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1.
Yi Chuan ; 43(2): 134-141, 2021 Feb 16.
Artigo em Inglês | MEDLINE | ID: mdl-33724216

RESUMO

The mechanisms underlying the establishment of left-right (L-R) asymmetry in bilaterians is one of the central enigmas in developmental biology. Amphioxus is an important model in studying the mechanisms of animal asymmetry specification due to its particular phylogenetic position, vertebrate-like embryogenesis and body plan. Recently, with the establishments of artificial breeding technology, high-efficiency microinjection method and gene knockout technology, researchers have successfully dissected the mechanisms of amphioxus L-R asymmetry development. In this review, we summarize the major progress in understanding L-R asymmetry specification in amphioxus and propose a model of regulation of L-R asymmetry in this species. Hh protein is transported dominantly to the right side by cilia movement, leading to R>L Hh signaling andCerexpression. Cer inhibits expression of Nodal, leading to the asymmetric expression of Nodal-dependent genes. The L-R differences in the propagation of the Nodal pathway result in the correct morphological L-R asymmetry development in amphioxus embryo. BMP signaling probably does not provide the asymmetric cue, but is necessary for correct expression ofCer andNodal.


Assuntos
Anfioxos , Animais , Padronização Corporal/genética , Regulação da Expressão Gênica no Desenvolvimento , Anfioxos/genética , Anfioxos/metabolismo , Proteína Nodal/genética , Proteína Nodal/metabolismo , Filogenia
3.
Zoolog Sci ; 38(1): 26-35, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33639715

RESUMO

The spatiotemporal expression of zygotic genes is regulated by transcription factors, which mediate cell fate decision and morphogenesis. Investigation of the expression patterns and their transcriptional regulatory relationships is crucial to understand embryonic development. Staged RNA-seq of the ascidian Halocynthia roretzi has previously shown that nine genes encoding transcription factors are transiently expressed at the blastula stage, which is the stage at which cell fates are specified and differentiation starts. Six of these transcription factors have already been found to play important roles during early development. However, the functions of the other transcription factors (FoxJ-r, SoxF, and SP8/9) remain unknown. The study of the spatial and temporal expression patterns showed that all three genes were expressed in the animal hemisphere as early as the 16-cell stage. This is likely due to transcription factor genes that are expressed in the vegetal hemisphere, which have been extensively and comprehensively analyzed in previous studies of ascidians. Functional analyses using FoxJ-r morphants showed that they resulted in the disruption of laterality and the absence of epidermal mono-cilia, suggesting FoxJ-r functions in cilia formation and, consequently, in the generation of left-right asymmetry, as observed in vertebrates. SoxF knockdown resulted in incomplete epiboly by the ectoderm during gastrulation, while SP8/9 knockdown showed no phenotype until the tailbud stage in the present study, although it was expressed during blastula stages. Our results indicate that transcription factor genes expressed at the cleavage stages play roles in diverse functions, and are not limited to cell fate specification.


Assuntos
Fatores de Transcrição/genética , Urocordados/embriologia , Urocordados/genética , Animais , Padronização Corporal/genética , Embrião não Mamífero/embriologia , Desenvolvimento Embrionário/genética , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Silenciamento de Genes , Fatores de Transcrição/metabolismo , Urocordados/metabolismo
4.
Methods Mol Biol ; 2258: 105-116, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33340357

RESUMO

Pluripotent stem cells (PSCs) possess the ability to self-organize into complex tissue-like structures; however, the genetic mechanisms and multicellular dynamics that direct such patterning are difficult to control. Here, we pair live imaging with controlled induction of gene knockdown by CRISPR interference (CRISPRi) to generate changes within subpopulations of human PSCs, allowing for control over organization and analysis of emergent behaviors. Specifically, we use forced aggregation of mixtures of cells with and without an inducible CRISPRi system to knockdown molecular regulators of tissue symmetry. We then track the resulting multicellular organization through fluorescence live imaging concurrent with the induction of knockdown. Overall, this technique allows for controlled initiation of symmetry breaking by CRISPRi to produce changes in cellular behavior that can be tracked over time within high-density pluripotent stem cell colonies.


Assuntos
Padronização Corporal , Sistemas CRISPR-Cas , Edição de Genes , Células-Tronco Pluripotentes/fisiologia , Proteínas Associadas a CRISPR/genética , Proteínas Associadas a CRISPR/metabolismo , Células Cultivadas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Regulação da Expressão Gênica no Desenvolvimento , Microscopia de Fluorescência , Microscopia de Vídeo , RNA Guia/genética , RNA Guia/metabolismo , Transdução de Sinais , Fatores de Tempo , Imagem com Lapso de Tempo
5.
Methods Mol Biol ; 2258: 131-147, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33340359

RESUMO

Gastruloids are embryonic organoids made from small, defined numbers of mouse embryonic stem cells (mESCs) aggregated in suspension culture, which over time form 3D structures that mimic many of the features of early mammalian development. Unlike embryoid bodies that are usually disorganized when grown over several days, gastruloids display distinct, well-organized gene expression domains demarcating the emergence of the three body axes, anteroposterior axial elongation, and implementation of collinear Hox transcriptional patterns over 5-7 days of culture. As such gastruloids represent a useful experimental system that is complementary to in vivo approaches in studying early developmental patterning mechanisms regulating the acquisition of cell fates. In this protocol, we describe the most recent method for generating gastruloids with high reproducibility, and provide a comprehensive list of possible challenges as well as steps for protocol optimization.


Assuntos
Padronização Corporal , Diferenciação Celular , Linhagem da Célula , Gastrulação , Células-Tronco Embrionárias Murinas/fisiologia , Animais , Técnicas de Cultura de Células , Células Cultivadas , Regulação da Expressão Gênica no Desenvolvimento , Camundongos , Microscopia , Organoides , Transdução de Sinais , Fatores de Tempo
6.
Nature ; 589(7842): 431-436, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33361814

RESUMO

Gene expression is an inherently stochastic process1,2; however, organismal development and homeostasis require cells to coordinate the spatiotemporal expression of large sets of genes. In metazoans, pairs of co-expressed genes often reside in the same chromosomal neighbourhood, with gene pairs representing 10 to 50% of all genes, depending on the species3-6. Because shared upstream regulators can ensure correlated gene expression, the selective advantage of maintaining adjacent gene pairs remains unknown6. Here, using two linked zebrafish segmentation clock genes, her1 and her7, and combining single-cell transcript counting, genetic engineering, real-time imaging and computational modelling, we show that gene pairing boosts correlated transcription and provides phenotypic robustness for the formation of developmental patterns. Our results demonstrate that the prevention of gene pairing disrupts oscillations and segmentation, and the linkage of her1 and her7 is essential for the development of the body axis in zebrafish embryos. We predict that gene pairing may be similarly advantageous in other organisms, and our findings could lead to the engineering of precise synthetic clocks in embryos and organoids.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Padronização Corporal/genética , Proteínas CLOCK/genética , Fatores de Transcrição/genética , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Animais , Relógios Biológicos/genética , Mutação , Análise de Célula Única
7.
PLoS Genet ; 16(12): e1008948, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33320862

RESUMO

During metazoan development, the cell cycle is remodelled to coordinate proliferation with differentiation. Developmental cues cause dramatic changes in the number and timing of replication initiation events, but the mechanisms and physiological importance of such changes are poorly understood. Cyclin-dependent kinases (CDKs) are important for regulating S-phase length in many metazoa, and here we show in the nematode Caenorhabditis elegans that an essential function of CDKs during early embryogenesis is to regulate the interactions between three replication initiation factors SLD-3, SLD-2 and MUS-101 (Dpb11/TopBP1). Mutations that bypass the requirement for CDKs to generate interactions between these factors is partly sufficient for viability in the absence of Cyclin E, demonstrating that this is a critical embryonic function of this Cyclin. Both SLD-2 and SLD-3 are asymmetrically localised in the early embryo and the levels of these proteins inversely correlate with S-phase length. We also show that SLD-2 asymmetry is determined by direct interaction with the polarity protein PKC-3. This study explains an essential function of CDKs for replication initiation in a metazoan and provides the first direct molecular mechanism through which polarization of the embryo is coordinated with DNA replication initiation factors.


Assuntos
Padronização Corporal , Proteínas de Caenorhabditis elegans/genética , Quinases Ciclina-Dependentes/metabolismo , Replicação do DNA , Animais , Caenorhabditis elegans , Proteínas de Caenorhabditis elegans/metabolismo , Quinases Ciclina-Dependentes/genética , Mutação , Proteína Quinase C/genética , Proteína Quinase C/metabolismo
8.
Science ; 370(6512): 113-116, 2020 10 02.
Artigo em Inglês | MEDLINE | ID: mdl-33004519

RESUMO

Animal development entails the organization of specific cell types in space and time, and spatial patterns must form in a robust manner. In the zebrafish spinal cord, neural progenitors form stereotypic patterns despite noisy morphogen signaling and large-scale cellular rearrangements during morphogenesis and growth. By directly measuring adhesion forces and preferences for three types of endogenous neural progenitors, we provide evidence for the differential adhesion model in which differences in intercellular adhesion mediate cell sorting. Cell type-specific combinatorial expression of different classes of cadherins (N-cadherin, cadherin 11, and protocadherin 19) results in homotypic preference ex vivo and patterning robustness in vivo. Furthermore, the differential adhesion code is regulated by the sonic hedgehog morphogen gradient. We propose that robust patterning during tissue morphogenesis results from interplay between adhesion-based self-organization and morphogen-directed patterning.


Assuntos
Padronização Corporal/fisiologia , Caderinas/metabolismo , Adesão Celular/fisiologia , Células-Tronco Neurais/fisiologia , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/crescimento & desenvolvimento , Animais , Padronização Corporal/genética , Caderinas/genética , Adesão Celular/genética , Medula Espinal/crescimento & desenvolvimento , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genética
9.
Science ; 370(6514): 321-327, 2020 10 16.
Artigo em Inglês | MEDLINE | ID: mdl-33060356

RESUMO

Morphogen gradients provide positional information during development. To uncover the minimal requirements for morphogen gradient formation, we have engineered a synthetic morphogen in Drosophila wing primordia. We show that an inert protein, green fluorescent protein (GFP), can form a detectable diffusion-based gradient in the presence of surface-associated anti-GFP nanobodies, which modulate the gradient by trapping the ligand and limiting leakage from the tissue. We next fused anti-GFP nanobodies to the receptors of Dpp, a natural morphogen, to render them responsive to extracellular GFP. In the presence of these engineered receptors, GFP could replace Dpp to organize patterning and growth in vivo. Concomitant expression of glycosylphosphatidylinositol (GPI)-anchored nonsignaling receptors further improved patterning, to near-wild-type quality. Theoretical arguments suggest that GPI anchorage could be important for these receptors to expand the gradient length scale while at the same time reducing leakage.


Assuntos
Padronização Corporal , Drosophila melanogaster/crescimento & desenvolvimento , Proteínas de Fluorescência Verde/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Animais , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Glicosilfosfatidilinositóis/metabolismo , Proteínas de Fluorescência Verde/genética , Discos Imaginais/crescimento & desenvolvimento , Engenharia de Proteínas , Proteínas Recombinantes de Fusão/genética , Asas de Animais/crescimento & desenvolvimento
10.
Science ; 370(6514): 327-331, 2020 10 16.
Artigo em Inglês | MEDLINE | ID: mdl-33060357

RESUMO

In metazoan tissues, cells decide their fates by sensing positional information provided by specialized morphogen proteins. To explore what features are sufficient for positional encoding, we asked whether arbitrary molecules (e.g., green fluorescent protein or mCherry) could be converted into synthetic morphogens. Synthetic morphogens expressed from a localized source formed a gradient when trapped by surface-anchoring proteins, and they could be sensed by synthetic receptors. Despite their simplicity, these morphogen systems yielded patterns reminiscent of those observed in vivo. Gradients could be reshaped by altering anchor density or by providing a source of competing inhibitor. Gradient interpretation could be altered by adding feedback loops or morphogen cascades to receiver cell response circuits. Orthogonal cell-cell communication systems provide insight into morphogen evolution and a platform for engineering tissues.


Assuntos
Padronização Corporal , Proteínas de Fluorescência Verde/metabolismo , Engenharia Tecidual/métodos , Animais , Drosophila melanogaster/crescimento & desenvolvimento , Fibroblastos , Proteínas de Fluorescência Verde/genética , Engenharia de Proteínas , Receptores Notch/genética , Receptores Notch/metabolismo
11.
Nature ; 585(7826): 574-578, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32939089

RESUMO

Epithelial organoids, such as those derived from stem cells of the intestine, have great potential for modelling tissue and disease biology1-4. However, the approaches that are used at present to derive these organoids in three-dimensional matrices5,6 result in stochastically developing tissues with a closed, cystic architecture that restricts lifespan and size, limits experimental manipulation and prohibits homeostasis. Here, by using tissue engineering and the intrinsic self-organization properties of cells, we induce intestinal stem cells to form tube-shaped epithelia with an accessible lumen and a similar spatial arrangement of crypt- and villus-like domains to that in vivo. When connected to an external pumping system, the mini-gut tubes are perfusable; this allows the continuous removal of dead cells to prolong tissue lifespan by several weeks, and also enables the tubes to be colonized with microorganisms for modelling host-microorganism interactions. The mini-intestines include rare, specialized cell types that are seldom found in conventional organoids. They retain key physiological hallmarks of the intestine and have a notable capacity to regenerate. Our concept for extrinsically guiding the self-organization of stem cells into functional organoids-on-a-chip is broadly applicable and will enable the attainment of more physiologically relevant organoid shapes, sizes and functions.


Assuntos
Homeostase , Intestinos/embriologia , Morfogênese , Organoides/embriologia , Tecidos Suporte , Animais , Padronização Corporal , Diferenciação Celular , Linhagem da Célula , Cryptosporidium parvum/patogenicidade , Células-Tronco Embrionárias Humanas/citologia , Células Endoteliais da Veia Umbilical Humana , Humanos , Intestinos/citologia , Intestinos/parasitologia , Intestinos/patologia , Camundongos , Modelos Biológicos , Organoides/citologia , Organoides/parasitologia , Organoides/patologia , Regeneração , Medicina Regenerativa , Células-Tronco , Técnicas de Cultura de Tecidos/métodos , Engenharia Tecidual
12.
Nat Commun ; 11(1): 4399, 2020 09 02.
Artigo em Inglês | MEDLINE | ID: mdl-32879319

RESUMO

In cnidarians, axial patterning is not restricted to embryogenesis but continues throughout a prolonged life history filled with unpredictable environmental changes. How this developmental capacity copes with fluctuations of food availability and whether it recapitulates embryonic mechanisms remain poorly understood. Here we utilize the tentacles of the sea anemone Nematostella vectensis as an experimental paradigm for developmental patterning across distinct life history stages. By analyzing over 1000 growing polyps, we find that tentacle progression is stereotyped and occurs in a feeding-dependent manner. Using a combination of genetic, cellular and molecular approaches, we demonstrate that the crosstalk between Target of Rapamycin (TOR) and Fibroblast growth factor receptor b (Fgfrb) signaling in ring muscles defines tentacle primordia in fed polyps. Interestingly, Fgfrb-dependent polarized growth is observed in polyp but not embryonic tentacle primordia. These findings show an unexpected plasticity of tentacle development, and link post-embryonic body patterning with food availability.


Assuntos
Padronização Corporal , Anêmonas-do-Mar , Animais , Padronização Corporal/genética , Padronização Corporal/fisiologia , Desenvolvimento Embrionário/efeitos dos fármacos , Comportamento Alimentar , Regulação da Expressão Gênica no Desenvolvimento , Receptores de Fatores de Crescimento de Fibroblastos/genética , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Anêmonas-do-Mar/embriologia , Anêmonas-do-Mar/genética , Anêmonas-do-Mar/crescimento & desenvolvimento , Transdução de Sinais , Sirolimo/farmacologia , Serina-Treonina Quinases TOR/metabolismo
13.
Nat Commun ; 11(1): 4477, 2020 09 08.
Artigo em Inglês | MEDLINE | ID: mdl-32901019

RESUMO

Individual cells detach from cohesive ensembles during development and can inappropriately separate in disease. Although much is known about how cells separate from epithelia, it remains unclear how cells disperse from clusters lacking apical-basal polarity, a hallmark of advanced epithelial cancers. Here, using live imaging of the developmental migration program of Drosophila primordial germ cells (PGCs), we show that cluster dispersal is accomplished by stabilizing and orienting migratory forces. PGCs utilize a G protein coupled receptor (GPCR), Tre1, to guide front-back migratory polarity radially from the cluster toward the endoderm. Posteriorly positioned myosin-dependent contractile forces pull on cell-cell contacts until cells release. Tre1 mutant cells migrate randomly with transient enrichment of the force machinery but fail to separate, indicating a temporal contractile force threshold for detachment. E-cadherin is retained on the cell surface during cell separation and augmenting cell-cell adhesion does not impede detachment. Notably, coordinated migration improves cluster dispersal efficiency by stabilizing cell-cell interfaces and facilitating symmetric pulling. We demonstrate that guidance of inherent migratory forces is sufficient to disperse cell clusters under physiological settings and present a paradigm for how such events could occur across development and disease.


Assuntos
Drosophila melanogaster/embriologia , Células Germinativas Embrionárias/fisiologia , Animais , Animais Geneticamente Modificados , Fenômenos Biomecânicos , Padronização Corporal/fisiologia , Caderinas/metabolismo , Adesão Celular/fisiologia , Movimento Celular/fisiologia , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citologia , Drosophila melanogaster/fisiologia , Células Germinativas Embrionárias/citologia , Microscopia de Fluorescência por Excitação Multifotônica , Miosina Tipo II/metabolismo , Transdução de Sinais , Análise de Célula Única , Proteínas rho de Ligação ao GTP/metabolismo
14.
Nat Commun ; 11(1): 4360, 2020 08 31.
Artigo em Inglês | MEDLINE | ID: mdl-32868762

RESUMO

The hypothalamus is a central regulator of many innate behaviors essential for survival, but the molecular mechanisms controlling hypothalamic patterning and cell fate specification are poorly understood. To identify genes that control hypothalamic development, we have used single-cell RNA sequencing (scRNA-Seq) to profile mouse hypothalamic gene expression across 12 developmental time points between embryonic day 10 and postnatal day 45. This identified genes that delineated clear developmental trajectories for all major hypothalamic cell types, and readily distinguished major regional subdivisions of the developing hypothalamus. By using our developmental dataset, we were able to rapidly annotate previously unidentified clusters from existing scRNA-Seq datasets collected during development and to identify the developmental origins of major neuronal populations of the ventromedial hypothalamus. We further show that our approach can rapidly and comprehensively characterize mutants that have altered hypothalamic patterning, identifying Nkx2.1 as a negative regulator of prethalamic identity. These data serve as a resource for further studies of hypothalamic development, physiology, and dysfunction.


Assuntos
Diferenciação Celular , Hipotálamo , Neurônios/metabolismo , Fator Nuclear 1 de Tireoide/metabolismo , Animais , Sequência de Bases , Padronização Corporal , Regulação da Expressão Gênica no Desenvolvimento , Hipotálamo/citologia , Hipotálamo/embriologia , Hipotálamo/crescimento & desenvolvimento , Hipotálamo/metabolismo , Camundongos , Mutação , Análise de Célula Única , Fator Nuclear 1 de Tireoide/genética
15.
Proc Natl Acad Sci U S A ; 117(35): 21459-21468, 2020 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-32817436

RESUMO

Animal development has traditionally been viewed as an autonomous process directed by the host genome. But, in many animals, biotic and abiotic cues, like temperature and bacterial colonizers, provide signals for multiple developmental steps. Hydra offers unique features to encode these complex interactions of developmental processes with biotic and abiotic factors, and we used it here to investigate the impact of bacterial colonizers and temperature on the pattern formation process. In Hydra, formation of the head organizer involves the canonical Wnt pathway. Treatment with alsterpaullone (ALP) results in acquiring characteristics of the head organizer in the body column. Intriguingly, germfree Hydra polyps are significantly more sensitive to ALP compared to control polyps. In addition to microbes, ß-catenin-dependent pattern formation is also affected by temperature. Gene expression analyses led to the identification of two small secreted peptides, named Eco1 and Eco2, being up-regulated in the response to both Curvibacter sp., the main bacterial colonizer of Hydra, and low temperatures. Loss-of-function experiments revealed that Eco peptides are involved in the regulation of pattern formation and have an antagonistic function to Wnt signaling in Hydra.


Assuntos
Hydra/genética , Hydra/metabolismo , beta Catenina/metabolismo , Animais , Bactérias/metabolismo , Padronização Corporal/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Interação Gene-Ambiente , Hydra/fisiologia , Peptídeos/metabolismo , Temperatura , Proteínas Wnt/metabolismo , Via de Sinalização Wnt/genética , Via de Sinalização Wnt/fisiologia
17.
Dev Genes Evol ; 230(4): 305-314, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32671457

RESUMO

Crinoids are considered as the most basal extant echinoderms. They retain aboral nervous system with a nerve center, which has been degraded in the eleutherozoan echinoderms. To investigate the evolution of patterning of the nervous systems in crinoids, we examined temporal and spatial expression patterns of three neural patterning-related homeobox genes, six3, pax6, and otx, throughout the development of a feather star Anneissia japonica. These genes were involved in the patterning of endomesodermal tissues instead of the ectodermal neural tissues in the early planktonic stages. In the stages after larval attachment, the expression of these genes was mainly observed in the podia and the oral nervous systems instead of the aboral nerve center. Our results indicate the involvement of these three genes in the formation of oral nervous system in the common ancestor of the echinoderms and suggest that the aboral nerve center is not evolutionally related to the brain of other bilaterians.


Assuntos
Equinodermos/crescimento & desenvolvimento , Proteínas do Olho/metabolismo , Proteínas de Homeodomínio/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Fatores de Transcrição Otx/metabolismo , Fator de Transcrição PAX6/metabolismo , Animais , Padronização Corporal/genética , Equinodermos/genética , Equinodermos/metabolismo , Evolução Molecular , Proteínas do Olho/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Proteínas de Homeodomínio/genética , Larva/genética , Larva/metabolismo , Proteínas do Tecido Nervoso/genética , Sistema Nervoso/crescimento & desenvolvimento , Sistema Nervoso/metabolismo , Neurônios , Fatores de Transcrição Otx/genética , Fator de Transcrição PAX6/genética
18.
PLoS Biol ; 18(7): e3000561, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32702011

RESUMO

Maternal ß-catenin activity is essential and critical for dorsal induction and its dorsal activation has been thoroughly studied. However, how the maternal ß-catenin activity is suppressed in the nondorsal cells remains poorly understood. Nanog is known to play a central role for maintenance of the pluripotency and maternal -zygotic transition (MZT). Here, we reveal a novel role of Nanog as a strong repressor of maternal ß-catenin signaling to safeguard the embryo against hyperactivation of maternal ß-catenin activity and hyperdorsalization. In zebrafish, knockdown of nanog at different levels led to either posteriorization or dorsalization, mimicking zygotic or maternal activation of Wnt/ß-catenin activities, and the maternal zygotic mutant of nanog (MZnanog) showed strong activation of maternal ß-catenin activity and hyperdorsalization. Although a constitutive activator-type Nanog (Vp16-Nanog, lacking the N terminal) perfectly rescued the MZT defects of MZnanog, it did not rescue the phenotypes resulting from ß-catenin signaling activation. Mechanistically, the N terminal of Nanog directly interacts with T-cell factor (TCF) and interferes with the binding of ß-catenin to TCF, thereby attenuating the transcriptional activity of ß-catenin. Therefore, our study establishes a novel role for Nanog in repressing maternal ß-catenin activity and demonstrates a transcriptional switch between ß-catenin/TCF and Nanog/TCF complexes, which safeguards the embryo from global activation of maternal ß-catenin activity.


Assuntos
Desenvolvimento Embrionário/genética , Proteína Homeobox Nanog/metabolismo , Transativadores/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/embriologia , Peixe-Zebra/genética , beta Catenina/metabolismo , Animais , Padronização Corporal/genética , Núcleo Celular/metabolismo , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Silenciamento de Genes , Masculino , Mutação/genética , Proteína Homeobox Nanog/química , Proteína Homeobox Nanog/genética , Ligação Proteica , Transporte Proteico , Proteínas Repressoras/metabolismo , Transcrição Genética , Via de Sinalização Wnt/genética , Proteínas de Peixe-Zebra/química , Proteínas de Peixe-Zebra/genética , Zigoto/metabolismo
19.
Nat Ecol Evol ; 4(9): 1247-1255, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32661406

RESUMO

In vertebrate embryos, Hedgehog (Hh) is expressed in some anterior basal plate domains and by notochord and floorplate cells, and ventral neural cells are patterned by the activities of Hh-regulated transcription factors. Hh signalling is antagonized by signals from the dorsal neural tube and loss of Hh leads to loss of ventral patterning as dorsal pattern expands. These mechanisms are critical for producing the neurons that implement motor responses to sensory inputs but understanding how they evolved has been hindered by lack of insight from commonly studied invertebrates where nervous system morphology and genetic mechanisms are non-conserved with vertebrates. The invertebrate chordate amphioxus, which expresses Hh in its notochord and floorplate, provides a window into the prevertebrate condition. We examined amphioxus neural development by manipulating Hh and downstream genes involved in neural pattern and cell identity. We show that Hh signalling regulates the differentiation of some neurons in amphioxus, including a subset of motor neurons. This demonstrates some conservation of mechanism between vertebrates and amphioxus. However, other aspects of neural patterning differ between the lineages. We suggest the complexity of Hh-dependent neural patterning in vertebrates evolved in a step-wise manner. Alongside other previously described regulatory changes, initial recruitment of Hh along the length of the axis occurred in an ancestor to the chordates to regulate the differentiation of a subset of neurons. This was followed, in the vertebrate lineage, by additional changes to the downstream gene regulatory network of transcription factors, giving Hh a broader role in dorsal-ventral neural patterning.


Assuntos
Padronização Corporal , Proteínas Hedgehog , Animais , Regulação da Expressão Gênica no Desenvolvimento , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Sistema Nervoso/metabolismo , Transdução de Sinais
20.
Nature ; 582(7812): 410-415, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32528178

RESUMO

The body plan of the mammalian embryo is shaped through the process of gastrulation, an early developmental event that transforms an isotropic group of cells into an ensemble of tissues that is ordered with reference to three orthogonal axes1. Although model organisms have provided much insight into this process, we know very little about gastrulation in humans, owing to the difficulty of obtaining embryos at such early stages of development and the ethical and technical restrictions that limit the feasibility of observing gastrulation ex vivo2. Here we show that human embryonic stem cells can be used to generate gastruloids-three-dimensional multicellular aggregates that differentiate to form derivatives of the three germ layers organized spatiotemporally, without additional extra-embryonic tissues. Human gastruloids undergo elongation along an anteroposterior axis, and we use spatial transcriptomics to show that they exhibit patterned gene expression. This includes a signature of somitogenesis that suggests that 72-h human gastruloids show some features of Carnegie-stage-9 embryos3. Our study represents an experimentally tractable model system to reveal and examine human-specific regulatory processes that occur during axial organization in early development.


Assuntos
Padronização Corporal , Gástrula/citologia , Células-Tronco Embrionárias Humanas/citologia , Organoides/citologia , Organoides/embriologia , Somitos/citologia , Somitos/embriologia , Padronização Corporal/genética , Gástrula/embriologia , Gástrula/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Técnicas In Vitro , Organoides/metabolismo , Transdução de Sinais , Somitos/metabolismo , Transcriptoma
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