RESUMO
Using the self-fertilizing mangrove killifish, we characterized two mutants, shorttail (stl) and balltail (btl). These mutants showed abnormalities in the posterior notochord and muscle development. Taking advantage of a highly inbred isogenic strain of the species, we rapidly identified the mutated genes, noto and msgn1 in the stl and btl mutants, respectively, using a single lane of RNA sequencing without the need of a reference genome or genetic mapping techniques. Next, we confirmed a conserved morphant phenotype in medaka and demonstrate a crucial role of noto and msgn1 in cell sorting between the axial and paraxial part of the tail mesoderm. This novel system could substantially accelerate future small-scale forward-genetic screening and identification of mutations. Therefore, the mangrove killifish could be used as a complementary system alongside existing models for future molecular genetic studies.
Assuntos
Desenvolvimento Embrionário/genética , Fundulidae/genética , Notocorda/crescimento & desenvolvimento , Cauda/crescimento & desenvolvimento , Animais , Mapeamento Cromossômico , Embrião não Mamífero , Fundulidae/crescimento & desenvolvimento , Testes Genéticos , Genoma/genética , Mutação/genética , Notocorda/metabolismo , Fenótipo , Filogenia , Autofertilização , Cauda/metabolismoRESUMO
The node-streak border region comprising notochord progenitor cells (NPCs) at the posterior node and neuro-mesodermal progenitor cells (NMPs) in the adjacent epiblast is the prime organizing center for axial elongation in mouse embryos. The T-box transcription factor brachyury (T) is essential for both formation of the notochord and maintenance of NMPs, and thus is a key regulator of trunk and tail development. The T promoter controlling T expression in NMPs and nascent mesoderm has been characterized in detail; however, control elements for T expression in the notochord have not been identified yet. We have generated a series of deletion alleles by CRISPR/Cas9 genome editing in mESCs, and analyzed their effects in mutant mouse embryos. We identified a 37â kb region upstream of T that is essential for notochord function and tailbud outgrowth. Within that region, we discovered a T-binding enhancer required for notochord cell specification and differentiation. Our data reveal a complex regulatory landscape controlling cell type-specific expression and function of T in NMP/nascent mesoderm and node/notochord, allowing proper trunk and tail development.
Assuntos
Desenvolvimento Embrionário/genética , Elementos Facilitadores Genéticos/genética , Proteínas Fetais/genética , Proteínas com Domínio T/genética , Cauda/crescimento & desenvolvimento , Sequência de Aminoácidos/genética , Animais , Sistemas CRISPR-Cas/genética , Edição de Genes/métodos , Regulação da Expressão Gênica no Desenvolvimento/genética , Mesoderma/crescimento & desenvolvimento , Mesoderma/metabolismo , Camundongos , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/metabolismo , Notocorda/crescimento & desenvolvimento , Notocorda/metabolismo , Regiões Promotoras Genéticas/genética , Sequências Reguladoras de Ácido Nucleico/genética , Cauda/metabolismoRESUMO
Generating properly differentiated embryonic structures in vitro from pluripotent stem cells remains a challenge. Here we show that instruction of aggregates of mouse embryonic stem cells with an experimentally engineered morphogen signalling centre, that functions as an organizer, results in the development of embryo-like entities (embryoids). In situ hybridization, immunolabelling, cell tracking and transcriptomic analyses show that these embryoids form the three germ layers through a gastrulation process and that they exhibit a wide range of developmental structures, highly similar to neurula-stage mouse embryos. Embryoids are organized around an axial chordamesoderm, with a dorsal neural plate that displays histological properties similar to the murine embryo neuroepithelium and that folds into a neural tube patterned antero-posteriorly from the posterior midbrain to the tip of the tail. Lateral to the chordamesoderm, embryoids display somitic and intermediate mesoderm, with beating cardiac tissue anteriorly and formation of a vasculature network. Ventrally, embryoids differentiate a primitive gut tube, which is patterned both antero-posteriorly and dorso-ventrally. Altogether, embryoids provide an in vitro model of mammalian embryo that displays extensive development of germ layer derivatives and that promises to be a powerful tool for in vitro studies and disease modelling.
Assuntos
Padronização Corporal/genética , Corpos Embrioides/metabolismo , Desenvolvimento Embrionário/genética , Células-Tronco Embrionárias Murinas/metabolismo , Transdução de Sinais/genética , Animais , Ectoderma/citologia , Ectoderma/crescimento & desenvolvimento , Ectoderma/metabolismo , Embrião de Mamíferos , Corpos Embrioides/citologia , Endoderma/citologia , Endoderma/crescimento & desenvolvimento , Endoderma/metabolismo , Fator de Transcrição GATA6/genética , Fator de Transcrição GATA6/metabolismo , Gástrula/citologia , Gástrula/crescimento & desenvolvimento , Gástrula/metabolismo , Gastrulação/genética , Regulação da Expressão Gênica no Desenvolvimento , Proteínas HMGB/genética , Proteínas HMGB/metabolismo , Camundongos , Células-Tronco Embrionárias Murinas/citologia , Proteína Homeobox Nanog/genética , Proteína Homeobox Nanog/metabolismo , Tubo Neural/citologia , Tubo Neural/crescimento & desenvolvimento , Tubo Neural/metabolismo , Notocorda/citologia , Notocorda/crescimento & desenvolvimento , Notocorda/metabolismo , Fatores de Transcrição SOXF/genética , Fatores de Transcrição SOXF/metabolismoRESUMO
BACKGROUND: The cylindromatosis (CYLD) tumor suppressor is a microtubule-associated deubiquitinase that plays a critical role in the regulation of cell signaling and contributes to a variety of physiological and pathological processes. However, the functions of CYLD in zebrafish are less well known, particularly with regard to their development and physiology. In this context, we investigated the loss of function of CYLD in zebrafish via transcription activator-like effector nuclease (TALEN)-based gene deletion. METHODS: Semi-quantitative RT-PCR was used to quantify CYLD mRNA expression in zebrafish embryos at various developmental stages. We also performed whole-mount in situ hybridization to further assess the dynamic expression and distribution of CYLD in the entire zebrafish embryos at different stages. In addition, we deleted CYLD in zebrafish with TALENs to investigate its potential impact on embryonic development. RESULTS: The expression of CYLD mRNA varied during early embryonic development. The CYLD mRNA localized to the brain and notochord of developing zebrafish embryos. Homozygous deletion of CYLD resulted in embryonic death before 8 h post-fertilization. CONCLUSIONS: CYLD appears to play an important role in central nervous system development in zebrafish. Although severe embryonic death restricted analysis of homozygous mutants, further research into the role of CYLD in central nervous system development is warranted.
Assuntos
Encéfalo/crescimento & desenvolvimento , Enzima Desubiquitinante CYLD/metabolismo , Notocorda/crescimento & desenvolvimento , Animais , Encéfalo/metabolismo , Enzima Desubiquitinante CYLD/genética , Humanos , Notocorda/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Peixe-ZebraRESUMO
MicroRNAs are frequently clustered in the genome and polycistronically transcribed, regulating targeted genes in diverse signaling pathways. The miR-17-92 cluster is a typical miRNA cluster, playing crucial roles in the organogenesis and homeostasis of physiological processes in vertebrates. Here, we identified three miRNAs (csa-miR-92a, csa-miR-92b, and csa-miR-92c) that belonged to the miR-92 family and formed a miRNA cluster in the genome of a urochordate marine ascidian Ciona savignyi. Except for miR-92a and miR-92b, other homologs of the vertebrate miR-17-92 cluster members could not be identified in the Ciona genome. We further found that the mature sequences of urochordate miR-92 family members were highly conserved compared with the vertebrate species. The expression pattern revealed that three miR-92 family members had consistent expression levels in adult tissues and were predominantly expressed in heart and muscle tissue. We further showed that, at the embryonic and larval stages, csa-miR-92c was expressed in the notochord of embryos during 18-31 h post fertilization (hpf) by in situ hybridization. Knockout of csa-miR-92c resulted in the disorganization of notochord cells and the block of lumen coalescence in the notochord. Fibroblast growth factor (FGF), mitogen-activated protein kinase (MAPK), and wingless/integrated (Wnt)/planar cell polarity (PCP) signaling pathways might be involved in the regulatory processes, since a large number of core genes of these pathways were the predicted target genes of the miR-92 family. Taken together, we identified a miR-92 cluster in urochordate Ciona and revealed the expression patterns and the regulatory roles of its members in organogenesis. Our results provide expression and phylogenetic data on the understanding of the miR-92 miRNA cluster's function during evolution.
Assuntos
Ciona/crescimento & desenvolvimento , Ciona/genética , MicroRNAs/genética , Notocorda/crescimento & desenvolvimento , Urocordados/crescimento & desenvolvimento , Urocordados/genética , Animais , Polaridade Celular/genética , Embrião não Mamífero , Regulação da Expressão Gênica no Desenvolvimento/genética , Genoma/genética , Larva/genética , Larva/crescimento & desenvolvimento , Filogenia , Transdução de Sinais/genética , Vertebrados/genética , Vertebrados/crescimento & desenvolvimentoRESUMO
The notochord is a defining feature of the chordates. The transcription factor Brachyury (Bra) is a key regulator of notochord fate but here we show that it is not a unitary master regulator in the model chordate Ciona Ectopic Bra expression only partially reprograms other cell types to a notochord-like transcriptional profile and a subset of notochord-enriched genes is unaffected by CRISPR Bra disruption. We identify Foxa.a and Mnx as potential co-regulators, and find that combinatorial cocktails are more effective at reprogramming other cell types than Bra alone. We reassess the network relationships between Bra, Foxa.a and other components of the notochord gene regulatory network, and find that Foxa.a expression in the notochord is regulated by vegetal FGF signaling. It is a direct activator of Bra expression and has a binding motif that is significantly enriched in the regulatory regions of notochord-enriched genes. These and other results indicate that Bra and Foxa.a act together in a regulatory network dominated by positive feed-forward interactions, with neither being a classically defined master regulator.
Assuntos
Ciona/genética , Ciona/metabolismo , Proteínas Fetais/genética , Proteínas Fetais/metabolismo , Notocorda/metabolismo , Proteínas com Domínio T/genética , Proteínas com Domínio T/metabolismo , Animais , Ciona intestinalis/genética , Ciona intestinalis/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Redes Reguladoras de Genes , Notocorda/crescimento & desenvolvimento , Transativadores , Fatores de Transcrição/metabolismoRESUMO
Many genes are regulated by two or more enhancers that drive similar expression patterns. Evolutionary theory suggests that these seemingly redundant enhancers must have functionally important differences. In the simple ascidian chordate Ciona, the transcription factor Brachyury is induced exclusively in the presumptive notochord downstream of lineage specific regulators and FGF-responsive Ets family transcription factors. Here we exploit the ability to finely titrate FGF signaling activity via the MAPK pathway using the MEK inhibitor U0126 to quantify the dependence of transcription driven by different Brachyury reporter constructs on this direct upstream regulator. We find that the more powerful promoter-adjacent proximal enhancer and a weaker distal enhancer have fundamentally different dose-response relationships to MAPK inhibition. The Distal enhancer is more sensitive to MAPK inhibition but shows a less cooperative response, whereas the Proximal enhancer is less sensitive and more cooperative. A longer construct containing both enhancers has a complex dose-response curve that supports the idea that the proximal and distal enhancers are moderately super-additive. We show that the overall expression loss from intermediate doses of U0126 is not only a function of the fraction of cells expressing these reporters, but also involves graded decreases in expression at the single-cell level. Expression of the endogenous gene shows a comparable dose-response relationship to the full length reporter, and we find that different notochord founder cells are differentially sensitive to MAPK inhibition. Together, these results indicate that although the two Brachyury enhancers have qualitatively similar expression patterns, they respond to FGF in quantitatively different ways and act together to drive high levels of Brachyury expression with a characteristic input/output relationship. This indicates that they are fundamentally not equivalent genetic elements.
Assuntos
Ciona intestinalis/genética , Elementos Facilitadores Genéticos/genética , Proteínas Fetais/genética , Fatores de Crescimento de Fibroblastos/genética , Proteínas com Domínio T/genética , Sequência de Aminoácidos/genética , Animais , Ciona intestinalis/crescimento & desenvolvimento , Regulação da Expressão Gênica no Desenvolvimento/genética , Sistema de Sinalização das MAP Quinases/genética , Notocorda/crescimento & desenvolvimento , Notocorda/metabolismo , Regiões Promotoras Genéticas/genética , Fatores de Transcrição/genéticaRESUMO
While it is well known that the notochord of bony fishes changes over developmental time, less is known about how it varies across different body regions. In the development of the Atlantic salmon, Salmo salar L., cranial and caudal ends of the notochord are overlaid by the formation of the bony elements of the neurocranium and caudal fin, respectively. To investigate, we describe how the notochord of the cranium and caudal fin changes from embryo to spawning adult, using light microscopy, SEM, TEM, dissection, and CT scanning. The differences are dramatic. In contrast to the abdominal and caudal regions, at the ends of the notochord vertebrae never develop. While the cranial notochord builds a tapering, unsegmented cone of chordal bone, the urostylic notochordal sheath never ossifies: adjacent, irregular bony elements form from the endoskeleton of the caudal fin. As development progresses, two previously undescribed processes occur. First, the bony cone of the cranial notochord, and its internal chordocytes, are degraded by chordoclasts, an undescribed function of the clastic cell type. Second, the sheath of the urostylic notochord creates transverse septae that partly traverse the lumen in an irregular pattern. By the adult stage, the cranial notochord is gone. In contrast, the urostylic notochord in adults is robust, reinforced with septae, covered by irregularly shaped pieces of cellular bone, and capped with an opistural cartilage that develops from the sheath of the urostylic notochord. A previously undescribed muscle, with its origin on the opistural cartilage, inserts on the lepidotrich ventral to it.
Assuntos
Nadadeiras de Animais/embriologia , Notocorda/embriologia , Salmo salar/embriologia , Crânio/embriologia , Nadadeiras de Animais/crescimento & desenvolvimento , Animais , Notocorda/crescimento & desenvolvimento , Salmo salar/crescimento & desenvolvimento , Crânio/crescimento & desenvolvimentoRESUMO
Ventral bending of the embryonic tail within the chorion is an evolutionarily conserved morphogenetic event in both invertebrates and vertebrates. However, the complexity of the anatomical structure of vertebrate embryos makes it difficult to experimentally identify the mechanisms underlying embryonic folding. This study investigated the mechanisms underlying embryonic tail bending in chordates. To further understand the mechanical role of each tissue, we also developed a physical model with experimentally measured parameters to simulate embryonic tail bending. Actomyosin asymmetrically accumulated at the ventral side of the notochord, and cell proliferation of the dorsal tail epidermis was faster than that in the ventral counterpart during embryonic tail bending. Genetic disruption of actomyosin activity and inhibition of cell proliferation dorsally caused abnormal tail bending, indicating that both asymmetrical actomyosin contractility in the notochord and the discrepancy of epidermis cell proliferation are required for tail bending. In addition, asymmetrical notochord contractility was sufficient to drive embryonic tail bending, whereas differential epidermis proliferation was a passive response to mechanical forces. These findings showed that asymmetrical notochord contractility coordinates with differential epidermis proliferation mechanisms to drive embryonic tail bending.This article has an associated 'The people behind the papers' interview.
Assuntos
Actomiosina/genética , Morfogênese/genética , Cauda/crescimento & desenvolvimento , Actomiosina/metabolismo , Animais , Proliferação de Células/genética , Ciona/embriologia , Ciona/genética , Ciona/crescimento & desenvolvimento , Células Epiteliais/metabolismo , Contração Muscular/fisiologia , Notocorda/embriologia , Notocorda/crescimento & desenvolvimento , Cauda/embriologiaRESUMO
The Brachyury gene encodes a transcription factor that is conserved across all animals. In non-chordate metazoans, brachyury is primarily expressed in ectoderm regions that are added to the endodermal gut during development, and often form a ring around the site of endoderm internalization in the gastrula, the blastopore. In chordates, this brachyury ring is conserved, but the gene has taken on a new role in the formation of the mesoderm. In this phylum, a novel type of mesoderm that develops into notochord and somites has been added to the ancestral lateral plate mesoderm. Brachyury contributes to a shift in cell fate from neural ectoderm to posterior notochord and somites during a major lineage segregation event that in Xenopus and in the zebrafish takes place in the early gastrula. In the absence of this brachyury function, impaired formation of posterior mesoderm indirectly affects the gastrulation movements of peak involution and convergent extension. These movements are confined to specific regions and stages, leaving open the question why brachyury expression in an extensive, coherent ring, before, during and after gastrulation, is conserved in the two species whose gastrulation modes differ considerably, and also in many other metazoan gastrulae of diverse structure.
Assuntos
Ectoderma/crescimento & desenvolvimento , Proteínas Fetais/genética , Gástrula/crescimento & desenvolvimento , Morfogênese/genética , Proteínas com Domínio T/genética , Animais , Endoderma/crescimento & desenvolvimento , Proteínas Fetais/ultraestrutura , Mesoderma/crescimento & desenvolvimento , Notocorda/crescimento & desenvolvimento , Proteínas com Domínio T/ultraestrutura , Xenopus laevis/genética , Xenopus laevis/crescimento & desenvolvimento , Peixe-Zebra/genética , Peixe-Zebra/crescimento & desenvolvimentoRESUMO
The notochord is a defining feature of chordates. During notochord formation in vertebrates and tunicates, notochord cells display dynamic morphogenetic movement, called convergent extension, in which cells intercalate and align at the dorsal midline. However, in cephalochordates, the most basal group of chordates, the notochord is formed without convergent extension. It is simply developed from mesodermal cells at the dorsal midline. This suggests that convergent extension movement of notochord cells is a secondarily acquired developmental attribute in the common ancestor of olfactores (vertebrates + tunicates), and that the chordate ancestor innovated the notochord upon a foundation of morphogenetic mechanisms independent of cell movement. Therefore, this review focuses on biological features specific to notochord cells, which have been well studied using clawed frogs, zebrafish, and tunicates. Attributes of notochord cells, such as vacuolation, membrane trafficking, extracellular matrix formation, and apoptosis, can be understood in terms of two properties: turgor pressure of vacuoles and strength of the notochord sheath. To maintain the straight rod-like structure of the notochord, these parameters must be counterbalanced. In the future, the turgor pressure-sheath strength model, proposed in this review, will be examined in light of quantitative molecular data and mathematical simulations, illuminating the evolutionary origin of the notochord.
Assuntos
Modelos Biológicos , Morfogênese , Notocorda/crescimento & desenvolvimento , Notocorda/metabolismo , Animais , Apoptose , Proliferação de Células , Notocorda/citologiaRESUMO
Developmental novelties often underlie the evolutionary origins of key metazoan features. The anuran urostyle, which evolved nearly 200 MYA, is one such structure. It forms as the tail regresses during metamorphosis, when locomotion changes from an axial-driven mode in larvae to a limb-driven one in adult frogs. The urostyle comprises of a coccyx and a hypochord. The coccyx forms by fusion of caudal vertebrae and has evolved repeatedly across vertebrates. However, the contribution of an ossifying hypochord to the coccyx in anurans is unique among vertebrates and remains a developmental enigma. Here, we focus on the developmental changes that lead to the anuran urostyle, with an emphasis on understanding the ossifying hypochord. We find that the coccyx and hypochord have two different developmental histories: First, the development of the coccyx initiates before metamorphic climax whereas the ossifying hypochord undergoes rapid ossification and hypertrophy; second, thyroid hormone directly affects hypochord formation and appears to have a secondary effect on the coccygeal portion of the urostyle. The embryonic hypochord is known to play a significant role in the positioning of the dorsal aorta (DA), but the reason for hypochordal ossification remains obscure. Our results suggest that the ossifying hypochord plays a role in remodeling the DA in the newly forming adult body by partially occluding the DA in the tail. We propose that the ossifying hypochord-induced loss of the tail during metamorphosis has enabled the evolution of the unique anuran bauplan.
Assuntos
Anuros , Evolução Biológica , Cóccix , Metamorfose Biológica/fisiologia , Animais , Anuros/anatomia & histologia , Anuros/embriologia , Anuros/crescimento & desenvolvimento , Cóccix/anatomia & histologia , Cóccix/embriologia , Cóccix/crescimento & desenvolvimento , Larva/anatomia & histologia , Larva/crescimento & desenvolvimento , Notocorda/anatomia & histologia , Notocorda/embriologia , Notocorda/crescimento & desenvolvimentoRESUMO
Anuran metamorphosis is perhaps the most dramatic developmental process regulated by thyroid hormone (TH). One of the unique processes that occur during metamorphosis is the complete resorption of the tail, including the notochord. Interestingly, recent gene knockout studies have shown that of the two known vertebrate TH receptors, TRα and TRß, TRß appears to be critical for notochord regression during tail resorption in Xenopus tropicalis. To determine the mechanisms underlying notochord regression, we carried out a comprehensive gene expression analysis in the notochord during metamorphosis by using RNA-Seq analyses of whole tail at stage 60 before any noticeable tail length reduction, whole tail at stage 63 when the tail length is reduced by about one half, and the rest of the tail at stage 63 after removing the notochord. This allowed us to identify many notochord-enriched, metamorphosis-induced genes at stage 63. Future studies on these genes should help to determine if they are regulated by TRß and play any roles in notochord regression.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento/genética , Notocorda/crescimento & desenvolvimento , RNA-Seq/métodos , Cauda/crescimento & desenvolvimento , Xenopus laevis/crescimento & desenvolvimento , Xenopus/genética , AnimaisRESUMO
Intervertebral disc degeneration (IDD) is a public health dilemma as it is associated with low back and neck pain, a frequent reason for patients to visit the physician. During IDD, nucleus pulposus (NP), the central compartment of intervertebral disc (IVD) undergo degeneration. Stem cells have been adopted as a promising biological source to regenerate the IVD and restore its function. Here, we describe a simple, two-step differentiation strategy using a cocktail of four factors (LDN, AGN, FGF, and CHIR) for efficient derivation of notochordal cells from human embryonic stem cells (hESCs). We employed a CRISPR/Cas9 based genome-editing approach to knock-in the mCherry reporter vector upstream of the 3' untranslated region of the Noto gene in H9-hESCs and monitored notochordal cell differentiation. Our data show that treatment of H9-hESCs with the above-mentioned four factors for 6 days successfully resulted in notochordal cells. These cells were characterized by morphology, immunostaining, and gene and protein expression analyses for established notochordal cell markers including FoxA2, SHH, and Brachyury. Additionally, pan-genomic high-throughput single cell RNA-sequencing revealed an efficient and robust notochordal differentiation. We further identified a key regulatory network consisting of eight candidate genes encoding transcription factors including PAX6, GDF3, FOXD3, TDGF1, and SOX5, which are considered as potential drivers of notochordal differentiation. This is the first single cell transcriptomic analysis of notochordal cells derived from hESCs. The ability to efficiently obtain notochordal cells from pluripotent stem cells provides an additional tool to develop new cell-based therapies for the treatment of IDD.
Assuntos
Diferenciação Celular/genética , Células-Tronco Embrionárias Humanas/metabolismo , Degeneração do Disco Intervertebral/genética , Transcriptoma/genética , Biomarcadores/metabolismo , Proteínas Fetais/genética , Fatores de Transcrição Forkhead/genética , Proteínas Ligadas por GPI/genética , Redes Reguladoras de Genes/genética , Fator 3 de Diferenciação de Crescimento/genética , Células-Tronco Embrionárias Humanas/citologia , Humanos , Células-Tronco Pluripotentes Induzidas , Peptídeos e Proteínas de Sinalização Intercelular/genética , Disco Intervertebral/crescimento & desenvolvimento , Degeneração do Disco Intervertebral/patologia , Proteínas de Neoplasias/genética , Notocorda/crescimento & desenvolvimento , Notocorda/metabolismo , Núcleo Pulposo/crescimento & desenvolvimento , Núcleo Pulposo/metabolismo , Fator de Transcrição PAX6/genética , Regeneração/genética , Fatores de Transcrição SOXD/genética , Análise de Célula Única , Proteínas com Domínio T/genéticaRESUMO
slc7a5 (also known as LAT1), largely accepted as an amino acid transporter, has been shown to play important roles in cancer and developmental processes. Because knockout mice of Slc7a5 are embryonically lethal due to placental defects, it is difficult to evaluate its role in early development. In this study, expression and function of slc7a5 were evaluated in Xenopus laevis embryos that develop without a placenta. Expression of slc7a5 was detected in the notochord and in the eye and it was not co-localized with slc3a2, which helps slc7a5 to localize at the plasma membrane, before the late neurula stage. Loss-of-function experiment with a morpholino antisense oligonucleotide led to defect of neural and non-neural patterning, inhibition of primary neurogenesis, and disruption of eye development. Disruption of neural development and primary neurogenesis was likely due to impaired notochord development as sonic hedgehog (shh) signaling pathway was compromised in slc7a5-inhibited embryos. These results suggest that slc7a5 is required for notochord development and subsequent primary neurogenesis via shh/gli signaling and for eye development. These novel developmental roles of slc7a5 appeared to be independent of transport function at least before the late neurula stage.
Assuntos
Olho/crescimento & desenvolvimento , Olho/metabolismo , Transportador 1 de Aminoácidos Neutros Grandes/metabolismo , Notocorda/crescimento & desenvolvimento , Notocorda/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus laevis/metabolismo , Animais , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Proteínas Hedgehog/metabolismo , Humanos , Neurogênese/fisiologia , Transdução de Sinais/fisiologiaRESUMO
The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is the formation of the transient organizers, the node and notochordal plate, from cells that have passed through the primitive streak. The proper formation of these signaling centers is essential for the development of the body plan and techniques to visualize them are of high interest to mouse developmental biologists. The node and notochordal plate lie on the ventral surface of gastrulating mouse embryos around embryonic day (E) 7.5 of development. The node is a cup-shaped structure whose cells possess a single slender cilium each. The proper subcellular localization and rotation of the cilia in the node pit determines left-right asymmetry. The notochordal plate cells also possess single cilia albeit shorter than those of the node cells. The notochordal plate forms the notochord which acts as an important signaling organizer for somitogenesis and neural patterning. Because the cells of the node and notochordal plate are transiently present on the surface and possess cilia, they can be visualized using scanning electron microscopy (SEM). Among other techniques used to visualize these structures at the cellular level is whole mount immunofluorescence (WMIF) using the antibodies against the proteins that are highly expressed in the node and notochordal plate. In this report, we describe our optimized protocols to perform SEM and WMIF of the node and notochordal plate in developing mouse embryos to help in the assessment of tissue shape and cellular organization in wild-type and gastrulation mutant embryos.
Assuntos
Desenvolvimento Embrionário/genética , Imunofluorescência/métodos , Gastrulação/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Microscopia Eletrônica de Varredura/métodos , Notocorda/crescimento & desenvolvimento , Animais , CamundongosRESUMO
This review covers recent advances in our understanding of the cell biology and morphogenesis of the ascidian notochord. In its development, the ascidian notochord undergoes a rapid series of cellular and morphogenic events that transform a group of 40 loosely packed cells in the neurula embryo into a tubular column with central lumen in the larva. The ascidian notochord has been a subject of intensive research in recent years, and particular focus in this review will be on events associated with the development and function of polarized cell properties, and the mechanism of lumen formation.
Assuntos
Ciona intestinalis/citologia , Notocorda/citologia , Animais , Linhagem da Célula , Polaridade Celular , Ciona intestinalis/embriologia , Ciona intestinalis/crescimento & desenvolvimento , Embrião não Mamífero/citologia , Técnicas de Silenciamento de Genes , Larva/citologia , Larva/ultraestrutura , Morfogênese/genética , Mosaicismo , Notocorda/embriologia , Notocorda/crescimento & desenvolvimento , Fenótipo , Cauda/embriologia , Cauda/crescimento & desenvolvimentoRESUMO
BACKGROUND: Mov10 is an RNA helicase that modulates access of Argonaute 2 to microRNA recognition elements in mRNAs. We examined the role of Mov10 in Xenopus laevis development and show a critical role for Mov10 in gastrulation and in the development of the central nervous system (CNS). RESULTS: Knockdown of maternal Mov10 in Xenopus embryos using a translation blocking morpholino led to defects in gastrulation and the development of notochord and paraxial mesoderm, and a failure to neurulate. RNA sequencing of the Mov10 knockdown embryos showed significant upregulation of many mRNAs when compared with controls at stage 10.5 (including those related to the cytoskeleton, adhesion, and extracellular matrix, which are involved in those morphogenetic processes). Additionally, the degradation of the miR-427 target mRNA, cyclin A1, was delayed in the Mov10 knockdowns. These defects suggest that Mov10's role in miRNA-mediated regulation of the maternal to zygotic transition could lead to pleiotropic effects that cause the gastrulation defects. Additionally, the knockdown of zygotic Mov10 showed that it was necessary for normal head, eye, and brain development in Xenopus consistent with a recent study in the mouse. CONCLUSIONS: Mov10 is essential for gastrulation and normal CNS development. Developmental Dynamics 247:660-671, 2018. © 2017 Wiley Periodicals, Inc.
Assuntos
Sistema Nervoso Central/crescimento & desenvolvimento , Gastrulação , RNA Helicases/fisiologia , Animais , Embrião não Mamífero , Mesoderma/crescimento & desenvolvimento , Notocorda/crescimento & desenvolvimento , Xenopus laevis/embriologiaRESUMO
The notochord gives rise to spinal segments during development, and it becomes embedded within the nucleus pulposus of the intervertebral disc (IVD) during maturation. The disruption of the notochord band has been observed with IVD degeneration. Since the mechanical competence of the IVD relies on its structural constituents, defining the structure of the notochord during aging is critical for investigations relating to IVD function and homeostasis. The assessment and imaging of the notochord has classically relied on histological techniques, which introduces sectioning artifacts during preparation and spatial biases. Magnetic resonance imaging (MRI) does not offer sufficient resolution to discriminate the notochord from the surrounding the nucleus pulposus, especially in murine models. Current X-ray based computed tomography systems provide imaging resolutions down to the single- and sub- micron scales, and when coupled with contrast-enhancing agents, enable the high-resolution three-dimensional imaging of relatively small features. Utilizing phosphomolybdic acid to preferentially bind to collagen cationic domains, we describe the structure of the notochord remnants with aging in the lumbar IVDs of BALB/c mice. These results provide a highly quantitative and sensitive approach to monitoring the IVD during postnatal development.
Assuntos
Notocorda/diagnóstico por imagem , Notocorda/crescimento & desenvolvimento , Intensificação de Imagem Radiográfica , Microtomografia por Raio-X , Animais , Processamento de Imagem Assistida por Computador , Imuno-Histoquímica , Camundongos , Notocorda/ultraestrutura , Intensificação de Imagem Radiográfica/métodos , Microtomografia por Raio-X/métodosRESUMO
During embryogenesis the spinal cord shifts position along the anterior-posterior axis relative to adjacent tissues. How motor neurons whose cell bodies are located in the spinal cord while their axons reside in adjacent tissues compensate for such tissue shift is not well understood. Using live cell imaging in zebrafish, we show that as motor axons exit from the spinal cord and extend through extracellular matrix produced by adjacent notochord cells, these cells shift several cell diameters caudally. Despite this pronounced shift, individual motoneuron cell bodies stay aligned with their extending axons. We find that this alignment requires myosin phosphatase activity within motoneurons, and that mutations in the myosin phosphatase subunit mypt1 increase myosin phosphorylation causing a displacement between motoneuron cell bodies and their axons. Thus, we demonstrate that spinal motoneurons fine-tune their position during axonogenesis and we identify the myosin II regulatory network as a key regulator.