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Recessive gene mutations underlie many developmental disorders and often lead to disabling neurological problems. Here, we report identification of a homozygous c.170G>A (p.Cys57Tyr or C57Y) mutation in the gene coding for protein disulfide isomerase A3 (PDIA3, also known as ERp57), an enzyme that catalyzes formation of disulfide bonds in the endoplasmic reticulum, to be associated with syndromic intellectual disability. Experiments in zebrafish embryos show that PDIA3C57Y expression is pathogenic and causes developmental defects such as axonal disorganization as well as skeletal abnormalities. Expression of PDIA3C57Y in the mouse hippocampus results in impaired synaptic plasticity and memory consolidation. Proteomic and functional analyses reveal that PDIA3C57Y expression leads to dysregulation of cell adhesion and actin cytoskeleton dynamics, associated with altered integrin biogenesis and reduced neuritogenesis. Biochemical studies show that PDIA3C57Y has decreased catalytic activity and forms disulfide-crosslinked aggregates that abnormally interact with chaperones in the endoplasmic reticulum. Thus, rare disease gene variant can provide insight into how perturbations of neuronal proteostasis can affect the function of the nervous system.
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Discapacidades del Desarrollo/genética , Retículo Endoplásmico/metabolismo , Proteína Disulfuro Isomerasas/genética , Proteostasis , Adolescente , Adulto , Animales , Axones/metabolismo , Axones/patología , Adhesión Celular , Células Cultivadas , Niño , Citoesqueleto/metabolismo , Discapacidades del Desarrollo/metabolismo , Discapacidades del Desarrollo/patología , Femenino , Hipocampo/metabolismo , Hipocampo/patología , Humanos , Integrinas/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Mutación Missense , Proyección Neuronal , Plasticidad Neuronal , Linaje , Proteína Disulfuro Isomerasas/metabolismo , Pez CebraRESUMEN
Nothobranchius furzeri is emerging as an exciting vertebrate organism in the field of biomedicine, developmental biology and ecotoxicology research. Its short generation time, compressed lifespan and accelerated ageing make it a versatile model for longitudinal studies with high traceability. Although in recent years the use of this model has increased enormously, there is still little information on the anatomy, morphology and histology of its main organs. In this paper, we present a description of the digestive system of N. furzeri, with emphasis on the intestine. We note that the general architecture of the intestinal tissue is shared with other vertebrates, and includes a folding mucosa, an outer muscle layer and a myenteric plexus. By immunohistochemical analysis, we reveal that the mucosa harbours the same type of epithelial cells observed in mammals, including enterocytes, goblet cells and enteroendocrine cells, and that the myenteric neurons express neurotransmitters common to other species, such as serotonin, substance P and tyrosine hydroxylase. In addition, we detect the presence of a proliferative compartment at the base of the intestinal folds. The description of the normal intestinal morphology provided here constitutes a baseline information to contrast with tissue alterations in future lines of research assessing pathologies, ageing-related diseases or damage caused by toxic agents.
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Envejecimiento , Intestinos , Animales , MamíferosRESUMEN
MOTIVATION: Quantitative descriptions of multi-cellular structures from optical microscopy imaging are prime to understand the variety of three-dimensional (3D) shapes in living organisms. Experimental models of vertebrates, invertebrates and plants, such as zebrafish, killifish, Drosophila or Marchantia, mainly comprise multilayer tissues, and even if microscopes can reach the needed depth, their geometry hinders the selection and subsequent analysis of the optical volumes of interest. Computational tools to "peel" tissues by removing specific layers and reducing 3D volume into planar images, can critically improve visualization and analysis. RESULTS: We developed VolumePeeler, a versatile FIJI plugin for virtual 3D "peeling" of image stacks. The plugin implements spherical and spline surface projections. We applied VolumePeeler to perform peeling in 3D images of spherical embryos, as well as non-spherical tissue layers. The produced images improve the 3D volume visualization and enable analysis and quantification of geometrically challenging microscopy datasets. AVAILABILITY: ImageJ/FIJI software, source code, examples, and tutorials are openly available in https://cimt.uchile.cl/mcerda.
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Drosophila , Pez Cebra , Animales , Microscopía , Programas InformáticosRESUMEN
Acute myeloid leukemia (AML) has a poor prognosis under the current standard of care. In recent years, venetoclax, a BCL-2 inhibitor, was approved to treat patients who are ineligible for intensive induction chemotherapy. However, complete remission rates with venetoclax-based therapies are hampered by minimal residual disease (MRD) in a proportion of patients, leading to relapse. MRD is a result of leukemic stem cells being retained in bone marrow protective environments; activation of the CXCL12-CXCR4 pathway was shown to be relevant to this process. An important role is also played by cell adhesion molecules such as CD44, which has been shown to be crucial for the development of AML. Here we show that CD44 is involved in CXCL12 promotion of resistance to venetoclax-induced apoptosis in human AML cell lines and AML patient samples, which could be abrogated by CD44 knock down, knockout, or blocking with an anti-CD44 antibody. Split-Venus bimolecular fluorescence complementation showed that CD44 and CXCR4 physically associate at the cell membrane upon CXCL12 induction. In the venetoclax-resistant OCI-AML3 cell line, CXCL12 promoted an increase in the proportion of cells expressing high levels of embryonic stem cell core transcription factors (ESC-TFs: Sox2, Oct4, Nanog) abrogated by CD44 knockdown. This ESC-TF-expressing subpopulation which could be selected by venetoclax treatment, exhibited a basally enhanced resistance to apoptosis and expressed higher levels of CD44. Finally, we developed a novel AML xenograft model in zebrafish, which showed that CD44 knockout sensitizes OCI-AML3 cells to venetoclax treatment in vivo. Our study shows that CD44 is a potential molecular target for sensitizing AML cells to venetoclax-based therapies.
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Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Quimiocina CXCL12 , Receptores de Hialuranos , Leucemia Mieloide Aguda , Mutación con Pérdida de Función , Proteínas Proto-Oncogénicas c-bcl-2 , Sulfonamidas/farmacología , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Quimiocina CXCL12/genética , Quimiocina CXCL12/metabolismo , Femenino , Humanos , Receptores de Hialuranos/genética , Receptores de Hialuranos/metabolismo , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Masculino , Proteínas Proto-Oncogénicas c-bcl-2/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Células Tumorales CultivadasRESUMEN
Transient receptor potential melastatin 4 (TRPM4) is a Ca2+ -activated nonselective cationic channel that regulates cell migration and contractility. Increased TRPM4 expression has been related to pathologies, in which cytoskeletal rearrangement and cell migration are altered, such as metastatic cancer. Here, we identify the K+ channel tetramerization domain 5 (KCTD5) protein, a putative adaptor of cullin3 E3 ubiquitin ligase, as a novel TRPM4-interacting protein. We demonstrate that KCTD5 is a positive regulator of TRPM4 activity by enhancing its Ca2+ sensitivity. We show that through its effects on TRPM4 that KCTD5 promotes cell migration and contractility. Finally, we observed that both TRPM4 and KCTD5 expression are increased in distinct patterns in different classes of breast cancer tumor samples. Together, these data support that TRPM4 activity can be regulated through expression levels of either TRPM4 or KCTD5, not only contributing to increased understanding of the molecular mechanisms involved on the regulation of these important ion channels, but also providing information that could inform treatments based on targeting these distinct molecules that define TRPM4 activity.
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Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Movimiento Celular/fisiología , Canales de Potasio/metabolismo , Canales Catiónicos TRPM/metabolismo , Animales , Mama/metabolismo , Mama/patología , Células COS , Línea Celular , Línea Celular Tumoral , Chlorocebus aethiops , Femenino , Células HEK293 , Humanos , Células MCF-7 , Pronóstico , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Disturbance of endoplasmic reticulum (ER) proteostasis is a common feature of amyotrophic lateral sclerosis (ALS). Protein disulfide isomerases (PDIs) areERfoldases identified as possibleALSbiomarkers, as well as neuroprotective factors. However, no functional studies have addressed their impact on the disease process. Here, we functionally characterized fourALS-linked mutations recently identified in two majorPDIgenes,PDIA1 andPDIA3/ERp57. Phenotypic screening in zebrafish revealed that the expression of thesePDIvariants induce motor defects associated with a disruption of motoneuron connectivity. Similarly, the expression of mutantPDIs impaired dendritic outgrowth in motoneuron cell culture models. Cellular and biochemical studies identified distinct molecular defects underlying the pathogenicity of thesePDImutants. Finally, targetingERp57 in the nervous system led to severe motor dysfunction in mice associated with a loss of neuromuscular synapses. This study identifiesERproteostasis imbalance as a risk factor forALS, driving initial stages of the disease.
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Esclerosis Amiotrófica Lateral/genética , Neuronas Motoras/patología , Procolágeno-Prolina Dioxigenasa/genética , Proteína Disulfuro Isomerasas/genética , Esclerosis Amiotrófica Lateral/patología , Animales , Animales Modificados Genéticamente , Electromiografía , Embrión no Mamífero , Estrés del Retículo Endoplásmico/genética , Humanos , Ratones Noqueados , Mutación , Neuritas/patología , Procolágeno-Prolina Dioxigenasa/metabolismo , Proteína Disulfuro Isomerasas/metabolismo , Pez Cebra/embriología , Pez Cebra/genéticaRESUMEN
BACKGROUND: Comparative studies beyond the traditional model organisms have been instrumental in enhancing our understanding of the conserved and derived features of gastrulation, a fundamental process in which the germ layers are specified and shaped to form the body axis. Here, we analyzed gastrulation in a vertebrate group with an extreme mode of early development, the annual killifish. RESULTS: Gastrulation in annual killifish of the genus Austrolebias takes place after the initially dispersed deep blastomeres congregate to form the so-called reaggregate. Cells from the early reaggregate do not appear to form part of any recognizable axial embryonic structure and are possibly extraembryonic. In contrast, later reaggregate cells become engaged in morphogenetic transformations indicative of a process of gastrulation and axis formation. The expression of brachyury and goosecoid suggests that gastrulation takes place in a compressed blastopore-like structure with an organizer region displaced to one end. No collective cell internalization proper of blastopore architecture is observed, though, and it appears that gastrulation primarily involves the reorganization of individual cells. CONCLUSIONS: The unique mode of gastrulation in annual killifish demonstrates that a process so ancient and fundamental to ontogenesis can have striking morphogenetic variations nonpredicted from the sole examination of model species. Developmental Dynamics 246:812-826, 2017. © 2017 Wiley Periodicals, Inc.
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Fundulidae/embriología , Gastrulación , Estratos Germinativos/citología , Animales , Embrión no Mamífero , Proteínas Fetales , Fundulidae/fisiología , Estratos Germinativos/crecimiento & desarrollo , Estratos Germinativos/metabolismo , Proteína Goosecoide , Proteínas de Dominio T BoxRESUMEN
Cell migration is a fundamental process that occurs during embryo development. Classic studies using in vitro culture systems have been instrumental in dissecting the principles of cell motility and highlighting how cells make use of topographical features of the substrate, cell-cell contacts, and chemical and physical environmental signals to direct their locomotion. Here, we review the guidance principles of in vitro cell locomotion and examine how they control directed cell migration in vivo during development. We focus on developmental examples in which individual guidance mechanisms have been clearly dissected, and for which the interactions among guidance cues have been explored. We also discuss how the migratory behaviours elicited by guidance mechanisms generate the stereotypical patterns of migration that shape tissues in the developing embryo.
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Movimiento Celular/fisiología , Desarrollo Embrionario , Animales , Comunicación Celular/fisiología , Células Cultivadas , Técnicas de Cultivo de Embriones , Matriz Extracelular/fisiología , Humanos , Técnicas de Cultivo de TejidosRESUMEN
Heterochrony is one proposed mechanism to explain how morphological variation and novelty arise during evolution. To experimentally approach heterochrony in a comprehensive manner, we must consider all three aspects of developmental time (sequence, timing, duration). This task is only possible in developmental models that allow the acquisition of high-quality temporal data in the context of normalized developmental time. Here we propose that epithalamic asymmetry of teleosts is one such model. Comparative studies among related teleost species have revealed heterochronic shifts in the timing of ontogenic events leading to the development of epithalamic asymmetry. Such temporal changes involve neural structures critical for tissue-tissue interactions underlying the generation of asymmetry and are concurrent with the appearance of morphological differences in the pattern of asymmetry between species. Based on these findings, we hypothesize that interspecies variation of epithalamic asymmetry results from changes in the timing of tissue-tissue interactions critical for the establishment of asymmetry during ontogeny. Importantly, this hypothesis can be tested by systematic comparative approaches among teleosts species based on normalized developmental time, combined with experimental manipulation of epithalamic asymmetry development.
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Evolución Biológica , Regulación del Desarrollo de la Expresión Génica , Animales , Tipificación del Cuerpo , Desarrollo Embrionario/genética , Desarrollo Embrionario/fisiologíaRESUMEN
Genetic and environmental factors control morphological and functional differences between the two sides of the nervous system. Neural asymmetries are proposed to have important roles in circuit physiology, cognition and species-specific behaviours. We propose two fundamentally different mechanisms for encoding left-right asymmetry in neural circuits. In the first, asymmetric circuits share common components; in the second, there are unique unilateral structures. Research in both vertebrates and invertebrates is helping to reveal the mechanisms underlying the development of neural lateralization, but less is known about the function of circuit asymmetries. Technical advances in the coming years are likely to revolutionize our understanding of left-right asymmetry in the nervous system.
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Cognición/fisiología , Lateralidad Funcional/fisiología , Red Nerviosa/fisiología , Sistema Nervioso/anatomía & histología , Animales , Humanos , Red Nerviosa/citología , Especificidad de la EspecieRESUMEN
Although progress has been made in resolving the genetic pathways that specify neuronal asymmetries in the brain, little is known about genes that mediate the development of structural asymmetries between neurons on left and right. In this study, we identify daam1a as an asymmetric component of the signalling pathways leading to asymmetric morphogenesis of the habenulae in zebrafish. Daam1a is a member of the Formin family of actin-binding proteins and the extent of Daam1a expression in habenular neuron dendrites mirrors the asymmetric growth of habenular neuropil between left and right. Local loss and gain of Daam1a function affects neither cell number nor subtype organisation but leads to a decrease or increase of neuropil, respectively. Daam1a therefore plays a key role in the asymmetric growth of habenular neuropil downstream of the pathways that specify asymmetric cellular domains in the habenulae. In addition, Daam1a mediates the development of habenular efferent connectivity as local loss and gain of Daam1a function impairs or enhances, respectively, the growth of habenular neuron terminals in the interpeduncular nucleus. Abrogation of Daam1a disrupts the growth of both dendritic and axonal processes and results in disorganised filamentous actin and α-tubulin. Our results indicate that Daam1a plays a key role in asymmetric habenular morphogenesis mediating the growth of dendritic and axonal processes in dorsal habenular neurons.
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Axones/metabolismo , Dendritas/metabolismo , Habénula/embriología , Habénula/metabolismo , Proteínas de Pez Cebra/metabolismo , Animales , Tipificación del Cuerpo/genética , Tipificación del Cuerpo/fisiología , Pez Cebra , Proteínas de Pez Cebra/genéticaRESUMEN
Organ formation requires the precise assembly of progenitor cells into a functional multicellular structure. Mechanical forces probably participate in this process but how they influence organ morphogenesis is still unclear. Here, we show that Wnt11- and Prickle1a-mediated planar cell polarity (PCP) signalling coordinates the formation of the zebrafish ciliated laterality organ (Kupffer's vesicle) by regulating adhesion properties between organ progenitor cells (the dorsal forerunner cells, DFCs). Combined inhibition of Wnt11 and Prickle1a reduces DFC cell-cell adhesion and impairs their compaction and arrangement during vesicle lumen formation. This leads to the formation of a mis-shapen vesicle with small fragmented lumina and shortened cilia, resulting in severely impaired organ function and, as a consequence, randomised laterality of both molecular and visceral asymmetries. Our results reveal a novel role for PCP-dependent cell adhesion in coordinating the supracellular organisation of progenitor cells during vertebrate laterality organ formation.
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Proteínas Portadoras/metabolismo , Adhesión Celular/fisiología , Polaridad Celular/fisiología , Embrión no Mamífero/embriología , Morfogénesis/fisiología , Transducción de Señal/fisiología , Proteínas Wnt/metabolismo , Proteínas de Pez Cebra/metabolismo , Pez Cebra/embriología , Proteínas Adaptadoras Transductoras de Señales , Animales , Epitelio/fisiología , Procesamiento de Imagen Asistido por Computador , Inmunohistoquímica , Hibridación in Situ , Proteínas con Dominio LIMRESUMEN
Comparative studies of the tetrapod raldh2 (aldh1a2) gene, which encodes a retinoic acid (RA) synthesis enzyme, have led to the identification of a dorsal spinal cord enhancer. Enhancer activity is directed dorsally to the roof plate and dorsal-most (dI1) interneurons through predicted Tcf- and Cdx-homeodomain binding sites and is repressed ventrally via predicted Tgif homeobox and ventral Lim-homeodomain binding sites. Raldh2 and Math1/Cath1 expression in mouse and chicken highlights a novel, transient, endogenous Raldh2 expression domain in dI1 interneurons, which give rise to ascending circuits and intraspinal commissural interneurons, suggesting roles for RA in the ontogeny of spinocerebellar and intraspinal proprioceptive circuits. Consistent with expression of raldh2 in the dorsal interneurons of tetrapods, we also found that raldh2 is expressed in dorsal interneurons throughout the agnathan spinal cord, suggesting ancestral roles for RA signaling in the ontogenesis of intraspinal proprioception.
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Aldehído Oxidorreductasas/fisiología , Médula Espinal/fisiología , Animales , Sitios de Unión , Pollos , Secuencia Conservada , Evolución Molecular , Factor Nuclear 1-alfa del Hepatocito , Proteínas de Homeodominio , Interneuronas , Proteínas con Homeodominio LIM , Ratones , Ratones Transgénicos , Proteínas Represoras , Factor 1 de Transcripción de Linfocitos T , Factores de Transcripción , Tretinoina/fisiologíaRESUMEN
Introduction: Deciphering the biological and physical requirements for the outset of multicellularity is limited to few experimental models. The early embryonic development of annual killifish represents an almost unique opportunity to investigate de novo cellular aggregation in a vertebrate model. As an adaptation to seasonal drought, annual killifish employs a unique developmental pattern in which embryogenesis occurs only after undifferentiated embryonic cells have completed epiboly and dispersed in low density on the egg surface. Therefore, the first stage of embryogenesis requires the congregation of embryonic cells at one pole of the egg to form a single aggregate that later gives rise to the embryo proper. This unique process presents an opportunity to dissect the self-organizing principles involved in early organization of embryonic stem cells. Indeed, the physical and biological processes required to form the aggregate of embryonic cells are currently unknown. Methods: Here, we developed an in silico, agent-based biophysical model that allows testing how cell-specific and environmental properties could determine the aggregation dynamics of early Killifish embryogenesis. In a forward engineering approach, we then proceeded to test two hypotheses for cell aggregation (cell-autonomous and a simple taxis model) as a proof of concept of modeling feasibility. In a first approach (cell autonomous system), we considered how intrinsic biophysical properties of the cells such as motility, polarity, density, and the interplay between cell adhesion and contact inhibition of locomotion drive cell aggregation into self-organized clusters. Second, we included guidance of cell migration through a simple taxis mechanism to resemble the activity of an organizing center found in several developmental models. Results: Our numerical simulations showed that random migration combined with low cell-cell adhesion is sufficient to maintain cells in dispersion and that aggregation can indeed arise spontaneously under a limited set of conditions, but, without environmental guidance, the dynamics and resulting structures do not recapitulate in vivo observations. Discussion: Thus, an environmental guidance cue seems to be required for correct execution of early aggregation in early killifish development. However, the nature of this cue (e.g., chemical or mechanical) can only be determined experimentally. Our model provides a predictive tool that could be used to better characterize the process and, importantly, to design informed experimental strategies.
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Teleost eggs have evolved a highly derived early developmental pattern within vertebrates as a result of the meroblastic cleavage pattern, giving rise to a polar stratified architecture containing a large acellular yolk and a small cellular blastoderm on top. Besides the acellular yolk, the teleost-specific yolk syncytial layer (YSL) and the superficial epithelial enveloping layer are recognized as extraembryonic structures that play critical roles throughout embryonic development. They provide enriched microenvironments in which molecular feedback loops, cellular interactions and mechanical signals emerge to sculpt, among other things, embryonic patterning along the dorsoventral and left-right axes, mesendodermal specification and the execution of morphogenetic movements in the early embryo and during organogenesis. An emerging concept points to a critical role of extraembryonic structures in reinforcing early genetic and morphogenetic programmes in reciprocal coordination with the embryonic blastoderm, providing the necessary boundary conditions for development to proceed. In addition, the role of the enveloping cell layer in providing mechanical, osmotic and immunological protection during early stages of development, and the autonomous nutritional support provided by the yolk and YSL, have probably been key aspects that have enabled the massive radiation of teleosts to colonize every ecological niche on the Earth. This article is part of the theme issue 'Extraembryonic tissues: exploring concepts, definitions and functions across the animal kingdom'.
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Peces , Pez Cebra , Animales , Embrión no Mamífero , Desarrollo Embrionario , MorfogénesisRESUMEN
Toxins of the OA-group (okadaic acid, OA; dinophysistoxin-1, DTX-1) are the most prevalent in the fjords of southern Chile, and are characterized by their potential harmful effects on aquatic organisms. The present study was carried out to determine the acute toxicity of OA/DTX-1 on oxidative stress parameters in medaka (Oryzias latipes) larvae. Medaka larvae were exposed to different concentrations (1.0-30 µg/mL) of OA/DTX-1 for 96 h to determine the median lethal concentration. The LC50 value after 96 h was 23.5 µg/mL for OA and 16.3 µg/mL for DTX-1 (95% confidence interval, CI was 22.56, 24.43 for OA and 15.42, 17.17 for DTX-1). Subsequently, larvae at 121 hpf were exposed to acute doses (10, 15 and 20 µg/mL OA and 5.0, 7.5 and 11.0 µg/mL DTX-1) for 96 h and every 6 h the corresponding group of larvae was euthanized in order to measure the activity levels of biochemical biomarkers (superoxide dismutase, SOD; catalase, CAT; glutathione peroxidase, GPx; and glutathione reductase, GR) as well as the levels of oxidative damage (malondialdehyde, MDA; and carbonyl content). Our results showed that acute doses caused a decrease in SOD (≈25%), CAT (≈55%), and GPx and GR (≈35%) activities, while MDA levels and carbonyl content increased significantly at the same OA/DTX-1 concentrations. This study shows that acute exposure to OA-group toxins tends to simultaneously alter the oxidative parameters that induce sustained morphological damage in medaka larvae. DTX-1 stands out as producing greater inhibition of the antioxidant system, leading to increased oxidative damage in medaka larvae. Considering that DTX-1 is the most prevalent HAB toxin in southern Chile, these findings raise the possibility of an important environmental impact on the larval stages of different fish species present in the southern fjords of the South Pacific.
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Several models have been proposed to describe the dynamics of epithelial tissues undergoing morphogenetic changes driven by apical constriction pulses, which differ in where the constriction is applied, either at the perimeter or in the medial regions. To help discriminate between these models, we analyse the impact of where constriction is applied on the final geometry of the active contracted cell, using the two-dimensional vertex model. We find that medial activity, characterized by a reduction in the reference area, generates anisotropic cell shapes, whereas isotropic cell shapes are produced when the reference perimeter is reduced. When plasticity is included, sufficiently slow processes of medial contractile activity, compared with the characteristic times of elasticity and plasticity, cells can achieve less elongated shapes. Similarly, for perimeter activity, the highest level of contraction is achieved. Finally, we apply the model to describe the apical contractile pulses observed within the epithelial enveloping cell layer during the pre-epiboly of the annual killifish Austrolebias nigripinnis. The analysis of the cell shape changes allowed a global fit of all parameters of the vertex model, with the pulses being quantitatively captured using perimeter activity and area plasticity.
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Células Epiteliales , Forma de la Célula , Epitelio , MorfogénesisRESUMEN
The parapineal organ is a midline-derived epithalamic structure that in zebrafish adopts a left-sided position at embryonic stages to promote the development of left-right asymmetries in the habenular nuclei. Despite extensive knowledge about its embryonic and larval development, it is still unknown whether the parapineal organ and its profuse larval connectivity with the left habenula are present in the adult brain or whether, as assumed from historical conceptions, this organ degenerates during ontogeny. This paper addresses this question by performing an ontogenetic analysis using an integrative morphological, ultrastructural and neurochemical approach. We find that the parapineal organ is lost as a morphological entity during ontogeny, while parapineal cells are incorporated into the posterior wall of the adult left dorsal habenular nucleus as small clusters or as single cells. Despite this integration, parapineal cells retain their structural, neurochemical and connective features, establishing a reciprocal synaptic connection with the more dorsal habenular neuropil. Furthermore, we describe the ultrastructure of parapineal cells using transmission electron microscopy and report immunoreactivity in parapineal cells with antibodies against substance P, tachykinin, serotonin and the photoreceptor markers arrestin3a and rod opsin. Our findings suggest that parapineal cells form an integral part of a neural circuit associated with the left habenula, possibly acting as local modulators of the circuit. We argue that the incorporation of parapineal cells into the habenula may be part of an evolutionarily relevant developmental mechanism underlying the presence/absence of the parapineal organ in teleosts, and perhaps in a broader sense in vertebrates.
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Habenulae asymmetries are widespread across vertebrates and analyses in zebrafish, the reference model organism for this process, have provided insight into their molecular nature, their mechanisms of formation and their important roles in the integration of environmental and internal cues with a variety of organismal adaptive responses. However, the generality of the characteristics identified in this species remains an open question, even on a relatively short evolutionary scale, in teleosts. To address this question, we have characterized the broad organization of habenulae in the Atlantic salmon and quantified the asymmetries in each of the identified subdomains. Our results show that a highly conserved partitioning into a dorsal and a ventral component is retained in the Atlantic salmon and that asymmetries are mainly observed in the former as in zebrafish. A remarkable difference is that a prominent left-restricted pax6 positive nucleus is observed in the Atlantic salmon, but undetectable in zebrafish. This nucleus is not observed outside teleosts, and harbors a complex presence/absence pattern in this group, retaining its location and cytoarchitectonic organization in an elopomorph, the European eel. These findings suggest an ancient origin and high evolvability of this trait in the taxon. Taken together, our data raise novel questions about the variability of asymmetries across teleosts and their biological significance depending on ecological contexts.
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The epithalamus of zebrafish presents the best-studied case of directional asymmetry in the vertebrate brain. Epithalamic asymmetries are coupled to visceral asymmetry and include left-sided migration of a single midline structure (the parapineal organ) and asymmetric differentiation of paired bilateral nuclei (habenulae). The mechanisms underlying the establishment of epithalamic asymmetry involve the interplay between anti-symmetry and laterality signals to guide asymmetric parapineal migration. This event triggers the amplification of habenular asymmetries and the subsequent organisation of lateralised circuits in the interpeduncular nucleus. This review will summarise our current understanding on these processes and propose a sequential modular organisation of the events controlling the development of asymmetry along the parapineal-habenular-interpeduncular axis.