A microfluidic chip for immobilization and imaging of Ciona intestinalis larvae.

Sea squirts (Tunicata) are chordates and develop a swimming larva with a small and defined number of individually identifiable cells. This offers the prospect of connecting specific stimuli to behavioral output and characterizing the neural activity that links these together. Here, we describe the development of a microfluidic chip that allows live larvae of the sea squirt Ciona intestinalis to be immobilized and recorded. By generating transgenic larvae expressing GCaAMP6m in defined cells, we show that calcium ion levels can be recorded from immobilized larvae, while microfluidic control allows larvae to be exposed to specific waterborne stimuli. We trial this on sea water carrying increased levels of carbon dioxide, providing evidence that larvae can sense this gas.

The evolutionary origins of the vertebrate olfactory system.

Vertebrates develop an olfactory system that detects odorants and pheromones through their interaction with specialized cell surface receptors on olfactory sensory neurons. During development, the olfactory system forms from the olfactory placodes, specialized areas of the anterior ectoderm that share cellular and molecular properties with placodes involved in the development of other cranial senses. The early-diverging chordate lineages amphioxus, tunicates, lampreys and hagfishes give insight into how this system evolved. Here, we review olfactory system development and cell types in these lineages alongside chemosensory receptor gene evolution, integrating these data into a description of how the vertebrate olfactory system evolved. Some olfactory system cell types predate the vertebrates, as do some of the mechanisms specifying placodes, and it is likely these two were already connected in the common ancestor of vertebrates and tunicates. In stem vertebrates, this evolved into an organ system integrating additional tissues and morphogenetic processes defining distinct olfactory and adenohypophyseal components, followed by splitting of the ancestral placode to produce the characteristic paired olfactory organs of most modern vertebrates.

The structure, splicing, synteny and expression of lamprey COE genes and the evolution of the COE gene family in chordates.

COE genes encode transcription factors that have been found in all metazoans examined to date. They possess a distinctive domain structure that includes a DNA-binding domain (DBD), an IPT/TIG domain and a helix-loop-helix (HLH) domain. An intriguing feature of the COE HLH domain is that in jawed vertebrates it is composed of three helices, compared to two in invertebrates. We report the isolation and expression of two COE genes from the brook lamprey Lampetra planeri and compare these to COE genes from the lampreys Lethenteron japonicum and Petromyzon marinus. Molecular phylogenetic analyses do not resolve the relationship of lamprey COE genes to jawed vertebrate paralogues, though synteny mapping shows that they all derive from duplication of a common ancestral genomic region. All lamprey genes encode conserved DBD, IPT/TIG and HLH domains; however, the HLH domain of lamprey COE-A genes encodes only two helices while COE-B encodes three helices. We also identified COE-B splice variants encoding either two or three helices in the HLH domain, along with other COE-A and COE-B splice variants affecting the DBD and C-terminal transactivation regions. In situ hybridisation revealed expression in the lamprey nervous system including the brain, spinal cord and cranial sensory ganglia. We also detected expression of both genes in mesenchyme in the pharyngeal arches and underlying the notochord. This allows us to establish the primitive vertebrate expression pattern for COE genes and compare this to that of invertebrate chordates and other animals to develop a model for COE gene evolution in chordates.