Assessing the Effects of Curcumin and 450 nm Photodynamic Therapy on Oxidative Metabolism and Cell Cycle in Head and Neck Squamous Cell Carcinoma: An In Vitro Study.

Oral cancer is the 16th most common malignant tumor worldwide. The risk of recurrence and mortality is high, and the survival rate is low over the following five years. Recent studies have shown that curcumin causes apoptosis in tumor cells by affecting FoF1-ATP synthase (ATP synthase) activity, which, in turn, hinders cell energy production, leading to a loss of cell viability. Additionally, irradiation of curcumin within cells can intensify its detrimental effects on cancer cell viability and proliferation (photodynamic therapy). We treated the OHSU-974 cell line, a model for human head and neck squamous cell carcinoma (HNSCC), and primary human fibroblasts. The treatment involved a 1 h exposure of cells to 0.1, 1.0, and 10 µM curcumin, followed or not by irradiation or the addition of the same concentration of pre-irradiated curcumin. Both instances involved a diode laser with a wavelength of 450 nm (0.25 W, 15 J, 60 s, 1 cm2, continuous wave mode). The treatment with non-irradiated 1 and 10 µM curcumin caused ATP synthase inhibition and a consequent reduction in the oxygen consumption rate (OCR) and the ATP/AMP ratio, which was associated with a decrement in lipid peroxidation accumulation and a slight increase in glutathione reductase and catalase activity. By contrast, 60 s curcumin irradiation with 0.25 W-450 nm caused a further oxidative phosphorylation (OxPhos) metabolism impairment that induced an uncoupling between respiration and energy production, leading to increased oxidative damage, a cellular growth and viability reduction, and a cell cycle block in the G1 phase. These effects appeared to be more evident when the curcumin was irradiated after cell incubation. Since cells belonging to the HNSCC microenvironment support tumor development, curcumin's effects have been analyzed on primary human fibroblasts, and a decrease in cell energy status has been observed with both irradiated and non-irradiated curcumin and an increase in oxidative lipid damage and a slowing of cell growth were observed when the curcumin was irradiated before or after cellular administration. Thus, although curcumin displays an anti-cancer role on OHSU-974 in its native form, photoactivation seems to enhance its effects, making it effective even at low dosages.

New Insights into Geometric Morphometry Applied to Fish Scales for Species Identification.

The possibility of quick and cheap recognition of a fish species from a single dermal scale would be interesting in a wide range of contexts. The methods of geometric morphometry appear to be quite promising, although wide studies comparing different approaches are lacking. We aimed to apply two methods of geometric morphometry, landmark-based and outline-based, on a dataset of scales from five different teleost species: Danio rerio, Dicentrarchus labrax, Mullus surmuletus, Sardina pilchardus, and Sparus aurata. For the landmark-based method the R library "geomorph" was used. Some issues about landmark selection and positioning were addressed and, for the first time on fish scales, an approach with both landmarks and semilandmarks was set up. For the outline-based method the R library "Momocs" was used. Despite the relatively low number of scales analyzed (from 11 to 81 for each species), both methods achieved quite good clustering of all the species. In particular, the landmark-based method used here gave generally higher R2 values in testing species clustering than the outline-based method, but it failed to distinguish between a few couples of species; on the other hand, the outline-based method seemed to catch the differences among all the couples except one. Larger datasets have the potential to achieve better results with outline-based geometric morphometry. This latter method, being free from the problem of recognizing and positioning landmarks, is also the most suitable for being automatized in future applications.

Serotonin Receptors and Their Involvement in Melanization of Sensory Cells in Ciona intestinalis.

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.

Prevention of Dry Socket with Ozone Oil-Based Gel after Inferior Third Molar Extraction: A Double-Blind Split-Mouth Randomized Placebo-Controlled Clinical Trial.

Tooth extraction is followed by a sequence of elaborate local changes affecting hard and soft tissues. Dry socket (DS) can occur as intense pain around and in the extraction site, with an incidence from 1-4% after generic tooth extraction to 45% for mandibular third molars. Ozone therapy has gained attention in the medical field because of its success in the treatment of various diseases, its biocompatible properties and its fewer side effects or discomfort than drugs. To investigate the preventive effect of the sunflower oil-based ozone gel Ozosan® (Sanipan srl, Clivio (VA), Italy) on DS, a double-blind split-mouth randomized placebo-controlled clinical trial was conducted according to the CONSORT guidelines. Ozosan® or the placebo gel were put in the socket, and the gels were washed off 2 min later. In total, 200 patients were included in our study. The patient population comprised 87 Caucasian males and 113 Caucasian females. The mean age of the included patients was 33.1 ± 12.4 years. Ozosan reduced the incidence of DS after inferior third molar extraction from 21.5% of the control to 2% (p < 0.001). Concerning the dry socket epidemiology, the incidence was not significantly correlated with gender, smoking or mesioangular, vertical or distoangular Winter's classification. Post hoc power calculation showed a power of 99.8% for this data, with alpha = 0.001.

Retinoic Acid and POU Genes in Developing Amphioxus: A Focus on Neural Development.

POU genes are a family of evolutionarily conserved transcription factors with key functions in cell type specification and neurogenesis. In vitro experiments have indicated that the expression of some POU genes is controlled by the intercellular signaling molecule retinoic acid (RA). In this work, we aimed to characterize the roles of RA signaling in the regulation of POU genes in vivo. To do so, we studied POU genes during the development of the cephalochordate amphioxus, an animal model crucial for understanding the evolutionary origins of vertebrates. The expression patterns of amphioxus POU genes were assessed at different developmental stages by chromogenic in situ hybridization and hybridization chain reaction. Expression was further assessed in embryos subjected to pharmacological manipulation of endogenous RA signaling activity. In addition to a detailed description of the effects of these treatments on amphioxus POU gene expression, our survey included the first description of Pou2 and Pou6 expression in amphioxus embryos. We found that Pit-1, Pou2, Pou3l, and Pou6 expression are not affected by alterations of endogenous RA signaling levels. In contrast, our experiments indicated that Brn1/2/4 and Pou4 expression are regulated by RA signaling in the endoderm and the nerve cord, respectively. The effects of the treatments on Pou4 expression in the nerve cord revealed that, in developing amphioxus, RA signaling plays a dual role by (1) providing anteroposterior patterning information to neural cells and (2) specifying neural cell types. This finding is coherent with a terminal selector function of Pou4 for GABAergic neurons in amphioxus and represents the first description of RA-induced changes in POU gene expression in vivo.

The Arrangement of the Peripheral Olfactory System of Pleuragramma antarcticum: A Well-Exploited Small Sensor, an Aided Water Flow, and a Prominent Effort in Primary Signal Elaboration.

The olfactory system is constituted in a consistent way across vertebrates. Nasal structures allow water/air to enter an olfactory cavity, conveying the odorants to a sensory surface. There, the olfactory neurons form, with their axons, a sensory nerve projecting to the telencephalic zone-named the olfactory bulb. This organization comes with many different arrangements, whose meaning is still a matter of debate. A morphological description of the olfactory system of many teleost species is present in the literature; nevertheless, morphological investigations rarely provide a quantitative approach that would help to provide a deeper understanding of the structures where sensory and elaborating events happen. In this study, the peripheral olfactory system of the Antarctic silverfish, which is a keystone species in coastal Antarctica ecosystems, has also been described, employing some quantitative methods. The olfactory chamber of this species is connected to accessory nasal sacs, which probably aid water movements in the chamber; thus, the head of the Antarctic silverfish is specialized to assure that the olfactory organ keeps in contact with a large volume of water-even when the fish is not actively swimming. Each olfactory organ, shaped like an asymmetric rosette, has, in adult fish, a sensory surface area of about 25 mm2, while each olfactory bulb contains about 100,000 neurons. The sensory surface area and the number of neurons in the primary olfactory brain region show that this fish invests energy in the detection and elaboration of olfactory signals and allow comparisons among different species. The mouse, for example-which is considered a macrosmatic vertebrate-has a sensory surface area of the same order of magnitude as that of the Antarctic silverfish, but ten times more neurons in the olfactory bulb. Catsharks, on the other hand, have a sensory surface area that is two orders of magnitude higher than that of the Antarctic silverfish, while the number of neurons has the same order of magnitude. The Antarctic silverfish is therefore likely to rely considerably on olfaction.

Case Report: A Peculiar Case of Inflammatory Colitis After SARS-CoV-2 Infection.

We report a case of inflammatory colitis after SARS-CoV-2 infection in a patient with no additional co-morbidity who died within three weeks of hospitalization. As it is becoming increasingly clear that SARS-CoV-2 infection can cause immunological alterations, we investigated the expression of the inhibitory checkpoint PD-1 and its ligand PD-L1 to explore the potential role of this axis in the break of self-tolerance. The presence of the SARS-CoV-2 virus in colon tissue was demonstrated by qRT-PCR and immunohistochemical localization of the nucleocapsid protein. Expression of lymphocyte markers, PD-1, and PD-L1 in colon tissue was investigated by IHC. SARS-CoV-2-immunoreactive cells were detected both in the ulcerated and non-ulcerated mucosal areas. Compared to healthy tissue, where PD-1 is weakly expressed and PD-L1 is absent, PD-1 and PD-L1 expression appears in the inflamed mucosal tissue, as expected, but was mainly confined to non-ulcerative areas. At the same time, these markers were virtually undetectable in areas of mucosal ulceration. Our data show an alteration of the PD-1/PD-L1 axis and suggest a link between SARS-CoV-2 infection and an aberrant autoinflammatory response due to concomitant breakdown of the PD-1/PD-L1 interaction leading to early death of the patient.

Heterodimer of A2A and Oxytocin Receptors Regulating Glutamate Release in Adult Striatal Astrocytes.

BACKGROUND: Roles of astrocytes in the modulatory effects of oxytocin (OT) in central nervous system are increasingly considered. Nevertheless, OT effects on gliotransmitter release have been neglected. METHODS: In purified astrocyte processes from adult rat striatum, we assessed OT receptor (OTR) and adenosine A2A receptor expression by confocal analysis. The effects of receptors activation on glutamate release from the processes were evaluated; A2A-OTR heteromerization was assessed by co-immunoprecipitation and PLA. Structure of the possible heterodimer of A2A and OT receptors was estimated by a bioinformatic approach. RESULTS: Both A2A and OT receptors were expressed on the same astrocyte processes. Evidence for A2A-OTR receptor-receptor interaction was obtained by measuring the release of glutamate: OT inhibited the evoked glutamate release, while activation of A2A receptors, per se ineffective, abolished the OT effect. Biochemical and biophysical evidence for A2A-OTR heterodimers on striatal astrocytes was also obtained. The residues in the transmembrane domains 4 and 5 of both receptors are predicted to be mainly involved in the heteromerization. CONCLUSIONS: When considering effects of OT in striatum, modulation of glutamate release from the astrocyte processes and of glutamatergic synapse functioning, and the interaction with A2A receptors on the astrocyte processes should be taken into consideration.

Functional Conservation and Genetic Divergence of Chordate Glycinergic Neurotransmission: Insights from Amphioxus Glycine Transporters.

Glycine is an important neurotransmitter in vertebrates, performing both excitatory and inhibitory actions. Synaptic levels of glycine are tightly controlled by the action of two glycine transporters, GlyT1 and GlyT2, located on the surface of glial cells and neurons, respectively. Only limited information is available on glycinergic neurotransmission in invertebrates, and the evolution of glycinergic neurotransmission is poorly understood. Here, by combining phylogenetic and gene expression analyses, we characterized the glycine transporter complement of amphioxus, an important invertebrate model for studying the evolution of chordates. We show that amphioxus possess three glycine transporter genes. Two of these (GlyT2.1 and GlyT2.2) are closely related to GlyT2 of vertebrates, whereas the third (GlyT) is a member of an ancestral clade of deuterostome glycine transporters. GlyT2.2 expression is predominantly non-neural, whereas GlyT and GlyT2.1 are widely expressed in the amphioxus nervous system and are differentially expressed, respectively, in neurons and glia. Vertebrate glycinergic neurons express GlyT2 and glia GlyT1, suggesting that the evolution of the chordate glycinergic system was accompanied by a paralog-specific inversion of gene expression. Despite this genetic divergence between amphioxus and vertebrates, we found strong evidence for conservation in the role glycinergic neurotransmission plays during larval swimming, the implication being that the neural networks controlling the rhythmic movement of chordate bodies may be homologous.

Beneficial Effect of Phenytoin and Carbamazepine on GFAP Gene Expression and Mutant GFAP Folding in a Cellular Model of Alexander's Disease.

Alexander's disease (AxD) is a rare, usually relentlessly progressive disorder of astroglial cells in the central nervous system related to mutations in the gene encoding the type III intermediate filament protein, glial fibrillary acidic protein (GFAP). The pathophysiology of AxD is only partially understood. Available data indicate that an excessive GFAP gene expression may play a role. In particular, a "threshold hypothesis" has been reported, suggesting that mutant GFAP representing about 20% of the total cellular GFAP should be sufficient to cause disease. Thus, strategies based on reducing cellular mutant GFAP protein levels and/or activating biological processes involved in the correct protein folding could be effective in counteracting the toxic effect of misfolded GFAP. Considering that clomipramine (CLM), which has been selected by a wide small molecules screening as the greatest inhibitory potential drug against GFAP expression, is contraindicated because of its proconvulsant activity in the infantile form of AxD, which is also characterized by the occurrence of epileptic seizures, two powerful antiepileptic agents, carbamazepine (CBZ) and phenytoin (PHT), which share specific stereochemical features in common with CLM, were taken into consideration in a reliable in vitro model of AxD. In the present work, we document for the first time that CBZ and PHT have a definite inhibitory effect on pathological GFAP cellular expression and folding. Moreover, we confirm previous results of a similar beneficial effect of CLM. In addition, we have demonstrated that CBZ and CLM play a refolding effect on mutant GFAP proteins, likely ascribed at the induction of CRYAB expression, resulting in the decrease of mutant GFAP aggregates formation. As CBZ and PHT are currently approved for use in humans, their documented effects on pathological GFAP cellular expression and folding may indicate a potential therapeutic role as disease-modifying agents of these drugs in the clinical management of AxD, particularly in AxD patients with focal epilepsy with and without secondary generalization.

Post-Transplant Nivolumab Plus Unselected Autologous Lymphocytes in Refractory Hodgkin Lymphoma: A Feasible and Promising Salvage Therapy Associated With Expansion and Maturation of NK Cells.

Immune checkpoint inhibitors (CI) have demonstrated clinical activity in Hodgkin Lymphoma (HL) patients relapsing after autologous stem cell transplantation (ASCT), although only 20% complete response (CR) rate was observed. The efficacy of CI is strictly related to the host immune competence, which is impaired in heavily pre-treated HL patients. Here, we aimed to enhance the activity of early post-ASCT CI (nivolumab) administration with the infusion of autologous lymphocytes (ALI). Twelve patients with relapse/refractory (R/R) HL (median age 28.5 years; range 18-65), underwent lymphocyte apheresis after first line chemotherapy and then proceeded to salvage therapy. Subsequently, 9 patients with progressive disease at ASCT received early post-transplant CI supported with four ALI, whereas 3 responding patients received ALI alone, as a control cohort. No severe adverse events were recorded. HL-treated patients achieved negative PET scan CR and 8 are alive and disease-free after a median follow-up of 28 months. Four patients underwent subsequent allogeneic SCT. Phenotypic analysis of circulating cells showed a faster expansion of highly differentiated NK cells in ALI plus nivolumab-treated patients as compared to control patients. Our data show anti-tumor activity with good tolerability of ALI + CI for R/R HL and suggest that this setting may accelerate NK cell development/maturation and favor the expansion of the "adaptive" NK cell compartment in patients with HCMV seropositivity, in the absence of HCMV reactivation.

Amphioxus neuroglia: Molecular characterization and evidence for early compartmentalization of the developing nerve cord.

Glial cells play important roles in the development and homeostasis of metazoan nervous systems. However, while their involvement in the development and function in the central nervous system (CNS) of vertebrates is increasingly well understood, much less is known about invertebrate glia and the evolutionary history of glial cells more generally. An investigation into amphioxus glia is therefore timely, as this organism is the best living proxy for the last common ancestor of all chordates, and hence provides a window into the role of glial cell development and function at the transition of invertebrates and vertebrates. We report here our findings on amphioxus glia as characterized by molecular probes correlated with anatomical data at the transmission electron microscopy (TEM) level. The results show that amphioxus glial lineages express genes typical of vertebrate astroglia and radial glia, and that they segregate early in development, forming what appears to be a spatially separate cell proliferation zone positioned laterally, between the dorsal and ventral zones of neural cell proliferation. Our study provides strong evidence for the presence of vertebrate-type glial cells in amphioxus, while highlighting the role played by segregated progenitor cell pools in CNS development. There are implications also for our understanding of glial cells in a broader evolutionary context, and insights into patterns of precursor cell deployment in the chordate nerve cord.

Alexander Disease Modeling in Zebrafish: An In Vivo System Suitable to Perform Drug Screening.

Alexander disease (AxD) is a rare astrogliopathy caused by heterozygous mutations, either inherited or arising de novo, on the glial fibrillary acid protein (GFAP) gene (17q21). Mutations in the GFAP gene make the protein prone to forming aggregates which, together with heat-shock protein 27 (HSP27), αB-crystallin, ubiquitin, and proteasome, contribute to form Rosenthal fibers causing a toxic effect on the cell. Unfortunately, no pharmacological treatment is available yet, except for symptom reduction therapies, and patients undergo a progressive worsening of the disease. The aim of this study was the production of a zebrafish model for AxD, to have a system suitable for drug screening more complex than cell cultures. To this aim, embryos expressing the human GFAP gene carrying the most severe p.R239C under the control of the zebrafish gfap gene promoter underwent functional validation to assess several features already observed in in vitro and other in vivo models of AxD, such as the localization of mutant GFAP inclusions, the ultrastructural analysis of cells expressing mutant GFAP, the effects of treatments with ceftriaxone, and the heat shock response. Our results confirm that zebrafish is a suitable model both to study the molecular pathogenesis of GFAP mutations and to perform pharmacological screenings, likely useful for the search of therapies for AxD.

Cancer Immunotherapy by Blocking Immune Checkpoints on Innate Lymphocytes.

Immune checkpoints refer to a plethora of inhibitory pathways of the immune system that play a crucial role in maintaining self-tolerance and in tuning the duration and amplitude of physiological immune responses to minimize collateral tissue damages. The breakdown of this delicate balance leads to pathological conditions, including cancer. Indeed, tumor cells can develop multiple mechanisms to escape from immune system defense, including the activation of immune checkpoint pathways. The development of monoclonal antibodies, targeting inhibitory immune checkpoints, has provided an immense breakthrough in cancer therapy. Immune checkpoint inhibitors (ICI), initially developed to reverse functional exhaustion in T cells, recently emerged as important actors in natural killer (NK)-cell-based immunotherapy. Moreover, the discovery that also helper innate lymphoid cells (ILCs) express inhibitory immune checkpoints, suggests that these molecules might be targeted on ILCs, to modulate their functions in the tumor microenvironment. Recently, other strategies to achieve immune checkpoint blockade have been developed, including miRNA exploiting systems. Herein, we provide an overview of the current knowledge on inhibitory immune checkpoints on NK cells and ILCs and we discuss how to target these innate lymphocytes by ICI in both solid tumors and hematological malignancies.

The Diversity of Muscles and Their Regenerative Potential across Animals.

Cells with contractile functions are present in almost all metazoans, and so are the related processes of muscle homeostasis and regeneration. Regeneration itself is a complex process unevenly spread across metazoans that ranges from full-body regeneration to partial reconstruction of damaged organs or body tissues, including muscles. The cellular and molecular mechanisms involved in regenerative processes can be homologous, co-opted, and/or evolved independently. By comparing the mechanisms of muscle homeostasis and regeneration throughout the diversity of animal body-plans and life cycles, it is possible to identify conserved and divergent cellular and molecular mechanisms underlying muscle plasticity. In this review we aim at providing an overview of muscle regeneration studies in metazoans, highlighting the major regenerative strategies and molecular pathways involved. By gathering these findings, we wish to advocate a comparative and evolutionary approach to prompt a wider use of "non-canonical" animal models for molecular and even pharmacological studies in the field of muscle regeneration.

Whole mount in situ hybridization and immunohistochemistry for studying retinoic acid signaling in developing amphioxus.

Retinoic acid (RA) is a vitamin A-derived signaling molecule acting during development and in the adult. This chapter provides protocols to characterize the role of RA signaling during development of the invertebrate chordate amphioxus. As sister group to all other chordates and characterized by the most vertebrate-like RA signaling system of all invertebrates, amphioxus is an important model for studying the evolution of RA signaling. Focusing on the development of GABAergic neurons in the amphioxus central nervous system, we provide detailed protocols for maintaining and breeding adult animals, for performing pharmacological treatments of embryos and for analyzing the effects of these treatments by whole mount in situ hybridization and immunohistochemistry coupled to confocal microscopy.

Insights into the evolution of metazoan regenerative mechanisms: roles of TGF superfamily members in tissue regeneration of the marine sponge Chondrosia reniformis.

Tissue repair is an adaptive and widespread metazoan response. It is characterised by different cellular mechanisms and complex signalling networks that involve numerous growth factors and cytokines. In higher animals, transforming growth factor-ß (TGF-ß) signalling plays a fundamental role in wound healing. In order to evaluate the involvement of TGF superfamily members in lower invertebrate tissue regeneration, sequences for putative TGF ligands and receptors were isolated from the transcriptome of the marine sponge Chondrosia reniformis We identified seven transcripts that coded for TGF superfamily ligands and three for TGF superfamily receptors. Phylogenetically, C. reniformis TGF ligands were not grouped into any TGF superfamily clades and thus presumably evolved independently, whereas the TGF receptors clustered in the Type I receptor group. We performed gene expression profiling of these transcripts in sponge regenerating tissue explants. Data showed that three ligands (TGF1, TGF3 and TGF6) were mainly expressed during early regeneration and seemed to be involved in stem cell maintenance, whereas two others (TGF4 and TGF5) were strongly upregulated during late regeneration and thus were considered pro-differentiating factors. The presence of a strong TGF inhibitor, SB431542, blocked the restoration of the exopinacoderm layer in the sponge explants, confirming the functional involvement of the TGF pathway in tissue regeneration in these early evolved animals.

The HMGA gene family in chordates: evolutionary perspectives from amphioxus.

High mobility group A proteins of vertebrates, HMGA1 and 2, are chromatin architectural factors involved in development, cell differentiation, and neoplastic transformation. Here, we characterize an amphioxus HMGA gene ortholog and analyze its expression. As a basal chordate, amphioxus is well placed to provide insights into the evolution of the HMGA gene family, particularly in the transition from invertebrates to vertebrates. Our phylogenetic analysis supports the basal position of amphioxus, echinoderm, and hemichordate HMGA sequences to those of vertebrate HMGA1 and HMGA2. Consistent with this, the genomic landscape around amphioxus HMGA shares features with both. Whole mount in situ hybridization shows that amphioxus HMGA mRNA is detectable from neurula stage onwards in both nervous and non-nervous tissues. This correlates with protein expression monitored immunocytochemically using antibodies against human HMGA2 protein, revealing especially high levels of expression in cells of the lamellar body, the amphioxus homolog of the pineal, suggesting that the gene may have, among its many functions, an evolutionarily conserved role in photoreceptor differentiation.

Novel polyclonal antibodies as a useful tool for expression studies in amphioxus embryos.

Cephalochordates, commonly called amphioxus or lancelets, are widely regarded as a useful proxy for the chordate ancestor. In recent decades, expression patterns of important developmental genes have been used extensively to infer homologies between cephalochordate and vertebrate embryos. Such comparisons provided important insight into cephalochordate biology and the origin of vertebrate traits. Most of the developmental expression data are collected using whole-mount in situ hybridization that allows the distributions of specific transcripts to be detected in fixed embryos. Here, we describe an experimental pipeline for production of small amounts of functional antibodies directed against amphioxus antigens for use in immunohistochemical labelling. In this pilot study, we generated antibodies against ß-catenin and the transcription factors FoxA, Lhx1, Lhx3 and Pax6. We demonstrate the usefulness of antibodies by performing immunostainings on fixed specimens of B. lanceolatum and B. floridae. We anticipate that amphioxus-specific antibodies will provide a useful tool for high-resolution labelling of individual cells within the embryo and for determining the subcellular localization of the corresponding proteins.