The landscape of combining immune checkpoint inhibitors with novel Therapies: Secret alliances against breast cancer.

This review focuses on the immune checkpoint inhibitors (ICIs) in the context of breast cancer (BC) management. These innovative treatments, by targeting proteins expressed on both tumor and immune cells, aim to overcome tumor-induced immune suppression and reactivate the immune system. The potential of this approach is the subject of numerous clinical studies. Here, we explore the key studies and emerging therapies related to ICIs providing a detailed analysis of their specific and combined use in BC treatment.

NK cell receptors in anti-tumor and healthy tissue protection: Mechanisms and therapeutic advances.

Natural Killer (NK) cells are integral to the innate immune system, renowned for their ability to target and eliminate cancer cells without the need for antigen presentation, sparing normal tissues. These cells are crucial in cancer immunosurveillance due to their diverse array of activating and inhibitory receptors that modulate their cytotoxic activity. However, the tumor microenvironment can suppress NK cell function through various mechanisms. Over recent decades, research has focused on overcoming these tumor escape mechanisms. Initially, efforts concentrated on enhancing T cell activity, leading to impressive results with immunotherapeutic approaches aimed at boosting T cell responses. Nevertheless, a substantial number of patients do not benefit from these treatments and continue to seek effective alternatives. In this context, NK cells present a promising avenue for developing new treatments, given their potent cytotoxic capabilities, safety profile, and activity against T cell-resistant tumors, such as those lacking HLA-I expression. Recent advancements in immunotherapy include strategies to restore and amplify NK cell activity through immune checkpoint inhibitors, cytokines, adoptive NK cell therapy, and CAR-NK cell technology. This review provides a comprehensive overview of NK cell receptors, the tumor escape mechanisms that hinder NK cell function, and the evolving field of NK cell-based cancer immunotherapy, highlighting ongoing efforts to develop more effective and targeted cancer treatment strategies.

Control of Dopamine Signal in High-Order Receptor Complex on Striatal Astrocytes.

The receptor-receptor interaction (RRI) of G protein-coupled receptors (GPCRs) leads to new functional entities that are conceptually distinct from the simple addition of signals mediated by the activation of the receptors that form the heteromers. Focusing on astrocytes, there is evidence for the existence of inhibitory and facilitatory RRIs, including the heteromers formed by the adenosine A2A and the dopamine D2 receptors, by A2A and the oxytocin receptor (OTR), and the D2-OTR heteromers. The possible involvement of these receptors in mosaicism has never been investigated in striatal astrocytes. By biophysical and functional approaches, we focused our attention on the existence of an A2A-D2-OTR high-order receptor complex and its role in modulating cytosolic calcium levels and endogenous glutamate release, when striatal astrocyte processes were stimulated with 4-aminopyridine. Functional data indicate a permissive role of OTR on dopamine signaling in the regulation of the glutamatergic transmission, and an inhibitory control mediated by A2A on both the D2-mediated signaling and on the OTR-facilitating effect on D2. Imaging biochemical and bioinformatic evidence confirmed the existence of the A2A-D2-OTR complex and its ternary structure in the membrane. In conclusion, the D2 receptor appears to be a hotspot in the control of the glutamate release from the astrocytic processes and may contribute to the regulation and integration of different neurotransmitter-mediated signaling in the striatum by the A2A-D2-OTR heterotrimers. Considering the possible selectivity of allosteric interventions on GPCRs organized as receptor mosaics, A2A-D2-OTR heterotrimers may offer selective pharmacological targets in neuropsychiatric disorders and neurodegenerative diseases.

The Bright Side of Curcumin: A Narrative Review of Its Therapeutic Potential in Cancer Management.

Curcumin, a polyphenolic compound derived from Curcuma longa, exhibits significant therapeutic potential in cancer management. This review explores curcumin's mechanisms of action, the challenges related to its bioavailability, and its enhancement through modern technology and approaches. Curcumin demonstrates strong antioxidant and anti-inflammatory properties, contributing to its ability to neutralize free radicals and inhibit inflammatory mediators. Its anticancer effects are mediated by inducing apoptosis, inhibiting cell proliferation, and interfering with tumor growth pathways in various colon, pancreatic, and breast cancers. However, its clinical application is limited by its poor bioavailability due to its rapid metabolism and low absorption. Novel delivery systems, such as curcumin-loaded hydrogels and nanoparticles, have shown promise in improving curcumin bioavailability and therapeutic efficacy. Additionally, photodynamic therapy has emerged as a complementary approach, where light exposure enhances curcumin's anticancer effects by modulating molecular pathways crucial for tumor cell growth and survival. Studies highlight that combining low concentrations of curcumin with visible light irradiation significantly boosts its antitumor efficacy compared to curcumin alone. The interaction of curcumin with cytochromes or drug transporters may play a crucial role in altering the pharmacokinetics of conventional medications, which necessitates careful consideration in clinical settings. Future research should focus on optimizing delivery mechanisms and understanding curcumin's pharmacokinetics to fully harness its therapeutic potential in cancer treatment.

Amphioxus (Branchiostoma lanceolatum) in the North Adriatic Sea: ecological observations and spawning behavior.

The European amphioxus (Branchiostoma lanceolatum) is a member of the chordate subphylum Cephalochordata, and, as such, a key model organism for providing insights into the origin and evolution of vertebrates. Despite its significance and global distribution, detailed characterizations of natural populations of cephalochordates are still very limited. This study investigates the abundance, habitat, and spawning behavior of amphioxus in the North Adriatic Sea. Across 32 sampled sites, adult amphioxus were consistently present, reaching densities exceeding 300 individuals m- 2. DNA barcoding confirmed the species as B. lanceolatum, and environmental analyses revealed an amphioxus preference for slightly gravelly sand with low silt content and a correlation between amphioxus density and the presence of specific macroinvertebrate taxa. Remarkably, the amphioxus population was breeding in early spring and possibly late fall, in contrast to the typical late spring/early summer spawning season described for other populations of European amphioxus. Amphioxus adults kept in captivity maintained the spawning seasonality of their place of origin, suggesting the possibility of extending the overall spawning season of European amphioxus in laboratory settings by exploiting populations from diverse geographic origins. This study thus expands our understanding of B. lanceolatum ecology and reproduction in the Mediterranean Sea, emphasizing the role of the North Adriatic Sea as a substantial reservoir.

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.

Heteromerization of Dopamine D2 and Oxytocin Receptor in Adult Striatal Astrocytes.

The ability of oxytocin (OT) to interact with the dopaminergic system through facilitatory D2-OT receptor (OTR) receptor-receptor interaction in the limbic system is increasingly considered to play roles in social or emotional behavior, and suggested to serve as a potential therapeutic target. Although roles of astrocytes in the modulatory effects of OT and dopamine in the central nervous system are well recognized, the possibility of D2-OTR receptor-receptor interaction in astrocytes has been neglected. In purified astrocyte processes from adult rat striatum, we assessed OTR and dopamine D2 receptor expression by confocal analysis. The effects of activation of these receptors were evaluated in the processes through a neurochemical study of glutamate release evoked by 4-aminopyridine; D2-OTR heteromerization was assessed by co-immunoprecipitation and proximity ligation assay (PLA). The structure of the possible D2-OTR heterodimer was estimated by a bioinformatic approach. We found that both D2 and OTR were expressed on the same astrocyte processes and controlled the release of glutamate, showing a facilitatory receptor-receptor interaction in the D2-OTR heteromers. Biochemical and biophysical evidence confirmed D2-OTR heterodimers on striatal astrocytes. The residues in the transmembrane domains four and five of both receptors are predicted to be mainly involved in the heteromerization. In conclusion, roles for astrocytic D2-OTR in the control of glutamatergic synapse functioning through modulation of astrocytic glutamate release should be taken into consideration when considering interactions between oxytocinergic and dopaminergic systems in striatum.

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.

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.

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.

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.

A Focus on Regulatory Networks Linking MicroRNAs, Transcription Factors and Target Genes in Neuroblastoma.

Neuroblastoma (NB) is a tumor of the peripheral sympathetic nervous system that substantially contributes to childhood cancer mortality. NB originates from neural crest cells (NCCs) undergoing a defective sympathetic neuronal differentiation and although the starting events leading to the development of NB remain to be fully elucidated, the master role of genetic alterations in key oncogenes has been ascertained: (1) amplification and/or over-expression of MYCN, which is strongly associated with tumor progression and invasion; (2) activating mutations, amplification and/or over-expression of ALK, which is involved in tumor initiation, angiogenesis and invasion; (3) amplification and/or over-expression of LIN28B, promoting proliferation and suppression of neuroblast differentiation; (4) mutations and/or over-expression of PHOX2B, which is involved in the regulation of NB differentiation, stemness maintenance, migration and metastasis. Moreover, altered microRNA (miRNA) expression takes part in generating pathogenetic networks, in which the regulatory loops among transcription factors, miRNAs and target genes lead to complex and aberrant oncogene expression that underlies the development of a tumor. In this review, we have focused on the circuitry linking the oncogenic transcription factors MYCN and PHOX2B with their transcriptional targets ALK and LIN28B and the tumor suppressor microRNAs let-7, miR-34 and miR-204, which should act as down-regulators of their expression. We have also looked at the physiologic role of these genetic and epigenetic determinants in NC development, as well as in terminal differentiation, with their pathogenic dysregulation leading to NB oncogenesis.

The OSMR Gene Is Involved in Hirschsprung Associated Enterocolitis Susceptibility through an Altered Downstream Signaling.

Hirschsprung (HSCR) Associated Enterocolitis (HAEC) is a common life-threatening complication in HSCR. HAEC is suggested to be due to a loss of gut homeostasis caused by impairment of immune system, barrier defense, and microbiome, likely related to genetic causes. No gene has been claimed to contribute to HAEC occurrence, yet. Genetic investigation of HAEC by Whole-Exome Sequencing (WES) on 24 HSCR patients affected (HAEC) or not affected (HSCR-only) by enterocolitis and replication of results on a larger panel of patients allowed the identification of the HAEC susceptibility variant p.H187Q in the Oncostatin-M receptor (OSMR) gene (14.6% in HAEC and 5.1% in HSCR-only, p = 0.0024). Proteomic analysis on the lymphoblastoid cell lines from one HAEC patient homozygote for this variant and one HAEC patient not carrying the variant revealed two well distinct clusters of proteins significantly up or downregulated upon OSM stimulation. A marked enrichment in immune response pathways (q < 0.0001) was shown in the HAEC H187 cell line, while proteins upregulated in the HAEC Q187 lymphoblasts sustained pathways likely involved in pathogen infection and inflammation. In conclusion, OSMR p.H187Q is an HAEC susceptibility variant and perturbates the downstream signaling cascade necessary for the gut immune response and homeostasis maintenance.

Human primed ILCPs support endothelial activation through NF-κB signaling.

Innate lymphoid cells (ILCs) represent the most recently identified subset of effector lymphocytes, with key roles in the orchestration of early immune responses. Despite their established involvement in the pathogenesis of many inflammatory disorders, the role of ILCs in cancer remains poorly defined. Here we assessed whether human ILCs can actively interact with the endothelium to promote tumor growth control, favoring immune cell adhesion. We show that, among all ILC subsets, ILCPs elicited the strongest upregulation of adhesion molecules in endothelial cells (ECs) in vitro, mainly in a contact-dependent manner through the tumor necrosis factor receptor- and RANK-dependent engagement of the NF-κB pathway. Moreover, the ILCP-mediated activation of the ECs resulted to be functional by fostering the adhesion of other innate and adaptive immune cells. Interestingly, pre-exposure of ILCPs to human tumor cell lines strongly impaired this capacity. Hence, the ILCP-EC interaction might represent an attractive target to regulate the immune cell trafficking to tumor sites and, therefore, the establishment of an anti-tumor immune response.

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.

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.

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.

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.