. RESULTS: Here, we present an improved genome assembly (Tcas5.2) and an enhanced genome annotation resulting in a new official gene set (OGS3) for Tribolium castaneum, which significantly increase the quality of the genomic resources. By adding large-distance jumping library DNA sequencing to join scaffolds and fill small gaps, the gaps in the genome assembly were reduced and the N50 increased to 4753kbp. The precision of the gene models was enhanced by the use of a large body of RNA-Seq reads of different life history stages and tissue types, leading to the discovery of 1452 novel gene sequences. We also added new features such as alternative splicing, well defined UTRs and microRNA target predictions. For quality control, 399 gene models were evaluated by manual inspection. The current gene set was submitted to Genbank and accepted as a RefSeq genome by NCBI. CONCLUSIONS: The new genome assembly (Tcas5.2) and the official gene set (OGS3) provide enhanced genomic resources for genetic work in Tribolium castaneum. The much improved information on transcription start sites supports transgenic and gene editing approaches. Further, novel types of information such as splice variants and microRNA target genes open additional possibilities for analysis.
Asunto(s)
Genes de Insecto , Genoma de los Insectos , Genómica , Tribolium/genética , Animales , Sitios de Unión , Biología Computacional/métodos , Genómica/métodos , MicroARNs/genética , Anotación de Secuencia Molecular , Filogenia , Interferencia de ARN , Reproducibilidad de los ResultadosRESUMEN
Anterior patterning in animals is based on a gene regulatory network, which comprises highly conserved transcription factors like six3, pax6 and otx. More recently, foxQ2 was found to be an ancestral component of this network but its regulatory interactions showed evolutionary differences. In most animals, foxQ2 is a downstream target of six3 and knockdown leads to mild or no epidermal phenotypes. In contrast, in the red flour beetle Tribolium castaneum, foxQ2 gained a more prominent role in patterning leading to strong epidermal and brain phenotypes and being required for six3 expression. However, it has remained unclear which of these novel aspects were insect or arthropod specific. Here, we study expression and RNAi phenotype of the single foxQ2 ortholog of the spider Parasteatoda tepidariorum. We find early anterior expression similar to the one of insects. Further, we show an epidermal phenotype in the labrum similar to the insect phenotype. However, our data indicate that foxQ2 is positioned downstream of six3 like in other animals but unlike insects. Hence, the epidermal and neural pattering function of foxQ2 is ancestral for arthropods while the upstream role of foxQ2 may have evolved in the lineage leading to the insects.
Asunto(s)
Proteínas de Artrópodos/metabolismo , Embrión no Mamífero/metabolismo , Proteínas del Ojo/metabolismo , Factores de Transcripción Forkhead/metabolismo , Proteínas de Homeodominio/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Neurogénesis/genética , Transducción de Señal/genética , Arañas/crecimiento & desarrollo , Animales , Proteínas de Artrópodos/genética , Evolución Biológica , Tipificación del Cuerpo/genética , Encéfalo/crecimiento & desarrollo , Encéfalo/metabolismo , Proteínas del Ojo/genética , Factores de Transcripción Forkhead/genética , Regulación del Desarrollo de la Expresión Génica/genética , Proteínas de Homeodominio/genética , Hibridación in Situ , Proteínas del Tejido Nervioso/genética , Fenotipo , Filogenia , Interferencia de ARN , Proteína Homeobox SIX3RESUMEN
Spiders are equipped with a large number of innervated cuticular specializations, which respond to various sensory stimuli. The physiological function of mechanosensory organs has been analysed in great detail in some model spider species (e.g. Cupiennius salei); however, much less is known about the distribution and function of chemosensory organs. Furthermore, our knowledge on how the sense organ pattern develops on the spider appendages is limited. Here we analyse the development of the pattern and distribution of six different external mechano- and chemosensory organs in all postembryonic stages and in adult male and female spiders of the species Parasteatoda tepidariorum. We show that except for small mechanosensory setae, external sense organs appear in fixed positions on the pedipalps and first walking legs, arranged in longitudinal rows along the proximal-distal axis or in invariable positions relative to morphological landmarks (joints, distal tarsal tip). A comparison to other Entelegynae spiders shows that these features are conserved. We hope that this study lays the foundation for future molecular analysis to address the question how this conserved pattern is generated.
Asunto(s)
Extremidades/crecimiento & desarrollo , Órganos de los Sentidos/crecimiento & desarrollo , Sensilos/anatomía & histología , Sensilos/crecimiento & desarrollo , Arañas/crecimiento & desarrollo , Animales , Extremidades/anatomía & histología , Femenino , Fémur/anatomía & histología , Fémur/crecimiento & desarrollo , Masculino , Metatarso/anatomía & histología , Metatarso/crecimiento & desarrollo , Microscopía Electrónica de Rastreo , Órganos de los Sentidos/anatomía & histología , Sensilos/ultraestructura , Arañas/anatomía & histología , Tibia/anatomía & histología , Tibia/crecimiento & desarrolloRESUMEN
Anterior patterning of animals is based on a set of highly conserved transcription factors but the interactions within the protostome anterior gene regulatory network (aGRN) remain enigmatic. Here, we identify the red flour beetle Tribolium castaneum ortholog of foxQ2 (Tc-foxQ2) as a novel upstream component of the aGRN. It is required for the development of the labrum and higher order brain structures, namely the central complex and the mushroom bodies. We reveal Tc-foxQ2 interactions by RNAi and heat shock-mediated misexpression. Surprisingly, Tc-foxQ2 and Tc-six3 mutually activate each other, forming a novel regulatory module at the top of the aGRN. Comparisons of our results with those of sea urchins and cnidarians suggest that foxQ2 has acquired more upstream functions in the aGRN during protostome evolution. Our findings expand the knowledge on foxQ2 gene function to include essential roles in epidermal development and central brain patterning.
Asunto(s)
Proteínas de Insectos/metabolismo , Tribolium/embriología , Animales , Tipificación del Cuerpo/genética , Tipificación del Cuerpo/fisiología , Cnidarios/metabolismo , Regulación del Desarrollo de la Expresión Génica/genética , Regulación del Desarrollo de la Expresión Génica/fisiología , Proteínas de Insectos/genética , Erizos de Mar/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Tribolium/metabolismoRESUMEN
BACKGROUND: Two visual systems are present in most arthropod groups: median and lateral eyes. Most of our current knowledge about the developmental and molecular mechanisms involved in eye formation in arthropods comes from research in the model system Drosophila melanogaster. Here, a core set of retinal determination genes, namely, sine-oculis (so), eyes absent (eya), dachshund (dac), and the two pax6 orthologues eyeless (ey) and twin of eyeless (toy) govern early retinal development. By contrast, not much is known about the development of the up-to-eight eyes present in spiders. Therefore, we analyzed the embryonic expression of core retinal determination genes in the common house spider Parasteatoda tepidariorum. RESULTS: We show that the anlagen of the median and lateral eyes in P. tepidariorum originate from different regions of the non-neurogenic ectoderm in the embryonic head. The median eyes are specified as two individual anlagen in an anterior median position in the developing head and subsequently move to their final position following extensive morphogenetic movements of the non-neurogenic ectoderm. The lateral eyes develop from a more lateral position. Intriguingly, they are specified as a unique field of cells that splits into the three individual lateral eyes during late embryonic development. Using gene expression analyses, we identified a unique combination of determination gene expression in the anlagen of the lateral and median eyes, respectively. CONCLUSIONS: This study of retinal determination genes in the common house spider P. tepidariorum represents the first comprehensive analysis of the well-known retinal determination genes in arthropods outside insects. The development of the individual lateral eyes via the subdivision of one single eye primordium might be the vestige of a larger composite eye anlage, and thus supports the notion that the composite eye is the plesiomorphic state of the lateral eyes in arthropods. The molecular distinction of the two visual systems is similar to the one described for compound eyes and ocelli in Drosophila, suggesting that a unique core determination network for median and lateral eyes, respectively, might have been in place already in the last common ancestor of spiders and insects.