RESUMEN
Neurons on the left and right sides of the nervous system often show asymmetric properties, but how such differences arise is poorly understood. Genetic screening in zebrafish revealed that loss of function of the transmembrane protein Cachd1 resulted in right-sided habenula neurons adopting left-sided identity. Cachd1 is expressed in neuronal progenitors, functions downstream of asymmetric environmental signals, and influences timing of the normally asymmetric patterns of neurogenesis. Biochemical and structural analyses demonstrated that Cachd1 can bind simultaneously to Lrp6 and Frizzled family Wnt co-receptors. Consistent with this, lrp6 mutant zebrafish lose asymmetry in the habenulae, and epistasis experiments support a role for Cachd1 in modulating Wnt pathway activity in the brain. These studies identify Cachd1 as a conserved Wnt receptor-interacting protein that regulates lateralized neuronal identity in the zebrafish brain.
Asunto(s)
Canales de Calcio , Habénula , Neurogénesis , Neuronas , Vía de Señalización Wnt , Proteínas de Pez Cebra , Pez Cebra , Animales , Receptores Frizzled/metabolismo , Receptores Frizzled/genética , Habénula/metabolismo , Habénula/embriología , Mutación con Pérdida de Función , Proteína-6 Relacionada a Receptor de Lipoproteína de Baja Densidad/metabolismo , Proteína-6 Relacionada a Receptor de Lipoproteína de Baja Densidad/genética , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/genética , Neuronas/metabolismo , Receptores Wnt/metabolismo , Receptores Wnt/genética , Pez Cebra/embriología , Pez Cebra/genética , Proteínas de Pez Cebra/metabolismo , Proteínas de Pez Cebra/genética , Canales de Calcio/genética , Canales de Calcio/metabolismoRESUMEN
Zebrafish telencephalon acquires an everted morphology by a two-step process that occurs from 1 to 5 days post-fertilization (dpf). Little is known about how this process affects the positioning of discrete telencephalic cell populations, hindering our understanding of how eversion impacts telencephalic structural organization. In this study, we characterize the neurochemistry, cycle state and morphology of an EGFP positive (+) cell population in the telencephalon of Et(gata2:EGFP)bi105 transgenic fish during eversion and up to 20dpf. We map the transgene insertion to the early-growth-response-gene-3 (egr3) locus and show that EGFP expression recapitulates endogenous egr3 expression throughout much of the pallial telencephalon. Using the gata2:EGFP bi105 transgene, in combination with other well-characterized transgenes and structural markers, we track the development of various cell populations in the zebrafish telencephalon as it undergoes the morphological changes underlying eversion. These datasets were registered to reference brains to form an atlas of telencephalic development at key stages of the eversion process (1dpf, 2dpf, and 5dpf) and compared to expression in adulthood. Finally, we registered gata2:EGFPbi105 expression to the Zebrafish Brain Browser 6dpf reference brain (ZBB, see Marquart et al., 2015, 2017; Tabor et al., 2019), to allow comparison of this expression pattern with anatomical data already in ZBB.
RESUMEN
Mutations in the SNX14 gene cause spinocerebellar ataxia, autosomal recessive 20 (SCAR20) in both humans and dogs. Studies implicating the phenotypic consequences of SNX14 mutations to be consequences of subcellular disruption to autophagy and lipid metabolism have been limited to in vitro investigation of patient-derived dermal fibroblasts, laboratory engineered cell lines and developmental analysis of zebrafish morphants. SNX14 homologues Snz (Drosophila) and Mdm1 (yeast) have also been conducted, demonstrated an important biochemical role during lipid biogenesis. In this study we report the effect of loss of SNX14 in mice, which resulted in embryonic lethality around mid-gestation due to placental pathology that involves severe disruption to syncytiotrophoblast cell differentiation. In contrast to other vertebrates, zebrafish carrying a homozygous, maternal zygotic snx14 genetic loss-of-function mutation were both viable and anatomically normal. Whilst no obvious behavioural effects were observed, elevated levels of neutral lipids and phospholipids resemble previously reported effects on lipid homeostasis in other species. The biochemical role of SNX14 therefore appears largely conserved through evolution while the consequences of loss of function varies between species. Mouse and zebrafish models therefore provide valuable insights into the functional importance of SNX14 with distinct opportunities for investigating its cellular and metabolic function in vivo.
Asunto(s)
Viabilidad Fetal/genética , Metabolismo de los Lípidos/genética , Placenta/anomalías , Nexinas de Clasificación/genética , Ataxias Espinocerebelosas/genética , Animales , Animales Modificados Genéticamente , Diferenciación Celular/genética , Desarrollo Embrionario/genética , Femenino , Humanos , Ratones , Ratones Endogámicos C57BL , Modelos Animales , Fenotipo , Fosfolípidos/sangre , Embarazo , Trofoblastos/citología , Pez CebraRESUMEN
Efficient and accurate DNA replication is particularly critical in stem and progenitor cells for successful proliferation and survival. The replisome, an amalgam of protein complexes, is responsible for binding potential origins of replication, unwinding the double helix, and then synthesizing complimentary strands of DNA. According to current models, the initial steps of DNA unwinding and opening are facilitated by the CMG complex, which is composed of a GINS heterotetramer that connects Cdc45 with the mini-chromosome maintenance (Mcm) helicase. In this work, we provide evidence that in the absence of GINS function DNA replication is cell autonomously impaired, and we also show that gins1 and gins2 mutants exhibit elevated levels of apoptosis restricted to actively proliferating regions of the central nervous system (CNS). Intriguingly, our results also suggest that the rapid cell cycles during early embryonic development in zebrafish may not require the function of the canonical GINS complex as neither zygotic Gins1 nor Gins2 isoforms seem to be present during these stages.
RESUMEN
Toxoplasma gondii is an obligate intracellular parasite capable of invading any nucleated cell. Three main clonal lineages (type I, II, III) exist and murine models have driven the understanding of general and strain-specific immune mechanisms underlying Toxoplasma infection. However, murine models are limited for studying parasite-leukocyte interactions in vivo, and discrepancies exist between cellular immune responses observed in mouse versus human cells. Here, we developed a zebrafish infection model to study the innate immune response to Toxoplasma in vivo By infecting the zebrafish hindbrain ventricle, and using high-resolution microscopy techniques coupled with computer vision-driven automated image analysis, we reveal that Toxoplasma invades brain cells and replicates inside a parasitophorous vacuole to which type I and III parasites recruit host cell mitochondria. We also show that type II and III strains maintain a higher infectious burden than type I strains. To understand how parasites are cleared in vivo, we further analyzed Toxoplasma-macrophage interactions using time-lapse microscopy and three-dimensional correlative light and electron microscopy (3D CLEM). Time-lapse microscopy revealed that macrophages are recruited to the infection site and play a key role in Toxoplasma control. High-resolution 3D CLEM revealed parasitophorous vacuole breakage in brain cells and macrophages in vivo, suggesting that cell-intrinsic mechanisms may be used to destroy the intracellular niche of tachyzoites. Together, our results demonstrate in vivo control of Toxoplasma by macrophages, and highlight the possibility that zebrafish may be further exploited as a novel model system for discoveries within the field of parasite immunity.This article has an associated First Person interview with the first author of the paper.
Asunto(s)
Macrófagos/parasitología , Rombencéfalo/microbiología , Toxoplasma/crecimiento & desarrollo , Toxoplasmosis Animal/parasitología , Toxoplasmosis Cerebral/parasitología , Pez Cebra/parasitología , Animales , Modelos Animales de Enfermedad , Interacciones Huésped-Parásitos , Macrófagos/inmunología , Macrófagos/ultraestructura , Microscopía Confocal , Microscopía Electrónica de Rastreo , Microscopía Fluorescente , Microscopía por Video , Carga de Parásitos , Rombencéfalo/inmunología , Rombencéfalo/ultraestructura , Factores de Tiempo , Toxoplasma/inmunología , Toxoplasma/ultraestructura , Toxoplasmosis Animal/inmunología , Toxoplasmosis Animal/patología , Toxoplasmosis Cerebral/inmunología , Toxoplasmosis Cerebral/patologíaRESUMEN
The vertebrate CNS is surrounded by the meninges, a protective barrier comprised of the outer dura mater and the inner leptomeninges, which includes the arachnoid and pial layers. While the dura mater contains lymphatic vessels, no conventional lymphatics have been found within the brain or leptomeninges. However, non-lumenized cells called Brain/Mural Lymphatic Endothelial Cells or Fluorescent Granule Perithelial cells (muLECs/BLECs/FGPs) that share a developmental program and gene expression with peripheral lymphatic vessels have been described in the meninges of zebrafish. Here we identify a structurally and functionally similar cell type in the mammalian leptomeninges that we name Leptomeningeal Lymphatic Endothelial Cells (LLEC). As in zebrafish, LLECs express multiple lymphatic markers, containing very large, spherical inclusions, and develop independently from the meningeal macrophage lineage. Mouse LLECs also internalize macromolecules from the cerebrospinal fluid, including Amyloid-ß, the toxic driver of Alzheimer's disease progression. Finally, we identify morphologically similar cells co-expressing LLEC markers in human post-mortem leptomeninges. Given that LLECs share molecular, morphological, and functional characteristics with both lymphatics and macrophages, we propose they represent a novel, evolutionary conserved cell type with potential roles in homeostasis and immune organization of the meninges.
Asunto(s)
Encéfalo/patología , Células Endoteliales/patología , Células Endoteliales/fisiología , Sistema Linfático/patología , Meninges/patología , Adulto , Anciano , Anciano de 80 o más Años , Péptidos beta-Amiloides , Animales , Femenino , Humanos , Masculino , Ratones , Pez CebraRESUMEN
The vertebrate eye originates from the eye field, a domain of cells specified by a small number of transcription factors. In this study, we show that Tcf7l1a is one such transcription factor that acts cell-autonomously to specify the eye field in zebrafish. Despite the much-reduced eye field in tcf7l1a mutants, these fish develop normal eyes revealing a striking ability of the eye to recover from a severe early phenotype. This robustness is not mediated through genetic compensation at neural plate stage; instead, the smaller optic vesicle of tcf7l1a mutants shows delayed neurogenesis and continues to grow until it achieves approximately normal size. Although the developing eye is robust to the lack of Tcf7l1a function, it is sensitised to the effects of additional mutations. In support of this, a forward genetic screen identified mutations in hesx1, cct5 and gdf6a, which give synthetically enhanced eye specification or growth phenotypes when in combination with the tcf7l1a mutation.
Asunto(s)
Ojo/crecimiento & desarrollo , Morfogénesis , Proteína 1 Similar al Factor de Transcripción 7/metabolismo , Proteínas de Pez Cebra/metabolismo , Pez Cebra/crecimiento & desarrollo , Animales , Proliferación Celular , Embrión no Mamífero/metabolismo , Ojo/patología , Femenino , Regulación del Desarrollo de la Expresión Génica , Sitios Genéticos , Cinética , Masculino , Mutación/genética , Placa Neural/embriología , Neurogénesis , Penetrancia , Fenotipo , Prosencéfalo/embriología , Proteína 1 Similar al Factor de Transcripción 7/genética , Regulación hacia Arriba/genética , Pez Cebra/embriología , Pez Cebra/genética , Proteínas de Pez Cebra/genética , Cigoto/metabolismoRESUMEN
Congenital heart defects contribute to embryonic or neonatal lethality but due to the complexity of cardiac development, the molecular changes associated with such defects are not fully understood. Here, we report that transcription factors (TFs) Brn-3a (POU4F1) and Brn-3b (POU4F2) are important for normal cardiac development. Brn-3a directly represses Brn-3b promoter in cardiomyocytes and consequently Brn-3a knockout (KO) mutant hearts express increased Brn-3b mRNA during mid-gestation, which is linked to hyperplastic growth associated with elevated cyclin D1, a known Brn-3b target gene. However, during late gestation, Brn-3b can cooperate with p53 to enhance transcription of pro-apoptotic genes e.g. Bax, thereby increasing apoptosis and contribute to morphological defects such as non-compaction, ventricular wall/septal thinning and increased crypts/fissures, which may cause lethality of Brn-3a KO mutants soon after birth. Despite this, early embryonic lethality in e9.5 double KO (Brn-3a-/- : Brn-3b-/-) mutants indicate essential functions with partial redundancy during early embryogenesis. High conservation between mammals and zebrafish (ZF) Brn-3b (87%) or Brn-3a (76%) facilitated use of ZF embryos to study potential roles in developing heart. Double morphant embryos targeted with morpholino oligonucleotides to both TFs develop significant cardiac defects (looping abnormalities and valve defects) suggesting essential roles for Brn-3a and Brn-3b in developing hearts.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica/fisiología , Corazón/embriología , Proteínas de Homeodominio/biosíntesis , Factor de Transcripción Brn-3A/biosíntesis , Factor de Transcripción Brn-3B/biosíntesis , Animales , Cardiopatías Congénitas/embriología , Cardiopatías Congénitas/genética , Proteínas de Homeodominio/genética , Ratones , Ratones Noqueados , Factor de Transcripción Brn-3A/genética , Factor de Transcripción Brn-3B/genéticaRESUMEN
The lymphatic system controls fluid homeostasis and the clearance of macromolecules from interstitial compartments. In mammals brain lymphatics were only recently discovered, with significant implications for physiology and disease. We examined zebrafish for the presence of brain lymphatics and found loosely connected endothelial cells with lymphatic molecular signature covering parts of the brain without forming endothelial tubular structures. These brain lymphatic endothelial cells (BLECs) derive from venous endothelium, are distinct from macrophages, and are sensitive to loss of Vegfc. BLECs endocytose macromolecules in a selective manner, which can be blocked by injection of mannose receptor ligands. This first report on brain lymphatic endothelial cells in a vertebrate embryo identifies cells with unique features, including the uptake of macromolecules at a single cell level. Future studies will address whether this represents an uptake mechanism that is conserved in mammals and how these cells affect functions of the embryonic and adult brain.
Asunto(s)
Encéfalo/embriología , Endocitosis , Células Endoteliales/metabolismo , Sustancias Macromoleculares/metabolismo , Pez Cebra/embriología , AnimalesRESUMEN
The neuropeptide agouti-related protein (AgRP) is expressed in the arcuate nucleus of the mammalian hypothalamus and plays a key role in regulating food consumption and energy homeostasis. Fish express two agrp genes in the brain: agrp1, considered functionally homologous with the mammalian AgRP, and agrp2. The role of agrp2 and its relationship to agrp1 are not fully understood. Utilizing BAC transgenesis, we generated transgenic zebrafish in which agrp1- and agrp2-expressing cells can be visualized and manipulated. By characterizing these transgenic lines, we showed that agrp1-expressing neurons are located in the ventral periventricular hypothalamus (the equivalent of the mammalian arcuate nucleus), projecting throughout the hypothalamus and towards the preoptic area. The agrp2 gene was expressed in the pineal gland in a previously uncharacterized subgroup of cells. Additionally, agrp2 was expressed in a small group of neurons in the preoptic area that project directly towards the pituitary and form an interface with the pituitary vasculature, suggesting that preoptic AgRP2 neurons are hypophysiotropic. We showed that direct synaptic connection can exist between AgRP1 and AgRP2 neurons in the hypothalamus, suggesting communication and coordination between AgRP1 and AgRP2 neurons and, therefore, probably also between the processes they regulate.
Asunto(s)
Proteína Relacionada con Agouti/metabolismo , Cromosomas Artificiales Bacterianos/genética , Técnicas de Transferencia de Gen , Neuronas/metabolismo , Glándula Pineal/citología , Hipófisis/metabolismo , Pez Cebra/genética , Animales , Animales Modificados Genéticamente , Comunicación Celular , Área Preóptica/metabolismoRESUMEN
The habenulae are bilateral nuclei located in the dorsal diencephalon that are conserved across vertebrates. Here we describe the main afferents to the habenulae in larval and adult zebrafish. We observe afferents from the subpallium, nucleus rostrolateralis, posterior tuberculum, posterior hypothalamic lobe, median raphe; we also see asymmetric afferents from olfactory bulb to the right habenula, and from the parapineal to the left habenula. In addition, we find afferents from a ventrolateral telencephalic nucleus that neurochemical and hodological data identify as the ventral entopeduncular nucleus (vENT), confirming and extending observations of Amo et al. (2014). Fate map and marker studies suggest that vENT originates from the diencephalic prethalamic eminence and extends into the lateral telencephalon from 48 to 120 hour post-fertilization (hpf). No afferents to the habenula were observed from the dorsal entopeduncular nucleus (dENT). Consequently, we confirm that the vENT (and not the dENT) should be considered as the entopeduncular nucleus "proper" in zebrafish. Furthermore, comparison with data in other vertebrates suggests that the vENT is a conserved basal ganglia nucleus, being homologous to the entopeduncular nucleus of mammals (internal segment of the globus pallidus of primates) by both embryonic origin and projections, as previously suggested by Amo et al. (2014).
Asunto(s)
Vías Aferentes/fisiología , Mapeo Encefálico , Regulación del Desarrollo de la Expresión Génica/fisiología , Habénula/fisiología , Aminoácidos/metabolismo , Animales , Animales Modificados Genéticamente , Ganglios Basales , Calbindina 2/genética , Calbindina 2/metabolismo , Embrión no Mamífero , Lateralidad Funcional , Regulación del Desarrollo de la Expresión Génica/genética , Ácido Glutámico/metabolismo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Habénula/citología , Habénula/crecimiento & desarrollo , Larva , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Neuronas/metabolismo , Bulbo Olfatorio , Pez Cebra , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismoRESUMEN
Maintaining neurogenesis in growing tissues requires a tight balance between progenitor cell proliferation and differentiation. In the zebrafish retina, neuronal differentiation proceeds in two stages with embryonic retinal progenitor cells (RPCs) of the central retina accounting for the first rounds of differentiation, and stem cells from the ciliary marginal zone (CMZ) being responsible for late neurogenesis and growth of the eye. In this study, we analyse two mutants with small eyes that display defects during both early and late phases of retinal neurogenesis. These mutants carry lesions in gdf6a, a gene encoding a BMP family member previously implicated in dorsoventral patterning of the eye. We show that gdf6a mutant eyes exhibit expanded retinoic acid (RA) signalling and demonstrate that exogenous activation of this pathway in wild-type eyes inhibits retinal growth, generating small eyes with a reduced CMZ and fewer proliferating progenitors, similar to gdf6a mutants. We provide evidence that RA regulates the timing of RPC differentiation by promoting cell cycle exit. Furthermore, reducing RA signalling in gdf6a mutants re-establishes appropriate timing of embryonic retinal neurogenesis and restores putative stem and progenitor cell populations in the CMZ. Together, our results support a model in which dorsally expressed gdf6a limits RA pathway activity to control the transition from proliferation to differentiation in the growing eye.
Asunto(s)
Factor 6 de Diferenciación de Crecimiento/genética , Neurogénesis/genética , Retina/embriología , Tretinoina/metabolismo , Proteínas de Pez Cebra/genética , Pez Cebra/embriología , Animales , Proteínas Morfogenéticas Óseas/metabolismo , Ciclo Celular/genética , Proliferación Celular , Embrión no Mamífero/embriología , Neurogénesis/fisiología , Transducción de Señal/genética , Células Madre/citologíaRESUMEN
Protein-coding mutations in the transcription factor-encoding gene ARX cause various forms of intellectual disability (ID) and epilepsy. In contrast, variations in surrounding non-coding sequences are correlated with milder forms of non-syndromic ID and autism and had suggested the importance of ARX gene regulation in the etiology of these disorders. We compile data on several novel and some already identified patients with or without ID that carry duplications of ARX genomic region and consider likely genetic mechanisms underlying the neurodevelopmental defects. We establish the long-range regulatory domain of ARX and identify its brain region-specific autoregulation. We conclude that neurodevelopmental disturbances in the patients may not simply arise from increased dosage due to ARX duplication. This is further exemplified by a small duplication involving a non-functional ARX copy, but with duplicated enhancers. ARX enhancers are located within a 504-kb region and regulate expression specifically in the forebrain in developing and adult zebrafish. Transgenic enhancer-reporter lines were used as in vivo tools to delineate a brain region-specific negative and positive autoregulation of ARX. We find autorepression of ARX in the telencephalon and autoactivation in the ventral thalamus. Fluorescently labeled brain regions in the transgenic lines facilitated the identification of neuronal outgrowth and pathfinding disturbances in the ventral thalamus and telencephalon that occur when arxa dosage is diminished. In summary, we have established a model for how breakpoints in long-range gene regulation alter the expression levels of a target gene brain region-specifically, and how this can cause subtle neuronal phenotypes relating to the etiology of associated neuropsychiatric disease.
Asunto(s)
Variaciones en el Número de Copia de ADN , Duplicación de Gen , Proteínas de Homeodominio/genética , Discapacidad Intelectual/genética , Factores de Transcripción/genética , Adulto , Animales , Animales Modificados Genéticamente , Encéfalo/embriología , Encéfalo/metabolismo , Estudios de Casos y Controles , Embrión no Mamífero , Femenino , Dosificación de Gen , Regulación del Desarrollo de la Expresión Génica , Proteínas de Homeodominio/metabolismo , Humanos , Masculino , Factores de Transcripción/metabolismo , Pez CebraRESUMEN
Enhancers can regulate the transcription of genes over long genomic distances. This is thought to lead to selection against genomic rearrangements within such regions that may disrupt this functional linkage. Here we test this concept experimentally using the human X chromosome. We describe a scoring method to identify evolutionary maintenance of linkage between conserved noncoding elements and neighbouring genes. Chromatin marks associated with enhancer function are strongly correlated with this linkage score. We test >1,000 putative enhancers by transgenesis assays in zebrafish to ascertain the identity of the target gene. The majority of active enhancers drive a transgenic expression in a pattern consistent with the known expression of a linked gene. These results show that evolutionary maintenance of linkage is a reliable predictor of an enhancer's function, and provide new information to discover the genetic basis of diseases caused by the mis-regulation of gene expression.
Asunto(s)
Cromosomas Humanos X/genética , Elementos de Facilitación Genéticos/genética , Expresión Génica/genética , Ligamiento Genético/genética , Selección Genética/genética , Animales , Animales Modificados Genéticamente , Evolución Molecular , Reordenamiento Génico/genética , Humanos , Pez CebraRESUMEN
BACKGROUND: Although left-right asymmetries are common features of nervous systems, their developmental bases are largely unknown. In the zebrafish epithalamus, dorsal habenular neurons adopt medial (dHbm) and lateral (dHbl) subnuclear character at very different frequencies on the left and right sides. The left-sided parapineal promotes the elaboration of dHbl character in the left habenula, albeit by an unknown mechanism. Likewise, the genetic pathways acting within habenular neurons to control their asymmetric differentiated character are unknown. RESULTS: In a forward genetic screen for mutations that result in loss of habenular asymmetry, we identified two mutant alleles of tcf7l2, a gene that encodes a transcriptional regulator of Wnt signaling. In tcf7l2 mutants, most neurons on both sides differentiate with dHbl identity. Consequently, the habenulae develop symmetrically, with both sides adopting a pronounced leftward character. Tcf7l2 acts cell automously in nascent equipotential neurons, and on the right side, it promotes dHbm and suppresses dHbl differentiation. On the left, the parapineal prevents this Tcf7l2-dependent process, thereby promoting dHbl differentiation. CONCLUSIONS: Tcf7l2 is essential for lateralized fate selection by habenular neurons that can differentiate along two alternative pathways, thereby leading to major neural circuit asymmetries.
Asunto(s)
Diferenciación Celular , Habénula/embriología , Neuronas/fisiología , Proteína 2 Similar al Factor de Transcripción 7/genética , Proteínas de Pez Cebra/genética , Pez Cebra/embriología , Animales , Embrión no Mamífero/embriología , Embrión no Mamífero/fisiología , Regulación de la Expresión Génica , Habénula/citología , Neuronas/citología , Transducción de Señal , Proteína 2 Similar al Factor de Transcripción 7/metabolismo , Pez Cebra/fisiología , Proteínas de Pez Cebra/metabolismoRESUMEN
Degradation of the myelin sheath is a common pathology underlying demyelinating neurological diseases from Multiple Sclerosis to Leukodistrophies. Although large malformations of myelin ultrastructure in the advanced stages of Wallerian degradation is known, its subtle structural variations at early stages of demyelination remains poorly characterized. This is partly due to the lack of suitable and non-invasive experimental probes possessing sufficient resolution to detect the degradation. Here we report the feasibility of the application of an innovative non-invasive local structure experimental approach for imaging the changes of statistical structural fluctuations in the first stage of myelin degeneration. Scanning micro X-ray diffraction, using advances in synchrotron x-ray beam focusing, fast data collection, paired with spatial statistical analysis, has been used to unveil temporal changes in the myelin structure of dissected nerves following extraction of the Xenopus laevis sciatic nerve. The early myelin degeneration is a specific ordered compacted phase preceding the swollen myelin phase of Wallerian degradation. Our demonstration of the feasibility of the statistical analysis of SµXRD measurements using biological tissue paves the way for further structural investigations of degradation and death of neurons and other cells and tissues in diverse pathological states where nanoscale structural changes may be uncovered.
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Axones/patología , Vaina de Mielina/patología , Sistema Nervioso Periférico/patología , Degeneración Walleriana/patología , Animales , Células Cultivadas , Enfermedades Desmielinizantes/patología , Femenino , Humanos , Esclerosis Múltiple/patología , Nervio Ciático/patología , Sincrotrones , Difracción de Rayos X/métodos , Xenopus laevisRESUMEN
Zebrafishbrain.org is an online neuroanatomical atlas of the embryonic zebrafish. The atlas uses high-resolution confocal images and movies of transgenic lines to describe different brain structures. This chapter covers detail of materials and protocols that we employ to generate data for the atlas.
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Encéfalo/anatomía & histología , Encéfalo/embriología , Neuroanatomía/métodos , Pez Cebra/anatomía & histología , Pez Cebra/embriología , Animales , Animales Modificados Genéticamente , Encéfalo/metabolismo , Técnicas de Cultivo , Disección , Técnica del Anticuerpo Fluorescente , Colorantes Fluorescentes/química , Microscopía Confocal , Fijación del Tejido , Pez Cebra/genéticaRESUMEN
During tissue morphogenesis and differentiation, cells must self-renew while contemporaneously generating daughters that contribute to the growing tissue. How tissues achieve this precise balance between proliferation and differentiation is, in most instances, poorly understood. This is in part due to the difficulties in dissociating the mechanisms that underlie tissue patterning from those that regulate proliferation. In the migrating posterior lateral line primordium (PLLP), proliferation is predominantly localised to the leading zone. As cells emerge from this zone, they periodically organise into rosettes that subsequently dissociate from the primordium and differentiate as neuromasts. Despite this reiterative loss of cells, the primordium maintains its size through regenerative cell proliferation until it reaches the tail. In this study, we identify a null mutation in the Wnt-pathway transcription factor Lef1 and show that its activity is required to maintain proliferation in the progenitor pool of cells that sustains the PLLP as it undergoes migration, morphogenesis and differentiation. In absence of Lef1, the leading zone becomes depleted of cells during its migration leading to the collapse of the primordium into a couple of terminal neuromasts. We show that this behaviour resembles the process by which the PLLP normally ends its migration, suggesting that suppression of Wnt signalling is required for termination of neuromast production in the tail. Our data support a model in which Lef1 sustains proliferation of leading zone progenitors, maintaining the primordium size and defining neuromast deposition rate.
Asunto(s)
Proliferación Celular , Homeostasis/genética , Sistema de la Línea Lateral/embriología , Factores de Transcripción/fisiología , Proteínas Wnt/fisiología , Proteínas de Pez Cebra/fisiología , beta Catenina/fisiología , Aletas de Animales/embriología , Aletas de Animales/crecimiento & desarrollo , Aletas de Animales/metabolismo , Animales , Animales Modificados Genéticamente , Tipificación del Cuerpo/genética , Diferenciación Celular/genética , Embrión no Mamífero , Homeostasis/fisiología , Sistema de la Línea Lateral/metabolismo , Masculino , Morfogénesis/genética , Morfogénesis/fisiología , Mutación/fisiología , Transducción de Señal/genética , Transducción de Señal/fisiología , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Proteínas Wnt/genética , Proteínas Wnt/metabolismo , Pez Cebra/embriología , Pez Cebra/genética , Pez Cebra/metabolismo , Pez Cebra/fisiología , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismo , beta Catenina/genética , beta Catenina/metabolismoRESUMEN
Pan-vertebrate developmental cis-regulatory elements are discernible as highly conserved noncoding elements (HCNEs) and are often dispersed over large areas around the pleiotropic genes whose expression they control. On the loci of two developmental transcription factor genes, SOX3 and PAX6, we demonstrate that HCNEs conserved between human and zebrafish can be systematically and reliably tested for their regulatory function in multiple stable transgenes in zebrafish, and their genomic reach estimated with confidence using synteny conservation and HCNE density along these loci. HCNEs of both human and zebrafish function as specific developmental enhancers in zebrafish. We show that human HCNEs result in expression patterns in zebrafish equivalent to those in mouse, establishing zebrafish as a suitable model for large-scale testing of human developmental enhancers. Orthologous human and zebrafish enhancers underwent functional evolution within their sequence and often directed related but non-identical expression patterns. Despite an evolutionary distance of 450 million years, one pax6 HCNE drove expression in identical areas when comparing zebrafish vs. human HCNEs. HCNEs from the same area often drive overlapping patterns, suggesting that multiple regulatory inputs are required to achieve robust and precise complex expression patterns exhibited by developmental genes.
Asunto(s)
Proteínas del Ojo/metabolismo , Regulación del Desarrollo de la Expresión Génica , Proteínas de Homeodominio/metabolismo , Factores de Transcripción Paired Box/metabolismo , Elementos Reguladores de la Transcripción , Proteínas Represoras/metabolismo , Factores de Transcripción SOXB1/metabolismo , Proteínas de Pez Cebra/metabolismo , Pez Cebra/fisiología , Animales , Sitios de Unión , Proteínas del Ojo/genética , Proteínas de Homeodominio/genética , Humanos , Ratones , Factor de Transcripción PAX6 , Factores de Transcripción Paired Box/genética , Proteínas Represoras/genética , Factores de Transcripción SOXB1/genética , Transgenes , Pez Cebra/anatomía & histología , Proteínas de Pez Cebra/genéticaRESUMEN
BACKGROUND: Key molecules involved in notochord differentiation and function have been identified through genetic analysis in zebrafish and mice, but MEK1 and 2 have so far not been implicated in this process due to early lethality (Mek1-/-) and functional redundancy (Mek2-/-) in the knockout animals. RESULTS: Here, we reveal a potential role for Mek1/2 during notochord development by using the small molecule Mek1/2 inhibitor U0126 which blocks phosphorylation of the Mek1/2 target gene Erk1/2 in vivo. Applying the inhibitor from early gastrulation until the 18-somite stage produces a specific and consistent phenotype with lack of dark pigmentation, shorter tail and an abnormal, undulated notochord. Using morphological analysis, in situ hybridization, immunhistochemistry, TUNEL staining and electron microscopy, we demonstrate that in treated embryos the chordamesoderm to notochord transition is disrupted and identify disorganization in the medial layer of the perinotochordal basement mebrane as the probable cause of the undulations and bulges in the notochord. We also examined and excluded FGF as the upstream signal during this process. CONCLUSION: Using the small chemical U0126, we have established a novel link between MAPK-signaling and notochord differentiation. Our phenotypic analysis suggests a potential connection between the MAPK-pathway, the COPI-mediated intracellular transport and/or the copper-dependent posttranslational regulatory processes during notochord differentiation.