RESUMO
Epithelial-mesenchymal transition (EMT) encompasses dynamic changes in cellular organization from epithelial to mesenchymal phenotypes, which leads to functional changes in cell migration and invasion. EMT occurs in a diverse range of physiological and pathological conditions and is driven by a conserved set of inducing signals, transcriptional regulators and downstream effectors. With over 5,700 publications indexed by Web of Science in 2019 alone, research on EMT is expanding rapidly. This growing interest warrants the need for a consensus among researchers when referring to and undertaking research on EMT. This Consensus Statement, mediated by 'the EMT International Association' (TEMTIA), is the outcome of a 2-year-long discussion among EMT researchers and aims to both clarify the nomenclature and provide definitions and guidelines for EMT research in future publications. We trust that these guidelines will help to reduce misunderstanding and misinterpretation of research data generated in various experimental models and to promote cross-disciplinary collaboration to identify and address key open questions in this research field. While recognizing the importance of maintaining diversity in experimental approaches and conceptual frameworks, we emphasize that lasting contributions of EMT research to increasing our understanding of developmental processes and combatting cancer and other diseases depend on the adoption of a unified terminology to describe EMT.
Assuntos
Pesquisa Biomédica/normas , Transição Epitelial-Mesenquimal , Animais , Movimento Celular , Plasticidade Celular , Consenso , Biologia do Desenvolvimento/normas , Humanos , Neoplasias/patologia , Terminologia como AssuntoRESUMO
Dicer substrate interfering RNAs (DsiRNAs) destroy targeted transcripts using the RNA-Induced Silencing Complex (RISC) through a process called RNA interference (RNAi). This process is ubiquitous among eukaryotes. Here we report the utility of DsiRNA in embryos of the sea urchin Lytechinus variegatus (Lv). Specific knockdowns phenocopy known morpholino and inhibitor knockdowns, and DsiRNA offers a useful alternative to morpholinos. Methods are described for the design of specific DsiRNAs that lead to destruction of targeted mRNA. DsiRNAs directed against pks1, an enzyme necessary for pigment production, show how successful DsiRNA perturbations are monitored by RNA in situ analysis and by qPCR to determine relative destruction of targeted mRNA. DsiRNA-based knockdowns phenocopy morpholino- and drug-based inhibition of nodal and lefty. Other knockdowns demonstrate that the RISC operates early in development as well as on genes that are first transcribed hours after gastrulation is completed. Thus, DsiRNAs effectively mediate destruction of targeted mRNA in the sea urchin embryo. The approach offers significant advantages over other widely used methods in the urchin in terms of cost, and ease of procurement, and offers sizeable experimental advantages in terms of ease of handling, injection, and knockdown validation.
RESUMO
Using scRNA-seq coupled with computational approaches, we studied transcriptional changes in cell states of sea urchin embryos during development to the larval stage. Eighteen closely spaced time points were taken during the first 24â h of development of Lytechinus variegatus (Lv). Developmental trajectories were constructed using Waddington-OT, a computational approach to 'stitch' together developmental time points. Skeletogenic and primordial germ cell trajectories diverged early in cleavage. Ectodermal progenitors were distinct from other lineages by the 6th cleavage, although a small percentage of ectoderm cells briefly co-expressed endoderm markers that indicated an early ecto-endoderm cell state, likely in cells originating from the equatorial region of the egg. Endomesoderm cells also originated at the 6th cleavage and this state persisted for more than two cleavages, then diverged into distinct endoderm and mesoderm fates asynchronously, with some cells retaining an intermediate specification status until gastrulation. Seventy-nine out of 80 genes (99%) examined, and included in published developmental gene regulatory networks (dGRNs), are present in the Lv-scRNA-seq dataset and are expressed in the correct lineages in which the dGRN circuits operate.
Assuntos
Genômica/métodos , Lytechinus/genética , RNA-Seq/métodos , Análise de Célula Única/métodos , Transcriptoma , Animais , Linhagem da Célula , Endoderma/citologia , Mesoderma/citologiaRESUMO
Sea urchin larvae spend weeks to months feeding on plankton prior to metamorphosis. When handled in the laboratory they are easily injured, suggesting that in the plankton they are injured with some frequency. Fortunately, larval wounds are repaired through an efficient wound response with mesenchymal pigment cells and blastocoelar cells assisting as the epithelium closes. An injury to the epithelium leads to an immediate calcium transient that rapidly spreads around the entire larva and is necessary for activating pigment cell migration toward the wound. If calcium transport is blocked, the pigment cells fail to activate and remain in place. When activated, pigment cells initiate directed migration to the wound site from distances of at least 85 âµm. Upon arrival at the wound site they participate in an innate immune response. Blastocoelar cells are recruited to the injury site as well, though the calcium transient is unnecessary for activating these cells. At the wound site, blastocoelar cells participate in several functions including remodeling the skeleton if it protrudes through the epithelium.
Assuntos
Cálcio , Ouriços-do-Mar , Animais , Epitélio , Larva , Metamorfose BiológicaRESUMO
In the sea urchin larva, most neurons lie within an ectodermal region called the ciliary band. Our understanding of the mechanisms of specification and patterning of these peripheral ciliary band neurons is incomplete. Here, we first examine the gene regulatory landscape from which this population of neural progenitors arise in the neuroectoderm. We show that ciliary band neural progenitors first appear in a bilaterally symmetric pattern on the lateral edges of chordin expression in the neuroectoderm. Later in development, these progenitors appear in a salt-and-pepper pattern in the ciliary band where they express soxC, and prox, which are markers of neural specification, and begin to express synaptotagminB, a marker of differentiated neurons. We show that the ciliary band expresses the acid sensing ion channel gene asicl, which suggests that ciliary band neurons control the larva's ability to discern touch sensitivity. Using a chemical inhibitor of MAPK signaling, we show that this signaling pathway is required for proper specification and patterning of ciliary band neurons. Using live imaging, we show that these neural progenitors undergo small distance migrations in the embryo. We then show that the normal swimming behavior of the larvae is compromised if the neurogenesis pathway is perturbed. The developmental sequence of ciliary band neurons is very similar to that of neural crest-derived sensory neurons in vertebrates and may provide insights into the evolution of sensory neurons in deuterostomes.
Assuntos
Padronização Corporal/genética , Ectoderma/crescimento & desenvolvimento , Neurogênese/genética , Neurônios/metabolismo , Ouriços-do-Mar/embriologia , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Butadienos/farmacologia , Regulação da Expressão Gênica no Desenvolvimento , Glicoproteínas/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Larva/crescimento & desenvolvimento , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Nitrilas/farmacologia , Proteína Nodal/metabolismo , Fatores de Transcrição SOXC/metabolismo , Transdução de Sinais/genética , Sinaptotagminas/metabolismoRESUMO
Many marine larvae begin feeding within a day of fertilization, thus requiring rapid development of a nervous system to coordinate feeding activities. Here, we examine the patterning and specification of early neurogenesis in sea urchin embryos. Lineage analysis indicates that neurons arise locally in three regions of the embryo. Perturbation analyses showed that when patterning is disrupted, neurogenesis in the three regions is differentially affected, indicating distinct patterning requirements for each neural domain. Six transcription factors that function during proneural specification were identified and studied in detail. Perturbations of these proneural transcription factors showed that specification occurs differently in each neural domain prior to the Delta-Notch restriction signal. Though gene regulatory network state changes beyond the proneural restriction are largely unresolved, the data here show that the three neural regions already differ from each other significantly early in specification. Future studies that define the larval nervous system in the sea urchin must therefore separately characterize the three populations of neurons that enable the larva to feed, to navigate, and to move food particles through the gut.
Assuntos
Embrião não Mamífero/metabolismo , Lytechinus/embriologia , Lytechinus/metabolismo , Neurogênese , Animais , Padronização Corporal/genética , Proteínas Morfogenéticas Ósseas/metabolismo , Linhagem da Célula/genética , Fatores de Crescimento de Fibroblastos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Redes Reguladoras de Genes , Lytechinus/genética , Modelos Biológicos , Neurogênese/genética , Proteína Nodal/metabolismo , Transdução de Sinais , Fatores de Transcrição/metabolismoRESUMO
Correct patterning of the nervous system is essential for an organism's survival and complex behavior. Embryologists have used the sea urchin as a model for decades, but our understanding of sea urchin nervous system patterning is incomplete. Previous histochemical studies identified multiple neurotransmitters in the pluteus larvae of several sea urchin species. However, little is known about how, where and when neural subtypes are differentially specified during development. Here, we examine the molecular mechanisms of neuronal subtype specification in 3 distinct neural subtypes in the Lytechinus variegatus larva. We show that these subtypes are specified through Delta/Notch signaling and identify a different transcription factor required for the development of each neural subtype. Our results show achaete-scute and neurogenin are proneural for the serotonergic neurons of the apical organ and cholinergic neurons of the ciliary band, respectively. We also show that orthopedia is not proneural but is necessary for the differentiation of the cholinergic/catecholaminergic postoral neurons. Interestingly, these transcription factors are used similarly during vertebrate neurogenesis. We believe this study is a starting point for building a neural gene regulatory network in the sea urchin and for finding conserved deuterostome neurogenic mechanisms.
Assuntos
Ectoderma/citologia , Regulação da Expressão Gênica no Desenvolvimento , Redes Reguladoras de Genes/genética , Lytechinus/embriologia , Proteínas do Tecido Nervoso/fisiologia , Neurogênese/fisiologia , Neurônios/citologia , Fatores de Transcrição/fisiologia , Região do Genoma do Complexo Achaete-Scute/fisiologia , Animais , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Lytechinus/citologia , Proteínas de Membrana/fisiologia , Morfolinos/farmacologia , Neurônios/classificação , RNA Antissenso/farmacologia , Receptores Notch/fisiologiaRESUMO
The ecologically significant shift in developmental strategy from planktotrophic (feeding) to lecithotrophic (nonfeeding) development in the sea urchin genus Heliocidaris is one of the most comprehensively studied life history transitions in any animal. Although the evolution of lecithotrophy involved substantial changes to larval development and morphology, it is not known to what extent changes in gene expression underlie the developmental differences between species, nor do we understand how these changes evolved within the context of the well-defined gene regulatory network (GRN) underlying sea urchin development. To address these questions, we used RNA-seq to measure expression dynamics across development in three species: the lecithotroph Heliocidaris erythrogramma, the closely related planktotroph H. tuberculata, and an outgroup planktotroph Lytechinus variegatus. Using well-established statistical methods, we developed a novel framework for identifying, quantifying, and polarizing evolutionary changes in gene expression profiles across the transcriptome and within the GRN. We found that major changes in gene expression profiles were more numerous during the evolution of lecithotrophy than during the persistence of planktotrophy, and that genes with derived expression profiles in the lecithotroph displayed specific characteristics as a group that are consistent with the dramatically altered developmental program in this species. Compared to the transcriptome, changes in gene expression profiles within the GRN were even more pronounced in the lecithotroph. We found evidence for conservation and likely divergence of particular GRN regulatory interactions in the lecithotroph, as well as significant changes in the expression of genes with known roles in larval skeletogenesis. We further use coexpression analysis to identify genes of unknown function that may contribute to both conserved and derived developmental traits between species. Collectively, our results indicate that distinct evolutionary processes operate on gene expression during periods of life history conservation and periods of life history divergence, and that this contrast is even more pronounced within the GRN than across the transcriptome as a whole.
Assuntos
Redes Reguladoras de Genes , Ouriços-do-Mar/crescimento & desenvolvimento , Animais , Linhagem da Célula , Evolução Molecular , Comportamento Alimentar , Trato Gastrointestinal/crescimento & desenvolvimento , Perfilação da Expressão Gênica , Larva/crescimento & desenvolvimento , Sistema Nervoso/crescimento & desenvolvimento , Filogenia , Ouriços-do-Mar/genética , Ouriços-do-Mar/metabolismo , Seleção Genética , TranscriptomaRESUMO
Most bilaterians exhibit a left-right asymmetric distribution of their internal organs. The sea urchin larva is notable in this regard since most adult structures are generated from left sided embryonic structures. The gene regulatory network governing this larval asymmetry is still a work in progress but involves several conserved signaling pathways including Nodal, and BMP. Here we provide a comprehensive analysis of Hedgehog signaling and it's contribution to left-right asymmetry. We report that Hh signaling plays a conserved role to regulate late asymmetric expression of Nodal and that this regulation occurs after Nodal breaks left-right symmetry in the mesoderm. Thus, while Hh functions to maintain late Nodal expression, the molecular asymmetry of the future coelomic pouches is locked in. Furthermore we report that cilia play a role only insofar as to transduce Hh signaling and do not have an independent effect on the asymmetry of the mesoderm. From this, we are able to construct a more complete regulatory network governing the establishment of left-right asymmetry in the sea urchin.
Assuntos
Proteínas Hedgehog/fisiologia , Ouriços-do-Mar/embriologia , Ouriços-do-Mar/fisiologia , Animais , Padronização Corporal , Cílios/fisiologia , Embrião não Mamífero/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Hibridização In Situ , Cinesinas/química , Mesoderma/metabolismo , Proteína Nodal/fisiologia , Oligonucleotídeos Antissenso/genética , Reação em Cadeia da Polimerase , Transdução de Sinais , Fator de Crescimento Transformador beta/metabolismoRESUMO
Epithelial-mesenchymal transition (EMT) is a fundamental cell state change that transforms epithelial to mesenchymal cells during embryonic development, adult tissue repair and cancer metastasis. EMT includes a complex series of intermediate cell state changes including remodeling of the basement membrane, apical constriction, epithelial de-adhesion, directed motility, loss of apical-basal polarity, and acquisition of mesenchymal adhesion and polarity. Transcriptional regulatory state changes must ultimately coordinate the timing and execution of these cell biological processes. A well-characterized gene regulatory network (GRN) in the sea urchin embryo was used to identify the transcription factors that control five distinct cell changes during EMT. Single transcription factors were perturbed and the consequences followed with in vivo time-lapse imaging or immunostaining assays. The data show that five different sub-circuits of the GRN control five distinct cell biological activities, each part of the complex EMT process. Thirteen transcription factors (TFs) expressed specifically in pre-EMT cells were required for EMT. Three TFs highest in the GRN specified and activated EMT (alx1, ets1, tbr) and the 10 TFs downstream of those (tel, erg, hex, tgif, snail, twist, foxn2/3, dri, foxb, foxo) were also required for EMT. No single TF functioned in all five sub-circuits, indicating that there is no EMT master regulator. Instead, the resulting sub-circuit topologies suggest EMT requires multiple simultaneous regulatory mechanisms: forward cascades, parallel inputs and positive-feedback lock downs. The interconnected and overlapping nature of the sub-circuits provides one explanation for the seamless orchestration by the embryo of cell state changes leading to successful EMT.
Assuntos
Transição Epitelial-Mesenquimal/genética , Redes Reguladoras de Genes/fisiologia , Lytechinus/embriologia , Animais , Padronização Corporal/genética , Adesão Celular/genética , Movimento Celular/genética , Polaridade Celular/genética , Embrião não Mamífero , Lytechinus/genética , Fatores de Transcrição da Família Snail , Fatores de Transcrição/fisiologia , Proteína 1 Relacionada a Twist/fisiologiaRESUMO
The border between the posterior ectoderm and the endoderm is a location where two germ layers meet and establish an enduring relationship that also later serves, in deuterostomes, as the anatomical site of the anus. In the sea urchin, a prototypic deuterostome, the ectoderm-endoderm boundary is established before gastrulation, and ectodermal cells at the boundary are thought to provide patterning inputs to the underlying mesenchyme. Here we show that a short-range Wnt5 signal from the endoderm actively patterns the adjacent boundary ectoderm. This signal activates a unique subcircuit of the ectoderm gene regulatory network, including the transcription factors IrxA, Nk1, Pax2/5/8 and Lim1, which are ultimately restricted to subregions of the border ectoderm (BE). Surprisingly, Nodal and BMP2/4, previously shown to be activators of ectodermal specification and the secondary embryonic axis, instead restrict the expression of these genes to subregions of the BE. A detailed examination showed that endodermal Wnt5 functions as a short-range signal that activates only a narrow band of ectodermal cells, even though all ectoderm is competent to receive the signal. Thus, cells in the BE integrate positive and negative signals from both the primary and secondary embryonic axes to correctly locate and specify the border ectoderm.
Assuntos
Padronização Corporal/fisiologia , Ectoderma/metabolismo , Endoderma/metabolismo , Ouriços-do-Mar/embriologia , Proteínas Wnt/metabolismo , Animais , Padronização Corporal/genética , Ectoderma/embriologia , Embrião não Mamífero/metabolismo , Endoderma/embriologia , Gastrulação , Regulação da Expressão Gênica no Desenvolvimento , Proteínas com Homeodomínio LIM/genética , Proteínas com Homeodomínio LIM/metabolismo , Ouriços-do-Mar/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Via de Sinalização WntRESUMO
In many embryos specification toward one cell fate can be diverted to a different cell fate through a reprogramming process. Understanding how that process works will reveal insights into the developmental regulatory logic that emerged from evolution. In the sea urchin embryo, cells at gastrulation were found to reprogram and replace missing cell types after surgical dissections of the embryo. Non-skeletogenic mesoderm (NSM) cells reprogrammed to replace missing skeletogenic mesoderm cells and animal caps reprogrammed to replace all endomesoderm. In both cases evidence of reprogramming onset was first observed at the early gastrula stage, even if the cells to be replaced were removed earlier in development. Once started however, the reprogramming occurred with compressed gene expression dynamics. The NSM did not require early contact with the skeletogenic cells to reprogram, but the animal cap cells gained the ability to reprogram early in gastrulation only after extended contact with the vegetal halves prior to that time. If the entire vegetal half was removed at early gastrula, the animal caps reprogrammed and replaced the vegetal half endomesoderm. If the animal caps carried morpholinos to either hox11/13b or foxA (endomesoderm specification genes), the isolated animal caps failed to reprogram. Together these data reveal that the emergence of a reprogramming capability occurs at early gastrulation in the sea urchin embryo and requires activation of early specification components of the target tissues.
Assuntos
Desenvolvimento Ósseo/fisiologia , Reprogramação Celular , Gastrulação/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Lytechinus/embriologia , Animais , Osso e Ossos/embriologia , Diferenciação Celular , Embrião não Mamífero/citologia , Embrião não Mamífero/embriologia , Gástrula , Mesoderma/citologia , Mesoderma/embriologia , Transdução de SinaisRESUMO
A relatively small number of signaling pathways govern the early patterning processes of metazoan development. The architectural changes over time to these signaling pathways offer unique insights into their evolution. In the case of Hedgehog (Hh) signaling, two very divergent mechanisms of pathway transduction have evolved. In vertebrates, signaling relies on trafficking of Hh pathway components to nonmotile specialized primary cilia. In contrast, protostomes do not use cilia of any kind for Hh signal transduction. How these divergent lineages adapted such dramatically different ways of activating the signaling pathway is an unanswered question. Here, we present evidence that in the sea urchin, a basal deuterostome, motile cilia are required for embryonic Hh signal transduction, and the Hh receptor Smoothened (Smo) localizes to cilia during active Hh signaling. This is the first evidence that Hh signaling requires motile cilia and the first case of an organism requiring cilia outside of the vertebrate lineage.
Assuntos
Cílios/metabolismo , Proteínas Hedgehog/metabolismo , Ouriços-do-Mar/genética , Transdução de Sinais , Animais , Evolução Molecular , Proteínas Hedgehog/genética , Microscopia Eletrônica de Varredura , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismoRESUMO
In sea urchins, the nuclear accumulation of ß-catenin in micromeres and macromeres at 4th and 5th cleavage activates the developmental gene regulatory circuits that specify all of the vegetal tissues (i.e. skeletogenic mesoderm, endoderm and non-skeletogenic mesoderm). Here, through the analysis of maternal Frizzled receptors as potential contributors to these processes, we found that, in Paracentrotus lividus, the receptor Frizzled1/2/7 is required by 5th cleavage for ß-catenin nuclearisation selectively in macromere daughter cells. Perturbation analyses established further that Frizzled1/2/7 signaling is required subsequently for the specification of the endomesoderm and then the endoderm but not for that of the non-skeletogenic mesoderm, even though this cell type also originates from the endomesoderm lineage. Complementary analyses on Wnt6 showed that this maternal ligand is similarly required at 5th cleavage for the nuclear accumulation of ß-catenin exclusively in the macromeres and for endoderm but not for non-skeletogenic mesoderm specification. In addition, Wnt6 misexpression reverses Frizzled1/2/7 downregulation-induced phenotypes. Thus, the results indicate that Wnt6 and Frizzled1/2/7 are likely to behave as the ligand-receptor pair responsible for initiating ß-catenin nuclearisation in macromeres at 5th cleavage and that event is necessary for endoderm specification. They show also that ß-catenin nuclearisation in micromeres and macromeres takes place through a different mechanism, and that non-skeletogenic mesoderm specification occurs independently of the nuclear accumulation of ß-catenin in macromeres at the 5th cleavage. Evolutionarily, this analysis outlines further the conserved involvement of the Frizzled1/2/7 subfamily, but not of specific Wnts, in the activation of canonical Wnt signaling during early animal development.
Assuntos
Desenvolvimento Embrionário/fisiologia , Endoderma/fisiologia , Receptores Frizzled/metabolismo , Paracentrotus/citologia , Paracentrotus/embriologia , Transdução de Sinais/fisiologia , beta Catenina/metabolismo , Animais , Embrião não Mamífero/citologia , Embrião não Mamífero/fisiologia , Endoderma/citologia , Receptores Frizzled/genética , Regulação da Expressão Gênica no Desenvolvimento , Paracentrotus/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Células-Tronco/citologia , Células-Tronco/fisiologia , beta Catenina/genéticaRESUMO
Bilateral animals, including humans and most metazoans, are not perfectly symmetrical. Some internal structures are distributed asymmetrically to the right or left side. A conserved Nodal and BMP signaling system directs molecular pathways that impart the sidedness to those asymmetric structures. In the sea urchin embryo, one such asymmetrical structure, oddly enough, is the entire adult, which grows out of left sided structures produced in the larva. In a paper just published in PLOS Biology, BMP signaling is shown to be necessary early in larval development to initiate the asymmetric specification of one of those left-sided structures, called the left coelomic pouch. This study reports that BMP signaling activates a group of transcription factors asymmetrically in the left coelomic pouch only, which launch the pathway that eventually leads to the formation of the adult that emerges from the larva at metamorphosis.
Assuntos
Padronização Corporal/fisiologia , Proteínas Morfogenéticas Ósseas/metabolismo , Embrião não Mamífero/metabolismo , Ouriços-do-Mar/embriologia , Animais , Ouriços-do-Mar/metabolismoRESUMO
It is a challenge to understand how the information encoded in DNA is used to build a three-dimensional structure. To explore how this works the assembly of a relatively simple skeleton has been examined at multiple control levels. The skeleton of the sea urchin embryo consists of a number of calcite rods produced by 64 skeletogenic cells. The ectoderm supplies spatial cues for patterning, essentially telling the skeletogenic cells where to position themselves and providing the factors for skeletal growth. Here, we describe the information known about how this works. First the ectoderm must be patterned so that the signaling cues are released from precise positions. The skeletogenic cells respond by initiating skeletogenesis immediately beneath two regions (one on the right and the other on the left side). Growth of the skeletal rods requires additional signaling from defined ectodermal locations, and the skeletogenic cells respond to produce a membrane-bound template in which the calcite crystal grows. Important in this process are three signals, fibroblast growth factor, vascular endothelial growth factor, and Wnt5. Each is necessary for explicit tasks in skeleton production.
Assuntos
Evolução Biológica , Padronização Corporal/fisiologia , Modelos Biológicos , Ouriços-do-Mar/anatomia & histologia , Ouriços-do-Mar/embriologia , Transdução de Sinais/fisiologia , Animais , Carbonato de Cálcio/metabolismo , Movimento Celular/fisiologia , Ectoderma/metabolismo , Fatores de Crescimento de Fibroblastos/metabolismo , Larva/anatomia & histologia , Fator A de Crescimento do Endotélio Vascular/metabolismo , Proteínas Wnt/metabolismoRESUMO
Embryos of the echinoderms, especially those of sea urchins and sea stars, have been studied as model organisms for over 100 years. The simplicity of their early development, and the ease of experimentally perturbing this development, provides an excellent platform for mechanistic studies of cell specification and morphogenesis. As a result, echinoderms have contributed significantly to our understanding of many developmental mechanisms, including those that govern the structure and design of gene regulatory networks, those that direct cell lineage specification, and those that regulate the dynamic morphogenetic events that shape the early embryo.
Assuntos
Evolução Biológica , Biologia do Desenvolvimento/métodos , Animais , Redes Reguladoras de Genes , Ouriços-do-Mar/genética , Ouriços-do-Mar/crescimento & desenvolvimentoRESUMO
Early development requires well-organized temporal and spatial regulation of transcription factors that are assembled into gene regulatory networks (GRNs). In the sea urchin, an endomesoderm GRN model explains much of the specification in the endoderm and mesoderm prior to gastrulation, yet some GRN connections remain incomplete. Here, we characterize FoxN2/3 in the primary mesenchyme cell (PMC) GRN state. Expression of foxN2/3 mRNA begins in micromeres at the hatched blastula stage and then is lost from micromeres at the mesenchyme blastula stage. foxN2/3 expression then shifts to the non-skeletogenic mesoderm and, later, to the endoderm. Here, we show that Pmar1, Ets1 and Tbr are necessary for activation of foxN2/3 in micromeres. The later endomesoderm expression of foxN2/3 is independent of the earlier expression of foxN2/3 in micromeres and is independent of signals from PMCs. FoxN2/3 is necessary for several steps in the formation of the larval skeleton. Early expression of genes for the skeletal matrix is dependent on FoxN2/3, but only until the mesenchyme blastula stage as foxN2/3 mRNA disappears from PMCs at that time and we assume that the protein is not abnormally long-lived. Knockdown of FoxN2/3 inhibits normal PMC ingression and foxN2/3 morphant PMCs do not organize in the blastocoel and fail to join the PMC syncytium. In addition, without FoxN2/3, the PMCs fail to repress the transfating of other mesodermal cells into the skeletogenic lineage. Thus, FoxN2/3 is necessary for normal ingression, for expression of several skeletal matrix genes, for preventing transfating and for fusion of the PMC syncytium.
Assuntos
Calcificação Fisiológica/genética , Redes Reguladoras de Genes/fisiologia , Fatores de Transcrição/genética , Animais , Blástula , Embrião não Mamífero , Endoderma , Matriz Extracelular/genética , Células Gigantes , Mesoderma , RNA Mensageiro/análise , Ouriços-do-MarRESUMO
In the sea urchin, entry of ß-catenin into the nuclei of the vegetal cells at 4th and 5th cleavages is necessary for activation of the endomesoderm gene regulatory network. Beyond that, little is known about how the embryo uses maternal information to initiate specification. Here, experiments establish that of the three maternal Wnts in the egg, Wnt6 is necessary for activation of endodermal genes in the endomesoderm GRN. A small region of the vegetal cortex is shown to be necessary for activation of the endomesoderm GRN. If that cortical region of the egg is removed, addition of Wnt6 rescues endoderm. At a molecular level, the vegetal cortex region contains a localized concentration of Dishevelled (Dsh) protein, a transducer of the canonical Wnt pathway; however, Wnt6 mRNA is not similarly localized. Ectopic activation of the Wnt pathway, through the expression of an activated form of ß-catenin, of a dominant-negative variant of GSK-3ß or of Dsh itself, rescues endomesoderm specification in eggs depleted of the vegetal cortex. Knockdown experiments in whole embryos show that absence of Wnt6 produces embryos that lack endoderm, but those embryos continue to express a number of mesoderm markers. Thus, maternal Wnt6 plus a localized vegetal cortical molecule, possibly Dsh, is necessary for endoderm specification; this has been verified in two species of sea urchin. The data also show that Wnt6 is only one of what are likely to be multiple components that are necessary for activation of the entire endomesoderm gene regulatory network.