Small plastic particles, named microplastics, are abundant in the marine environment and can be ingested by marine organisms. Species with different feeding strategies can be differently affected by the presence of microplastics. Moreover, the impact of these particles can depend on their size. In this study, we analyzed the effects of 1 µm polystyrene particles on larval and juvenile development in the ascidian Ciona intestinalis. As previously reported for 10 µm beads, smaller particles caused a delay in the growth of juveniles, even if this delay was registered only at the highest concentration tested. Instead, larval development was not affected by the presence of microplastics. Histological analysis of juveniles revealed that 1 µm particles, after ingestion, can translocate from the gut to the hemocoelic cavity in just 8 days. As a defense mechanism, plastic spheres can also be phagocytized from specific circulating cells with phagocytic activity. Microplastics confirmed their potential as a threat to marine wildlife, interfering with food uptake and growth.

The evolutionary origin of vertebrates included innovations in sensory processing associated with the acquisition of a predatory lifestyle 1 . Vertebrates perceive external stimuli through sensory systems serviced by cranial sensory ganglia (CSG) which develop from cranial placodes; however understanding the evolutionary origin of placodes and CSGs is hampered by the gulf between living lineages and difficulty in assigning homology between cell types and structures. Here we use the Hmx gene family to address this question. We show Hmx is a constitutive component of vertebrate CSG development and that Hmx in the tunicate Ciona is able to drive the differentiation program of Bipolar Tail Neurons (BTNs), cells previously thought neural crest homologs 2,3 . Using Ciona and lamprey transgenesis we demonstrate that a unique, tandemly duplicated enhancer pair regulated Hmx in the stem-vertebrate lineage. Strikingly, we also show robust vertebrate Hmx enhancer function in Ciona, demonstrating that deep conservation of the upstream regulatory network spans the evolutionary origin of vertebrates. These experiments demonstrate regulatory and functional conservation between Ciona and vertebrate Hmx, and confirm BTNs as CSG homologs. Our analysis also identifies derived evolutionary changes, including a genetic basis for secondary simplicity in Ciona and unique regulatory complexity in vertebrates.CSG, including the trigeminal, vestibuloacoustic and epibranchial ganglia, relay information from sensory cells to the brain. CSG neurons derive from two sources: cranial placodes provide neurons that delaminate from the cranial ectoderm, and cranial neural crest cells migrate into ganglia providing all of the glia and some neurons of the trigeminal ganglia. The evolution of neural crest, placodes and CSG form part of the influential 'New Head Hypothesis' which posits that these and other innovations underlie the transformation of an ancestral chordate filter feeder into the ancestral vertebrate-type predator 1 . Our molecular and genetic understanding of this transformation has been limited, however, by the substantial anatomical gulf between vertebrates and their nearest living relatives, amphioxus and tunicates, which lack most or all of these characters (Fig. 1A) 4 .To address this gap we focused on the Hmx gene family, which encodes homeodomain transcription factors. We previously used transcriptomics to find markers for placode-derived CSG neurons, identifying Hmx3 as one such gene 5 . Jawed vertebrates have 4 Hmx family genes named Hmx1, Hmx2, Hmx3 and SOHo 6 , with expression in mouse, chicken, Xenopus

Serotonin (5-hydroxytryptamine (5-HT)) is a biogenic monoamine with pleiotropic functions. It exerts its roles by binding to specific 5-HT receptors (5HTRs) classified into different families and subtypes. Homologs of 5HTRs are widely present in invertebrates, but their expression and pharmacological characterization have been scarcely investigated. In particular, 5-HT has been localized in many tunicate species but only a few studies have investigated its physiological functions. Tunicates, including ascidians, are the sister group of vertebrates, and data about the role of 5-HTRs in these organisms are thus important for understanding 5-HT evolution among animals. In the present study, we identified and described 5HTRs in the ascidian Ciona intestinalis. During development, they showed broad expression patterns that appeared consistent with those reported in other species. Then, we investigated 5-HT roles in ascidian embryogenesis exposing C. intestinalis embryos to WAY-100635, an antagonist of the 5HT1A receptor, and explored the affected pathways in neural development and melanogenesis. Our results contribute to unraveling the multifaceted functions of 5-HT, revealing its involvement in sensory cell differentiation in ascidians.

The evolutionary origin of vertebrates included innovations in sensory processing associated with the acquisition of a predatory lifestyle. Vertebrates perceive external stimuli through sensory systems serviced by cranial sensory ganglia (CSG) which develop from cranial placodes; however understanding the evolutionary origin of placodes and CSGs is hampered by the gulf between living lineages and difficulty in assigning homology between cell types and structures. Here we use the Hmx gene family to address this question. We show Hmx is a constitutive component of vertebrate CSG development and that Hmx in the tunicate Ciona is able to drive the differentiation program of Bipolar Tail Neurons (BTNs), cells previously thought neural crest homologs. Using Ciona and lamprey transgenesis we demonstrate that a unique, tandemly duplicated enhancer pair regulated Hmx in the stem-vertebrate lineage. Strikingly, we also show robust vertebrate Hmx enhancer function in Ciona, demonstrating that deep conservation of the upstream regulatory network spans the evolutionary origin of vertebrates. These experiments demonstrate regulatory and functional conservation between Ciona and vertebrate Hmx, and confirm BTNs as CSG homologs. Our analysis also identifies derived evolutionary changes, including a genetic basis for secondary simplicity in Ciona and unique regulatory complexity in vertebrates.