The evolution of the knirps family of transcription factors in arthropods.

The orphan nuclear receptor gene knirps and its relatives encode a small family of highly conserved proteins. We take advantage of the conservation of the family, using the recent prevalence of genomic data, to reconstruct its evolutionary history, identifying duplication events and tracing the intron-exon structure of the genes over evolution. Many arthropod species have two or three members of this family, but the orthology between members is unclear. We have analyzed the protein coding sequences of members of this family from 15 arthropod species covering all four main arthropod classes, including a total of 28 genes. All members of the family encode a highly conserved 94 amino acid core sequence, part of which is encoded by a single invariant exon. We find that many of the automated predictions of these genes contain errors, while some copies of the gene were not uncovered by automated pipelines, requiring manual corrections and curation. We use the coding sequences to present a phylogenetic analysis of the knirps family. Our analysis indicates that there was a duplication of a single ancestral gene in the lineage leading to insects, which gave rise to two paralogs, eagle and knirps-related. Descendants of this duplication can be identified by the presence or absence of a short protein-coding motif. Independent, lineage-specific duplications occurred in the two crustaceans we sampled. Within the insects, the knirps-related gene underwent further lineage-specific duplications, giving rise to--among others--the Drosophila gap gene knirps.

Plasticity in mRNA expression and localization of orthodenticle within higher Diptera.

orthodenticle (otd) genes are found throughout the animal kingdom and encode well-studied homeodomain transcription factors that share conserved functions in cephalization, head segmentation, brain patterning, and the differentiation of photoreceptors. Otd proteins have been proposed as ancestral key players in anterior determination despite a high level of variation in gene expression at early developmental stages: otd is expressed strictly zygotically in the dipteran Drosophila melanogaster, while otd1 mRNA is contributed maternally to the embryo in the coleopteran Tribolium castaneum and maternal otd1 mRNA is localized to the anterior and posterior pole of the oocyte in the hymopteran Nasonia vitripennis. Here we demonstrate that such changes in otd mRNA expression and localization do not need to represent large phylogenetic distances but can occur even within closely related taxa. We show maternal otd expression in the medfly Ceratitis capitata and maternally localized otd mRNA in the caribfly Anastrepha suspensa, two cyclorrhaphan species closely related to Drosophila. This indicates considerable plasticity in expression and mRNA localization of key developmental genes even within short evolutionary distances.

Expression of otd orthologs in the amphipod crustacean, Parhyale hawaiensis.

The arthropod head is a complex metameric structure. In insects, orthodenticle (otd) functions as a 'head gap gene' and plays a significant role in patterning and development of the anterior head ectoderm, the protocerebrum, and the ventral midline. In this study, we characterize the structure and developmental deployment of two otd paralogs in the amphipod crustacean, Parhyale hawaiensis. Photd1 is initially expressed at gastrulation through germband stages in a bilaterally symmetric, restricted region of the anterior head ectoderm and also in a single column of cells along the ventral midline. Late in embryogenesis, Photd1 is expressed within the developing anterior brain and the expression along the embryonic midline has become restricted to a stereotypic group of segmentally reiterated cells. The second ortholog Photd2, however, has a unique temporal-spatial expression pattern and is not detected until after the head lobes have been organized in the developing ectoderm of the germband during late germband stages. Anteriorly, Photd2 is coincident with the Photd1 head expression domain; however, Photd2 is not detected along the ventral midline during formation of the germband and only appears in the ventral midline late in embryonic development in a restricted group of cells distinct from those expressing Photd1. The early expression of Photd1 in the anterior head ectoderm is consistent with a role as a head gap gene. The more posterior expression of Photd1 is suggestive of a role in patterning the embryonic ventral midline. Photd2 expression appears too late to play a role in early head patterning but may contribute to latter patterning in restricted regions of both the head and the ventral midline. The comparative analysis of otd reveals the divergence of gene expression and gene function associated with duplication of this important developmental gene.

Fluorescent transformation markers for insect transgenesis.

The first effectively achieved germ-line transformations of non-drosophilid insects were based on mutant rescue of eye color phenotypes. However, for most insect species neither visible mutants nor corresponding cloned genes are available. Therefore, the development of broadly applicable and reliable transformation markers will be of great importance to fully exploit the enormous potential transgenic insect technology has to offer. Here we review transposon-mediated germ-line transformation approaches that employ green fluorescent protein (GFP) variants to identify successful gene transfer. Furthermore, we provide novel data on the use of DsRed as an additional red fluorescent transformation marker for insect transgenesis. In conclusion, fluorescent proteins controlled by suitable strong promoters possess ideal characteristics to serve as transformation markers for a wide range of insect species.