System Of Vertebrates Research Articles

Assessment of Acetylcholinesterase Enzyme (AChE) Activity in the Developing Brain of TSD Lizard, Calotes Versicolor (Daud.)

Acetylcholinesterase (AChE) is a critical enzyme in the neuronal cholinergic system of vertebrates. It is one of the efficient cholinesterases that is involved in the termination of acetylcholine-mediated neurotransmission, rapidly hydrolyzing acetylcholine into acetate and choline. Barring a few reports on the distribution/presence of AChE enzyme activity in the adult reptilian species there are nil reports on the presence of this enzyme activity in the developing brain. Hence, the present investigation aims to evaluate AChE enzyme activity in the developing brain of an oviparous lizard, Calotes versicolor which exhibits a novel Female-Male-Female-Male (FMFM) pattern of temperature-dependent sex determination (TSD). The eggs were collected during breeding season, padded with moist cotton, and incubated in an incubator at 30o C. The whole brain from the embryos was collected during different developmental stages from oviposition (Stage 27) to hatching (stage 42). The AChE enzyme activity was quantified in the brain, according to Ellman’s protocol using a spectrophotometer. The findings reveal that the onset of AChE enzyme activity is observed as early as at oviposition, which reflects the early action of AChE activity in the developing brain. The observed results albeit indirectly suggest the involvement of AChE enzyme activity in the morphogenetic process. Further, an exponential increase in AChE activity during the post-gonadal differentiation phase indicates age-related elevation in AChE enzyme activity which in turn unveils its involvement in neuronal transmission of the embryonic brain. Besides, this specific AChE enzyme activity in the developing brain of Calotes versicolor represents a conserved pattern for the cholinergic system in vertebrates.

Znf687 recruits Brd4-Smrt complex to regulate gfi1aa during neutrophil development.

Neutrophils are key component of the innate immune system in vertebrates. Diverse transcription factors and cofactors act in a well-coordinated manner to ensure proper neutrophil development. Dysregulation of the transcriptional program triggering neutrophil differentiation is associated with various human hematologic disorders such as neutropenia, neutrophilia, and leukemia. In the current study we show the zinc finger protein Znf687 is a lineage-preferential transcription factor, whose deficiency leads to an impaired neutrophil development in zebrafish. Mechanistically, Znf687 functions as a negative regulator of gfi1aa, a pivotal modulator in terminal granulopoiesis, to regulate neutrophil maturation. Moreover, we found BRD4, an important epigenetic regulator, directly interacts with ZNF687 in neutrophils. Deficiency of brd4 results in similar defective neutrophil development as observed in znf687 mutant zebrafish. Biochemical and genetic analyses further reveal that instead of serving as a canonical transcriptional coactivator, Brd4 directly interacts and bridges Znf687 and Smrt nuclear corepressor on gfi1aa gene's promoter to exert transcription repression. In addition, the ZNF687-BRD4-SMRT-GFI1 transcriptional regulatory network is evolutionary conserved in higher vertebrate. Overall, our work indicates Znf687 and Brd4 are two novel master regulators in promoting terminal granulopoiesis.

The influence of acute dopamine transporter inhibition on manic-, depressive-like phenotypes, and brain oxidative status in adult zebrafish

Functional changes in dopamine transporter (DAT) are related to various psychiatric conditions, including bipolar disorder (BD) symptoms. In experimental research, the inhibition of DAT induces behavioral alterations that recapitulate symptoms found in BD patients, including mania and depressive mood. Thus, developing novel animal models that mimic BD-related conditions by pharmacologically modulating the dopaminergic signaling is relevant. The zebrafish (Danio rerio) has been considered a suitable vertebrate system for modeling BD-like responses, due to the well-characterized behavioral responses and evolutionarily conservation of the dopaminergic system of this species. Here, we investigate whether GBR 12909, a selective inhibitor of DAT, causes neurobehavioral alterations in zebrafish similar to those observed in BD patients. Behaviors were recorded after a single intraperitoneal (i.p.) administration of GBR 12909 at different doses (3.75, 7.5, 15 and 30 mg/kg). To observe temporal effects on behavior, swim path parameters were measured immediately after the administration period during 30 min. Locomotion, anxiety-like behavior, social preference, aggression, despair-like behavior, and oxidative stress-related biomarkers in the brain were measured 30 min post administration. GBR 12909 induced prominent effects on locomotor activity and vertical exploration during the 30-min period. Hyperactivity was observed in GBR 30 group after 25 min, while all doses markedly reduced vertical drifts. GBR 12909 elicited hyperlocomotion, anxiety-like behavior, decreased social preference, aggression, and induced depressive-like behavior in a behavioral despair task. Depending on the dose, GBR 12909 also decreased SOD activity and TBARS levels, as well as increased GR activity and NPSH content. Collectively, our novel findings show that a single GBR 12909 administration evokes neurobehavioral changes that recapitulate manic- and depressive-like states observed in rodents, fostering the use of zebrafish models to explore BD-like responses in translational neuroscience research.

Distribution of gamma-aminobutyric acid immunoreactivity in the brain of the Siberian sturgeon (Acipenser baeri): Comparison with other fishes.

Gamma-aminobutyric acid (GABA) is the main inhibitory neurotransmitter in the central nervous system (CNS) of vertebrates. Immunohistochemical techniques with specific antibodies against GABA or against its synthesizing enzyme, glutamic acid decarboxylase (GAD) allowed characterizing GABAergic neurons and fibers in the CNS. However, studies on the CNS distribution of GABAergic neurons and fibers of bony fishes are scant and were done in teleost species. With the aim of understanding the early evolution of this system in bony vertebrates, we analyzed the distribution of GABA-immunoreactive (-ir) and GAD-ir neurons and fibers in the CNS of a basal ray-finned fish, the Siberian sturgeon (Chondrostei, Acipenseriformes), using immunohistochemical techniques. Our results revealed the presence and distribution of GABA/GAD-ir cells in different regions of the CNS such as olfactory bulbs, pallium and subpallium, hypothalamus, thalamus, pretectum, optic tectum, tegmentum, cerebellum, central grey, octavolateralis area, vagal lobe, rhombencephalic reticular areas, and the spinal cord. Abundant GABAergic innervation was observed in most brain regions, and GABAergic fibers were very abundant in the hypothalamic floor along the hypothalamo-hypophyseal tract and neurohypophysis. In addition, GABA-ir cerebrospinal fluid-contacting cells were observed in the alar and basal hypothalamus, saccus vasculosus, and spinal cord central canal. The distribution of GABAergic systems in the sturgeon brain shows numerous similarities to that observed in lampreys, but also to those of teleosts and tetrapods.

Classification and Immune Function of Coelomocytes in Echinoderms

Abstract: The top taxonomic position among non-chordate invertebrates, which make up the evolutionary connection between invertebrates and vertebrates, is held by echinoderms. Their immune responses rely on coelomocyte activity functioning concurrently with a range of humoral components that directly interact with invasive pathogens. However, markedly lower numbers of systematic reviews of the classification and immune function of coelomocytes have been performed compared with those of vertebrates. Studying echinoderm coelomocytes continues to be an important evolutionary vantage point for determining the origins of bilaterian immunity as well as the principles behind the adaptive immune system of vertebrates. This article reviews the classifications and immune functions of coelomocytes (sea urchin, sea cucumber and sea star). It summarizes the research progress on immune-related genes/proteins, signal transduction pathways and effector molecules in echinoderms according to the recent literature. This summary provides a theoretical basis for studying coelomocytes and disease control in echinoderms. In the future, a variety of methods and techniques should be used and combined with the surface receptors of cells to study the classification and function of coelomocytes of echinoderms.

Basement membrane extract potentiates the endochondral ossification phenotype of bone marrow-derived mesenchymal stem cell-based cartilage organoids

Endochondral ossification is a developmental process in the skeletal system and bone marrow of vertebrates. During endochondral ossification, primitive cartilaginous anlages derived from mesenchymal stem cells (MSCs) undergo vascular invasion and ossification. In vitro regeneration of endochondral ossification is beneficial for research on the skeletal system and bone marrow development as well as their clinical aspects. However, to achieve the regeneration of endochondral ossification, a stem cell-based artificial cartilage (cartilage organoid, Cart-Org) that possesses an endochondral ossification phenotype is required. Here, we modified a conventional 3D culture method to create stem cell-based Cart-Org by mixing it with a basement membrane extract (BME) and further characterized its chondrogenic and ossification properties. BME enlarged and matured the bone marrow MSC-based Cart-Orgs without any shape abnormalities. Histological analysis using Alcian blue staining showed that the production of cartilaginous extracellular matrices was enhanced in Cart-Org treated with BME. Transcriptome analysis using RNA sequencing revealed that BME altered the gene expression pattern of Cart-Org to a dominant chondrogenic state. BME triggered the activation of the SMAD pathway and inhibition of the NK-κB pathway, which resulted in the upregulation of SOX9, COL2A1, and ACAN in Cart-Org. BME also facilitated the upregulation of genes associated with hypertrophic chondrocytes (IHH, PTH1R, and COL10A1) and ossification (SP7, ALPL, and MMP13). Our findings indicate that BME promotes cartilaginous maturation and further ossification of bone marrow MSC-based Cart-Org, suggesting that Cart-Org treated with BME possesses the phenotype of endochondral ossification.

The Relationship of Gametes and Somatic Cells of Male Gonads of Vertebrates: Evolutionary, Age and Seasonal Aspects, Adaptive and Reactive Transformations Under the Action of Destabilizing Factors

The aim of the study is a comparative analysis of the morphofunctional interactions of germ and somatic cells in the organs of the male reproductive system of vertebrates during ontogenesis, under conditions of changes in reproductive activity and under the influence of destabilizing factors. Material and methods. Using light and electron microscopy, immunohistochemistry and morphometry, the testes of representatives of various classes of vertebrates (amphibians, reptiles, birds and mammals) were studied. The collection of material was carried out in environmentally safe ecosystems and in ecosystems with high anthropogenic load in the Orenburg region of Russia and in the Aktobe region of the Republic of Kazakhstan. Results. Based on the material of our own studies of the gonads, as well as on the basis of information from modern domestic and foreign literature, the patterns of relationships between germ cells and somatic cells of their microenvironment in the testes of vertebrates are shown. The features of the structural organization of the germinal and endocrine components of the male gonads are considered. The features of the structural organization of intertitial endocrinocytes (Leydig cells) are shown, and their high resistance to the action of various destabilizing factors compared to spermatogenic epithelium is established. Pubertal generation of endocrinocytes in vertebrate testes during reproductive age is characterized by stability in cell numbers and belongs to cell populations of a stable type. The role and significance of the structures of the blood-testis barrier are shown and the morphological equivalents of changes in the permeability of this barrier are established; it is revealed that in the studied animals, under the influence of negative anthropogenic factors, the integrity of the blood-testis barrier in the testes is violated. The action of destabilizing factors leads to a decrease in the number of developing germ cells in the lumen of the epididymal tubules (up to their complete disappearance in individual individuals), which is probably due to the deterioration of the trophic influences on them from the structures of the epididymis. Conclusion. The principle of organization of the main compartments of the testes, the convoluted seminiferous tubules and the interstitium of the organ, formed during evolution, probably turned out to be evolutionarily advantageous, since it is represented in the testes of all amniotes - reptiles, birds and mammals. In the process of evolutionary development, several types of structural organization of convoluted seminiferous tubules and interstitium were formed in the testes of vertebrates. The need to protect developing germ cells from the effects of immunocytes, which led to the formation of a blood-testis barrier in the testes.

TLR5M and TLR5S Synergistically Sense Flagellin in Early Endosome in Lamprey Petromyzon marinus, Switched by the N-Glycosylation Site N239.

In mammals, TLR5 functions as a homodimer to recognize bacterial flagellin on the cytomembrane. The current investigations reveal the existence of two types of TLR5, a membrane-bound PmTLR5M, and a soluble variant PmTLR5S, in lamprey (Petromyzon marinus). Although both PmTLR5M and PmTLR5S can bind flagellin, only PmTLR5M is capable of eliciting a proinflammatory response, whereas PmTLR5S can detect the flagellin and facilitate the role of PmTLR5M in early endosomes. The trafficking chaperone UNC93B1 enhances the ligand-induced signaling via PmTLR5M or the combination of PmTLR5M and PmTLR5S. PmTLR5M recruits MyD88 as an adaptor. Furthermore, chimeric receptor studies demonstrate the indispensability of the intradomain of PmTLR5M in effective activation of the proinflammatory pathway upon flagellin stimulation, and the combination of PmTLR5S with a singular intradomain in both homodimer and heterodimer ectodomain arrangements can very significantly augment the immune response. Furthermore, the flagellin binding sites between PmTLR5M and PmTLR5S are conserved, which are essential for ligand binding and signal transduction. Moreover, investigations on N-linked glycosylation modifications reveal that the N239 site in PmTLR5M and PmTLR5S plays a switch role in both flagellin binding and immune responses. In addition, PmTLR5M exhibits the high-mannose-type and complex-type N-glycosylation modifications; however, PmTLR5S shows exclusive complex-type N-glycosylation modification. The key N239 site demonstrates complex-type N-glycosylation modification. The findings address the function and mechanism of TLR5 in ligand recognition, subcellular localization, and signaling pathway in lowest vertebrate and immune system transition species, highlight the regulatory role of N-glycosylation modification in TLRs, and augment immune evolutionary research on the TLR signaling pathway.

Ontogenetic changes in the tyrosine hydroxylase immunoreactive preoptic area in the small-spotted catshark Scyliorhinus canicula (L., 1758) females: catecholaminergic involvement in sexual maturation.

The catecholaminergic component of the brain-pituitary-gonadal axis, which mediates the influence of external and internal stimuli on the central nervous system and gonad development in vertebrates, is largely unexplored in Chondrichthyes. We considered Scyliorhinus canicula (L., 1758) females as a model for this vertebrate's class, to assess the involvement of the catecholaminergic system of the brain in its reproduction. Along the S. canicula reproductive cycle, we characterized and evaluated differences in somata morphometry and the number of putative catecholaminergic neurons in two brain nuclei: the periventricular preoptic nucleus, hypothesized to be a positive control for ovarian development, and the suprachiasmatic nucleus, examined as a negative control. 16 S. canicula wild females were sampled and grouped in maturity stages (immature, maturing, mature, and mature egg-laying). The ovary was histologically processed for the qualitative description of maturity stages. Anti-tyrosine hydroxylase immunofluorescence was performed on the diencephalic brain sections. The immunoreactive somata were investigated for morphometry and counted using the optical fractionator method, throughout the confocal microscopy. Qualitative and quantitative research confirmed two separate populations of immunoreactive neurons. The modifications detected in the preoptic nucleus revealed that somata were more numerous, significantly smaller in size, and more excitable during the maturing phase but decreased, becoming slightly bigger and less excitable in the egg-laying stage. This may indicate that the catecholaminergic preoptic nucleus is involved in the control of reproduction, regulating both the onset of puberty and the imminent spawning. In contrast, somata in the suprachiasmatic nucleus grew in size and underwent turnover in morphometry, increasing the total number from the immature-virgin to maturing stage, with similar values in the more advanced maturity stages. These changes were not linked to a reproductive role. These findings provide new valuable information on Chondrichthyes, suggesting the existence of an additional brain system implicated in the integration of internal and environmental cues for reproduction.

Mosquito ageing modulates the development, virulence and transmission potential of pathogens.

Host age variation is a striking source of heterogeneity that can shape the evolution and transmission dynamic of pathogens. Compared with vertebrate systems, our understanding of the impact of host age on invertebrate-pathogen interactions remains limited. We examined the influence of mosquito age on key life-history traits driving human malaria transmission. Females of Anopheles coluzzii, a major malaria vector, belonging to three age classes (4-, 8- and 12-day-old), were experimentally infected with Plasmodium falciparum field isolates. Our findings revealed reduced competence in 12-day-old mosquitoes, characterized by lower oocyst/sporozoite rates and intensities compared with younger mosquitoes. Despite shorter median longevities in older age classes, infected 12-day-old mosquitoes exhibited improved survival, suggesting that the infection might act as a fountain of youth for older mosquitoes specifically. The timing of sporozoite appearance in the salivary glands remained consistent across mosquito age classes, with an extrinsic incubation period of approximately 13 days. Integrating these results into an epidemiological model revealed a lower vectorial capacity for older mosquitoes compared with younger ones, albeit still substantial owing to extended longevity in the presence of infection. Considering age heterogeneity provides valuable insights for ecological and epidemiological studies, informing targeted control strategies to mitigate pathogen transmission.

The motor apparatus of head movements in the Oleander hawkmoth (Daphnis nerii, Lepidoptera).

Head movements of insects play a vital role in diverse locomotory behaviors including flying and walking. Because insect eyes move minimally within their sockets, their head movements are essential to reduce visual blur and maintain a stable gaze. As in most vertebrates, gaze stabilization behavior in insects requires the integration of both visual and mechanosensory feedback by the neck motor neurons. Although visual feedback is derived from the optic flow over the retina of their compound eyes, mechanosensory feedback is derived from their organs of balance, similar to the vestibular system in vertebrates. In Diptera, vestibular feedback is derived from the halteres-modified hindwings that evolved into mechanosensory organs-and is integrated with visual feedback to actuate compensatory head movements. However, non-Dipteran insects, including Lepidoptera, lack halteres. In these insects, vestibular feedback is obtained from the antennal Johnston's organs but it is not well-understood how it integrates with visual feedback during head movements. Indeed, although head movements are well-studied in flies, the underlying motor apparatus in non-Dipteran taxa has received relatively less attention. As a first step toward understanding compensatory head movements in the Oleander hawkmoth Daphnis nerii, we image the anatomy and architecture of their neck joint sclerites and muscles using X-ray microtomography, and the associated motor neurons using fluorescent dye fills and confocal microscopy. Based on these morphological data, we propose testable hypotheses about the putative function of specific neck muscles during head movements, which can shed light on their role in neck movements and gaze stabilization.

Physical presence of chemical synapses is necessary for turning behavior of anterograde synaptic vesicles at the branch point of PLM neurons in C. elegans.

Neurons exhibit complex branched axonal morphologies in both vertebrate and invertebrate systems, and show heterogeneity in the distribution of synaptic cargo across multiple synapses. It is possible that differences in transport across multiple branches contribute to the heterogeneity in cargo distribution across multiple synapses. However, the regulation of transport at axonal branch points is not well understood. We demonstrate that branch-specific transport of synaptic vesicles is dependent on the existence of a connection between the branch and synapses. The loss of this connection causes an immediate decrease in branch-specific transport of synaptic vesicles in the PLM neuron of C. elegans .

Vertebrate-conserved Schnurri zinc fingers restrain Drosophila vein patterning.

The Drosophila Smad-interacting co-factor, Schnurri (Shn) confers transcriptional repression in response to Decapentaplegic (Dpp) signaling. Shn zinc fingers 6-8 mediate this Smad interaction but are lacking in vertebrate Shn homologs. In contrast, the vertebrate-conserved zinc finger 1,2 and 4,5 pairs have been reported to engage in Smad-mediated transcriptional activation in fly and vertebrate systems, and to contribute to Dpp-dependent tissue repair in the fly retina. We report that mutation of zinc coordination residues within vertebrate-conserved Shn zinc finger pairs 1,2 and 4,5 results in ectopic venation that is sensitive to Dpp signaling.

The oral sensory organs in Bathochordaeus stygius (Tunicata Appendicularia) are unique in structure and homologous to the coronal organ

BackgroundAppendicularia consists of approximately 70 purely marine species that belong to Tunicata the probable sister taxon to Craniota. Therefore, Appendicularia plays a pivotal role for our understanding of chordate evolution. In addition, appendicularians are an important part of the epipelagic marine plankton. Nevertheless, little is known about appendicularian species, especially from deeper water.ResultsUsing µCT, scanning electron microscopy, and digital 3D-reconstruction techniques we describe three pairs of complex oral sensory organs in the mesopelagic appendicularian Bathochordaeus stygius. The oral sensory organs are situated at the anterior and lateral margin of the mouth and inside the mouth cavity. A single organ consists of 22–90 secondary receptor cells that project apical cilia through a narrow hole in the epidermis. The receptor cells are innervated by branches of the second brain nerve.ConclusionsBased on position, morphology, and innervation we suggest that the oral sensory organs are homologues of the coronal organs in other tunicates. We discuss the hypothesized homology of coronal organs and the lateral line system of primary aquatic vertebrates. The complex oral sensory organs of B. stygius are unique in tunicates and could be adaptations to the more muffled environment of the mesopelagic.

The ontogeny of lymphoid organs and IgM+ B-cells in ballan wrasse (Labrus bergylta) reveals a potential site for extrarenal B-cell lymphopoiesis: The pancreas

Vaccination of farmed fish is the most effective prophylactic measure against contagious diseases but requires specific knowledge on when the adaptive immune system is fully developed. The present work describes kidney and spleen morphogenesis as well as B-cell development in the ballan wrasse (Labrus bergylta). The kidney was present at hatching (0 days pot hatching, dph) but was not lymphoid before larvae was 50–60 dph (stage 5), containing abundant Igμ+ cells. The spleen anlage was first observed in larvae at 20–30 dph and was later populated with B-cells. Unexpectedly, we found strong RAG1 signal together with abundant Igμ+ and IgM + cells in the exocrine pancreas of larvae from when the kidney was lymphoid and onwards, suggesting that B-cell lymphopoiesis occurs not only in the head kidney (HK) but also in pancreatic tissue. In this agastric fish, the pancreas is diffused along the intestine and the early presence of IgM+ B-cells in pancreatic tissue might have a role in maintain immune homeostasis in the peritoneal cavity, making a substantial contribution to early protection. IgM-secreting cells in HK indicate the presence of systemic IgM at stage 5, before the first IgM+ cells were identified in mucosal sites. This work together with our previous study on T-cell development in this species indicates that although T- and B-cells start to develop around the same time, B-cells migrate to mucosal tissues ahead of T-cells. This early migration likely involves the production of natural antibodies, contributing significantly to early protection. Moreover, a diet composed of barnacle nauplii did not result in an earlier onset of B-cell lymphopoiesis, as seen in the previous study analysing T-cell development. Nevertheless, components for adaptive immunity indicating putative immunocompetence is likely achieved in early juveniles (>100 dph). Additionally, maternal transfer of IgM to the offspring is also described. These findings provide important insights into the development of the immune system in ballan wrasse and lay the foundation for optimizing prophylactic strategies in the future. Furthermore, this work adds valuable information to broaden the knowledge on the immune system in lower vertebrates.

The MA Helix Is Important for Receptor Assembly and Function in the α4β2 nACh Receptor.

Pentameric ligand-gated ion channels (pLGICs) are expressed throughout the central and peripheral nervous systems of vertebrates and modulate many aspects of human health and disease. Recent structural and computational data indicate that cation-selective pLGICs contain a long helical extension (MA) of one of the transmembrane helices. The MA helix has been shown to affect both the membrane expression of, and ion conductance levels through, these pLGICs. Here we probe the functional effects of 68 mutations in the MA region of the α4β2 nicotinic acetylcholine receptor (nAChR), using a voltage-sensitive membrane dye and radioligand binding to measure receptor function and expression/assembly. We found seven alanine mutations in a stretch of the MA helix that prevent correct receptor folding and/or assembly, as evidenced by the lack of both function and ligand binding. A further two alanine mutations resulted in receptors that were capable of binding ligand but showed no functional response, and we propose that, in these mutants, ligand binding is insufficient to trigger channel opening. The data clarify the effect of the MA helix, and as the effects of some of our mutations in the α4β2 nAChR differ from the effects of equivalent mutations in other cation-selective pLGICs, we suggest that residues in the MA helix may play subtly different roles in different receptors.

The antiviral effects of TRIM23 and TRIM32 proteins in rainbow trout (Oncorhynchus mykiss)

TRIM proteins play a crucial antiviral effector role in the innate immune system of vertebrates. In this study, we found that TRIM proteins exhibited the highest expression levels in immune organs such as spleen and kidney during IHNV infection in rainbow trout, meanwhile, we successfully amplified TRIM23 and TRIM32 from diseased rainbow trout and analyzed their gene sequences, revealing that rainbow trout TRIM23 and TRIM32 proteins are closely related to Atlantic salmon and Chinook salmon; In this experiment, the TRIM23 and TRIM32 protein genes were resoundingly constructed as a recombinant plasmids and expressed in CHSE-214 cells. Upon transfected with the recombinant plasmid, followed by viral infection, significant decreasion in the copy numbers of the virus was observed, indicating that the TRIM23 and TRIM32 proteins of rainbow trout play an important role in inhibiting virus replication, with the TRIM32 role being the most pronounced. These results provide a basis for subsequent in-depth study of the antiviral effects of TRIM proteins, and provide new ideas for immune enhancers.

Myosin XV is a negative regulator of signaling filopodia during long-range lateral inhibition

The self-organization of cells during development is essential for the formation of healthy tissues and requires the coordination of cell activities at local scales. Cytonemes, or signaling filopodia, are dynamic actin-based cellular protrusions that allow cells to engage in contact mediated signaling at a distance. While signaling filopodia have been shown to support several signaling paradigms during development, less is understood about how these protrusions are regulated. We investigated the role of the plus-end directed, unconventional MyTH4-FERM myosins in regulating signaling filopodia during sensory bristle patterning on the dorsal thorax of the fruit fly Drosophila melanogaster. We found that Myosin XV is required for regulating signaling filopodia dynamics and, as a consequence, lateral inhibition more broadly throughout the patterning epithelium. We found that Myosin XV is required for limiting the length and number of signaling filopodia generated by bristle precursor cells. Cells with additional and longer signaling filopodia due to loss of Myosin XV are not signaling competent, due to altered levels of Delta ligand and Notch receptor along their lengths. We conclude that Myosin XV acts to negatively regulate signaling filopodia, as well as promote the ability of signaling filopodia to engage in long-range Notch signaling. Since Myosin XV isoforms are present across several vertebrate and invertebrate systems, this may have significance for other long-range signaling mechanisms.

Radial polarity in the first cranial neuromast of selected teleost fishes.

The neuromast is a sensory structure of the lateral line system in aquatic vertebrates, which consists of hair cells and supporting cells. Hair cells are mechanosensory cells, generally arranged with bidirectional polarity. Here, we describe a neuromast with hair cells arranged radially instead of bidirectionally in the first cranial neuromast of four teleost species: red seabream (Pagrus major), spotted halibut (Verasper variegatus), brown sole (Pseudopleuronectes herzensteini), and marbled sole (Pseudopleuronectes yokohamae). In these four species, this polarity was identified only in the first cranial neuromast, where it appeared at the rostral edge of the otic vesicle before hatching. We investigated the initial appearance and fate of this unique neuromast using scanning electron microscopy. We also assessed characteristics of radial neuromast pertaining to morphogenesis, development, and innervation using a vital fluorescent marker and immunohistochemistry in V. variegatus. The kinocilium initially appears at the center of each hair cell, then moves to its outer perimeter to form radial polarity by around 7 days postfertilization. However, hair cells arranged radially disappear about 15 days after hatching. This is followed by the appearance of bidirectionally arranged hair cells, indicating that polarity replacement from radial to bidirectional has occurred. In P. herzensteini, both afferent and efferent synapses between the nerve fibers and hair cells were observed by transmission electron microscopy, suggesting that radial neuromast is functional. Our discovery suggests that neuromasts with radial polarity could enable larval fish to assimilate multiaxial stimuli during this life stage, potentially assisting them in detecting small water vibrations or water pressure changes.

Development of a New Enzyme-Linked Immunosorbent Assay (ELISA) for Measuring the Content of PACAP in Mammalian Tissue and Plasma.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a naturally occurring neuropeptide found in both the central and peripheral nervous systems of vertebrates. Recent studies have revealed the presence of PACAP and its corresponding receptors, namely, the pituitary adenylate cyclase-activating polypeptide type I receptor (PAC1R), vasoactive intestinal peptide receptor 1 (VIPR1), and vasoactive intestinal peptide receptor 2 (VIPR2), in various structures implicated in migraine pathophysiology, including sensory trigeminal neurons. Human studies have demonstrated that when infused, PACAP can cause dilation of cranial vessels and result in delayed migraine-like attacks. In light of this, we present a novel ELISA assay that has been validated for quantifying PACAP in tissue extracts and human plasma. Using two well characterized antibodies specifically targeting PACAP, we successfully developed a sandwich ELISA assay, capable of detecting and accurately quantifying PACAP without any cross-reactivity to closely related peptides. The quantification range was between 5.2 pmol/L and 400 pmol/L. The recovery in plasma ranged from 98.2% to 100%. The increasing evidence pointing to the crucial role of PACAP in migraine pathophysiology necessitates the availability of tools capable of detecting changes in the circulatory levels of PACAP and its potential application as a reliable biomarker.