RESUMEN
An important goal of many studies in molecular ecology is to utilize molecular tools to elucidate how critical traits like metabolism and growth are affected by environmental stressors and how organisms offset these stresses by adaptive molecular-level responses. Stress from food deprivation may be critical for early developmental stages that require a continued supply of substrates for energy metabolism and growth if development is to be completed. In a 'From the Cover' article in this issue of Molecular Ecology, Li et al. (2023) examined the effects of withholding food (unicellular algae) on 10 traits of larvae of the purple sea urchin (Strongylocentrotus purpuratus), ranging from the molecular level (gene expression) to morphology. Overall, this study sheds new light on the plasticity of larval development and the tight linkages that exist among traits as they respond to changes in food availability. Importantly, shifts in the sources of food utilized under different dietary treatments show the plasticity of these larvae to alter reliance on endogenous energy stores and dissolved organic matter (DOM) as algae deprivation continues. The effects of global change on the amounts and phenology of productivity in the seas make this type of integrated, multi-level analysis an important tool for predicting the future states of marine ecosystems.
Asunto(s)
Larva , Animales , Larva/crecimiento & desarrollo , Erizos de Mar/crecimiento & desarrollo , Abastecimiento de Alimentos , Strongylocentrotus purpuratus/genética , Strongylocentrotus purpuratus/crecimiento & desarrolloRESUMEN
Identifying the molecular fingerprint of organismal cell types is key for understanding their function and evolution. Here, we use single-cell RNA sequencing (scRNA-seq) to survey the cell types of the sea urchin early pluteus larva, representing an important developmental transition from non-feeding to feeding larva. We identify 21 distinct cell clusters, representing cells of the digestive, skeletal, immune, and nervous systems. Further subclustering of these reveal a highly detailed portrait of cell diversity across the larva, including the identification of neuronal cell types. We then validate important gene regulatory networks driving sea urchin development and reveal new domains of activity within the larval body. Focusing on neurons that co-express Pdx-1 and Brn1/2/4, we identify an unprecedented number of genes shared by this population of neurons in sea urchin and vertebrate endocrine pancreatic cells. Using differential expression results from Pdx-1 knockdown experiments, we show that Pdx1 is necessary for the acquisition of the neuronal identity of these cells. We hypothesize that a network similar to the one orchestrated by Pdx1 in the sea urchin neurons was active in an ancestral cell type and then inherited by neuronal and pancreatic developmental lineages in sea urchins and vertebrates.
Asunto(s)
Diferenciación Celular/genética , Sistema Nervioso/crecimiento & desarrollo , Strongylocentrotus purpuratus/crecimiento & desarrollo , Animales , Larva/genética , Larva/crecimiento & desarrollo , Fenómenos Fisiológicos del Sistema Nervioso , RNA-Seq , Análisis de la Célula Individual , Strongylocentrotus purpuratus/genéticaRESUMEN
Dissolved organic carbon (DOC) is known to ameliorate the toxicity of the trace metal nickel (Ni) to aquatic animals. In theory, this effect is mediated by the capacity of DOC to bind Ni, rendering it less bioavailable, with the resulting reduction in accumulation limiting toxicological effects. However, there is a lack of experimental data examining Ni accumulation in marine settings with natural sources of DOC. In the current study, radiolabelled Ni was used to examine the time- and concentration-dependence of Ni accumulation, using naturally sourced DOC, on developing larvae of the sea urchin Strongylocentrotus purpuratus. Contrary to prediction, the two tested natural DOC samples (collected from the eastern United States, DOC 2 (Seaview park, Rhode Island (SVP)) and DOC 7 (Aubudon Coastal Center, Connecticut)) which had previously been shown to protect against Ni toxicity, did not limit accumulation. The control (artificial seawater with no added DOC), and the DOC 2 sample could mostly be described as having saturable Ni uptake, whereas Ni uptake in the presence of DOC 7 was mostly linear. These data provide evidence that DOC modifies the bioavailability of Ni, through either indirect effects (e.g. membrane permeability) or by the absorption of DOC-Ni complexes. There was some evidence for regulation of Ni accumulation in later-stage embryos (96-h) where the bioconcentration factor for Ni declined with increasing Ni exposure concentration. These data have implications for predictive modelling approaches that rely on known relationships between Ni speciation, bioavailability and bioreactivity, by suggesting that these relationships may not hold for natural marine DOC samples in the developing sea urchin model system.
Asunto(s)
Materia Orgánica Disuelta/farmacología , Níquel/farmacocinética , Strongylocentrotus purpuratus/efectos de los fármacos , Animales , Larva , Strongylocentrotus purpuratus/crecimiento & desarrollo , Strongylocentrotus purpuratus/metabolismo , Contaminantes Químicos del Agua/farmacologíaRESUMEN
Metal accumulation, disturbance of Ca2+ homeostasis, and occurrence of abnormalities are well-established consequences of single metal exposure during early development stages of sea urchins. However, the effects caused by low concentrations of metals and metal mixtures need to be better understood in marine invertebrates. Therefore, the present study investigated the effects of environmentally relevant concentrations of Zn (9 µg/L), Cd (30 µg/L) and Ni (5 µg/L) in single and binary exposures (Zn + Cd, Cd + Ni and Ni + Zn) to the early life stages of the purple sea urchin Strongylocentrotus purpuratus. Endpoints checked in all treatments after 48-h exposure were unidirectional metal influx rates, bioaccumulation, and Ca2+ influx rates. Additionally, the presence of abnormal larvae and developmental delay was evaluated at 24 h, 48 h and 72 h of exposure. Unidirectional influx rates of all three metals were significantly higher than control background rates in all single exposures and binary mixtures, and were generally not different between them. Net accumulation (body burden) of both Zn and Cd increased significantly as a result of their respective single exposures, while Ni accumulation decreased considerably. When Zn or Cd were presented in binary exposures with other metals, the net accumulations of Zn or Cd were reduced relative to single exposures to these metals, whereas this did not occur for Ni accumulation. Thus, bioaccumulation proved to be a better metric than influx rate measurements to analyze metal competition at a whole organism level at these low metal concentrations. Short-term Ca2+ influx also did not appear to be a sensitive metric, as the measured rates did not vary among all single and binary exposures, with the exception of a lower rate in Ni + Zn binary exposure. A critical aspect observed was the relationship between bioaccumulation versus influx measurements, which proved positive for Cd, but negative for Zn and Ni, demonstrating possible capacities for both Zn and Ni regulation by sea urchin larvae. Increases in larval abnormalities relative to controls occurred only after binary mixtures, starting at 48 h exposure and maintained until 72 h. However, delay of the sea urchin development by the presence of gastrula stage at 72 h exposure occurred in Zn and Ni single exposures and all metal mixtures, with very high abnormal development when Ni was present.
Asunto(s)
Bioacumulación/efectos de los fármacos , Cadmio/toxicidad , Larva/efectos de los fármacos , Níquel/toxicidad , Strongylocentrotus purpuratus/efectos de los fármacos , Contaminantes Químicos del Agua/toxicidad , Zinc/toxicidad , Animales , Transporte Biológico , Cadmio/metabolismo , Relación Dosis-Respuesta a Droga , Larva/crecimiento & desarrollo , Larva/metabolismo , Níquel/metabolismo , Agua de Mar/química , Strongylocentrotus purpuratus/crecimiento & desarrollo , Strongylocentrotus purpuratus/metabolismo , Contaminantes Químicos del Agua/metabolismo , Zinc/metabolismoRESUMEN
Environmental variation experienced by a species across space and time can promote the maintenance of genetic diversity that may be adaptive in future global change conditions. Selection experiments have shown that purple sea urchin, Strongylocentrotus purpuratus, populations have adaptive genetic variation for surviving pH conditions at the "edge" (pH 7.5) of conditions experienced in nature. However, little is known about whether populations have genetic variation for surviving low-pH events beyond those currently experienced in nature or how variation in pH conditions affects organismal and genetic responses. Here, we quantified survival, growth, and allele frequency shifts in experimentally selected developing purple sea urchin larvae in static and variable conditions at three pH levels: pH 8.1 (control), pH 7.5 (edge-of-range), and pH 7.0 (extreme). Variable treatments recovered body size relative to static treatments, but resulted in higher mortality, suggesting a potential tradeoff between survival and growth under pH stress. However, within each pH level, allele frequency changes were overlapping between static and variable conditions, suggesting a shared genetic basis underlying survival to mean pH regardless of variability. In contrast, genetic responses to pH 7.5 (edge) versus pH 7.0 (extreme) conditions were distinct, indicating a unique genetic basis of survival. In addition, loci under selection were more likely to be in exonic regions than regulatory, indicating that selection targeted protein-coding variation. Loci under selection in variable pH 7.5 conditions, more similar to conditions periodically experienced in nature, performed functions related to lipid biosynthesis and metabolism, while loci under selection in static pH 7.0 conditions performed functions related to transmembrane and mitochondrial processes. While these results are promising in that purple sea urchin populations possess genetic variation for surviving extreme pH conditions not currently experienced in nature, they caution that increased acidification does not result in a linear response but elicits unique physiological stresses and survival mechanisms.
Asunto(s)
Genoma/fisiología , Fenotipo , Agua de Mar/química , Strongylocentrotus purpuratus/fisiología , Animales , Tamaño Corporal , Dióxido de Carbono/análisis , Cambio Climático , Concentración de Iones de Hidrógeno , Larva/genética , Larva/crecimiento & desarrollo , Larva/fisiología , Strongylocentrotus purpuratus/genética , Strongylocentrotus purpuratus/crecimiento & desarrolloRESUMEN
Oxidative stress parameters were evaluated during the first 72â¯h of embryonic development of purple sea urchin Strongylocentrotus purpuratus continuously exposed to control conditions, to cadmium alone (Cd, 30⯵g/L), to zinc alone (Zn, 9⯵g/L) or to a Cd (28⯵g/L) plus Zn (9⯵g/L) mixture. These sublethal concentrations represent â¼ 10% of the acute EC50. Bioaccumulation, antioxidant capacity against peroxyl radicals (ACAP), total glutathione (GSH) level, glutathione-S-transferase (GST), glucose-6-phosphate dehydrogenase (G6PDH) and superoxide dismutase (SOD) activity, and lipid peroxidation (LPO) were analyzed at 24â¯h (blastula), 48â¯h (gastrula), and 72â¯h (pluteus) stages of development. Zinc (an essential metal) was well-regulated, whereas Cd (non-essential) bioaccumulated and whole-body [Cd] increased from blastula to pluteus stage in sea urchin larvae. In controls, ACAP progressively declined from 24â¯h to 72â¯h, while LPO reciprocally increased, but other parameters did not change. Cd alone was more potent than Zn alone as a pro-oxidant, with the major effects being decreases in SOD activity and parallel increases in LPO throughout development; GST activity also increased at 24â¯h. Zn alone caused only biphasic disturbances of ACAP. In all cases, the simultaneous presence of the other metal prevented the effects, and there was no instance where the oxidative stress response in the presence of the Cd/Zn mixture was greater than in the presence of either Cd or Zn alone. Therefore the sublethal effects of joint exposures were always less than additive or even protective, in agreement with classical toxicity data. Furthermore, our results indicate that SOD and Zn can play important roles in protecting sea urchin embryos against Cd-induced lipid peroxidation.
Asunto(s)
Antioxidantes/metabolismo , Cadmio/toxicidad , Estrés Oxidativo/efectos de los fármacos , Strongylocentrotus purpuratus/efectos de los fármacos , Contaminantes Químicos del Agua/toxicidad , Zinc/toxicidad , Animales , Cadmio/análisis , Glutatión/metabolismo , Glutatión Transferasa/metabolismo , Peroxidación de Lípido/efectos de los fármacos , Strongylocentrotus purpuratus/crecimiento & desarrollo , Strongylocentrotus purpuratus/metabolismo , Contaminantes Químicos del Agua/análisis , Zinc/análisisRESUMEN
Standing genetic variation is important for population persistence in extreme environmental conditions. While some species may have the capacity to adapt to predicted average future global change conditions, the ability to survive extreme events is largely unknown. We used single-generation selection experiments on hundreds of thousands of Strongylocentrotus purpuratus sea urchin larvae generated from wild-caught adults to identify adaptive genetic variation responsive to moderate (pH 8.0) and extreme (pH 7.5) low-pH conditions. Sequencing genomic DNA from pools of larvae, we identified consistent changes in allele frequencies across replicate cultures for each pH condition and observed increased linkage disequilibrium around selected loci, revealing selection on recombined standing genetic variation. We found that loci responding uniquely to either selection regime were at low starting allele frequencies while variants that responded to both pH conditions (11.6% of selected variants) started at high frequencies. Loci under selection performed functions related to energetics, pH tolerance, cell growth and actin/cytoskeleton dynamics. These results highlight that persistence in future conditions will require two classes of genetic variation: common, pH-responsive variants maintained by balancing selection in a heterogeneous environment, and rare variants, particularly for extreme conditions, that must be maintained by large population sizes.
Asunto(s)
Cambio Climático , Variación Genética , Strongylocentrotus purpuratus/genética , Animales , Conservación de los Recursos Naturales , Frecuencia de los Genes , Concentración de Iones de Hidrógeno , Larva/genética , Larva/crecimiento & desarrollo , Larva/fisiología , Desequilibrio de Ligamiento , Polimorfismo de Nucleótido Simple , Selección Genética , Strongylocentrotus purpuratus/crecimiento & desarrollo , Strongylocentrotus purpuratus/fisiologíaRESUMEN
Yin-Yang 1 (YY1) is a highly conserved transcription factor possessing RNA-binding activity. A putative YY1 homologue was previously identified in the developmental model organism Strongylocentrotus purpuratus (the purple sea urchin) by genomic sequencing. We identified a high degree of sequence similarity with YY1 homologues of vertebrate origin which shared 100% protein sequence identity over the DNA- and RNA-binding zinc-finger region with high similarity in the N-terminal transcriptional activation domain. SpYY1 demonstrated identical DNA- and RNA-binding characteristics between Xenopus laevis and S. purpuratus indicating that it maintains similar functional and biochemical properties across widely divergent deuterostome species. SpYY1 binds to the consensus YY1 DNA element, and also to U-rich RNA sequences. Although we detected SpYY1 RNA-binding activity in ova lysates and observed cytoplasmic localization, SpYY1 was not associated with maternal mRNA in ova. SpYY1 expressed in Xenopus oocytes was excluded from the nucleus and associated with maternally expressed cytoplasmic mRNA molecules. These data demonstrate the existence of an YY1 homologue in S. purpuratus with similar structural and biochemical features to those of the well-studied vertebrate YY1; however, the data reveal major differences in the biological role of YY1 in the regulation of maternally expressed mRNA in the two species.
Asunto(s)
Oocitos/metabolismo , Óvulo/metabolismo , ARN Mensajero Almacenado/metabolismo , ARN/metabolismo , Strongylocentrotus purpuratus/metabolismo , Xenopus laevis/metabolismo , Factor de Transcripción YY1/metabolismo , Secuencia de Aminoácidos , Animales , Femenino , Regulación del Desarrollo de la Expresión Génica , Oocitos/citología , Filogenia , ARN/genética , ARN Mensajero Almacenado/genética , Homología de Secuencia , Strongylocentrotus purpuratus/genética , Strongylocentrotus purpuratus/crecimiento & desarrollo , Xenopus laevis/genética , Xenopus laevis/crecimiento & desarrolloRESUMEN
Settlement is a rapid process in many marine invertebrate species, transitioning a planktonic larva into a benthic juvenile. In indirectly developing sea urchins, this ecological transition correlates with a morphological, developmental and physiological transition (metamorphosis) during which apoptosis is essential for the resorption and remodelling of larval and juvenile structures. While settlement is initiated by environmental cues (i.e. habitat-specific or benthic substrate cues), metamorphosis is regulated by developmental endocrine signals, such as histamine (HA), thyroid hormones (THs) and nitric oxide (NO). In the purple sea urchin, Strongylocentrotus purpuratus, we found that suH1R mRNA levels increase during larval development and peak during metamorphic competence. SuH1R positive cell clusters are prominently visible in the mouth region of sea urchin larvae, but the protein appears to be expressed at low levels throughout the larval arms and epidermis. SuH1R knock-down experiments in larval stages show that the function of suH1R is in inhibiting apoptosis. Our results therefore suggest that suH1R is regulating the metamorphic transition by inhibiting apoptosis. These results provide new insights into metamorphic mechanisms and have implications for our understanding of settlement and metamorphosis in the marine environment.
Asunto(s)
Apoptosis/fisiología , Larva/citología , Receptores Histamínicos/fisiología , Strongylocentrotus purpuratus/crecimiento & desarrollo , Animales , Ecosistema , Técnicas de Silenciamiento del Gen , Metamorfosis Biológica/fisiología , Receptores Histamínicos/genética , Agua de Mar , Strongylocentrotus purpuratus/anatomía & histologíaRESUMEN
BACKGROUND: The developmental gene regulatory network (GRN) that underlies skeletogenesis in sea urchins and other echinoderms is a paradigm of GRN structure, function, and evolution. This transcriptional network is deployed selectively in skeleton-forming primary mesenchyme cells (PMCs) of the early embryo. To advance our understanding of this model developmental GRN, we used genome-wide chromatin accessibility profiling to identify and characterize PMC cis-regulatory modules (CRMs). RESULTS: ATAC-seq (Assay for Transposase-Accessible Chromatin using sequencing) analysis of purified PMCs provided a global picture of chromatin accessibility in these cells. We used both ATAC-seq and DNase-seq (DNase I hypersensitive site sequencing) to identify > 3000 sites that exhibited increased accessibility in PMCs relative to other embryonic cell lineages, and provide both computational and experimental evidence that a large fraction of these sites represent bona fide skeletogenic CRMs. Putative PMC CRMs were preferentially located near genes differentially expressed by PMCs and consensus binding sites for two key transcription factors in the PMC GRN, Alx1 and Ets1, were enriched in these CRMs. Moreover, a high proportion of candidate CRMs drove reporter gene expression specifically in PMCs in transgenic embryos. Surprisingly, we found that PMC CRMs were partially open in other embryonic lineages and exhibited hyperaccessibility as early as the 128-cell stage. CONCLUSIONS: Our work provides a comprehensive picture of chromatin accessibility in an early embryonic cell lineage. By identifying thousands of candidate PMC CRMs, we significantly enhance the utility of the sea urchin skeletogenic network as a general model of GRN architecture and evolution. Our work also shows that differential chromatin accessibility, which has been used for the high-throughput identification of enhancers in differentiated cell types, is a powerful approach for the identification of CRMs in early embryonic cells. Lastly, we conclude that in the sea urchin embryo, CRMs that control the cell type-specific expression of effector genes are hyperaccessible several hours in advance of gene activation.
Asunto(s)
Cromatina/genética , Embrión no Mamífero/metabolismo , Células Madre Mesenquimatosas/metabolismo , Secuencias Reguladoras de Ácidos Nucleicos , Strongylocentrotus purpuratus/genética , Animales , Embrión no Mamífero/citología , Regulación del Desarrollo de la Expresión Génica , Redes Reguladoras de Genes , Secuenciación de Nucleótidos de Alto Rendimiento , Células Madre Mesenquimatosas/citología , Strongylocentrotus purpuratus/citología , Strongylocentrotus purpuratus/crecimiento & desarrollo , Factores de Transcripción/metabolismoRESUMEN
Understanding the mechanisms with which organisms can respond to a rapidly changing ocean is an important research priority in marine sciences, especially in the light of recent predictions regarding the pace of ocean change in the coming decades. Transgenerational effects, in which the experience of the parental generation can shape the phenotype of their offspring, may serve as such a mechanism. In this study, adult purple sea urchins, Strongylocentrotus purpuratus, were conditioned to regionally and ecologically relevant pCO2 levels and temperatures representative of upwelling (colder temperature and high pCO2 ) and nonupwelling (average temperature and low pCO2 ) conditions typical of coastal upwelling regions in the California Current System. Following 4.5 months of conditioning, adults were spawned and offspring were raised under either high or low pCO2 levels, to examine the role of maternal effects. Using RNA-seq and comparative transcriptomics, our results indicate that differential conditioning of the adults had an effect on the gene expression patterns of the progeny during the gastrula stage of early development. For example, maternal conditioning under upwelling conditions intensified the transcriptomic response of the progeny when they were raised under high versus low pCO2 conditions. Additionally, mothers that experienced upwelling conditions produced larger progeny. The overall findings of this study are complex, but do suggest that transgenerational plasticity in situ could act as an important mechanism by which populations might keep pace with rapid environmental change.
Asunto(s)
Dióxido de Carbono/farmacología , Strongylocentrotus purpuratus/genética , Aclimatación , Animales , Cambio Climático , Frío , Femenino , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica/genética , Exposición Materna , Strongylocentrotus purpuratus/crecimiento & desarrollo , Strongylocentrotus purpuratus/fisiologíaRESUMEN
Sea urchins are dominant members of rocky temperate reefs around the world. They often occur in cavities within the rock, and fit so tightly, it is natural to assume they sculpted these "pits." However, there are no experimental data demonstrating they bore pits. If they do, what are the rates and consequences of bioerosion to nearshore systems? We sampled purple sea urchins, Strongylocentrotus purpuratus, from sites with four rock types, three sedimentary (two sandstones and one mudstone) and one metamorphic (granite). A year-long experiment showed urchins excavated depressions on sedimentary rocks in just months. The rate of pit formation varied with rock type and ranged from <5 yr for medium-grain sandstone to >100 yr for granite. In the field, there were differences in pit size and shapes of the urchins (height:diameter ratio). The pits were shallow and urchins flatter at the granite site, and the pits were deeper and urchins taller at the sedimentary sites. Although overall pit sizes were larger on mudstone than on sandstone, urchin size accounted for this difference. A second, short-term experiment, showed the primary mechanism for bioerosion was ingestion of the substratum. This experiment eliminated potential confounding factors of the year-long experiment and yielded higher bioerosion rates. Given the high densities of urchins, large amounts of rock can be converted to sediment over short time periods. Urchins on sandstone can excavate as much as 11.4 kg m-2 yr-1. On a broader geographic scale, sediment production can exceed 100 t ha-1 yr-1, and across their range, their combined bioerosion is comparable to the sediment load of many rivers. The phase shift between urchin barrens and kelp bed habitats in the North Pacific is controlled by the trophic cascade of sea otters. By limiting urchin populations, these apex predators also may indirectly control a substantial component of coastal rates of bioerosion.
Asunto(s)
Strongylocentrotus purpuratus/fisiología , Animales , California , Clima , Ecosistema , Conducta Alimentaria , Sedimentos Geológicos , Fenómenos Geológicos , Modelos Biológicos , Strongylocentrotus purpuratus/crecimiento & desarrolloRESUMEN
Accumulating evidence indicates that Krüppel-like factors (KLFs) play important roles in fat biology via the regulation of CCAAT/enhancer binding proteins (C/EBPs). However, KLFs and C/EBPs have not been identified from Strongylocentrotus nudus, and their roles in this species are not clear. In this study, the full-length cDNA of S. nudus KLF10 (SnKLF10) and three cDNA fragments of S. nudus C/EBPs (SnC/EBPs) were obtained. Examination of tissue distribution and expression patterns during gonadal development implied that SnKLF10 and SnC/EBPs play important roles in gonadal lipogenesis. The presence of transcription factor-binding sites (TFBSs) for KLFs in SnC/EBPs, and the results of an over-expression assay, revealed that SnKLF10 negatively regulates the transcription of SnC/EBPs. In addition, the core promoter regions of SnC/EBPs were determined, and multiple TFBSs for transcription factor (TFs) were identified, which are potential regulators of SnC/EBP transcription. Taken together, these results suggest that SnC/EBP genes are potential targets of SnKLF10, and that SnKLF10 plays a role in lipogenesis by repressing the transcription of SnC/EBPs. These findings provide information for further studies of KLF10 in invertebrates and provide new insight into the regulatory mechanisms of C/EBP transcription.
Asunto(s)
Proteínas Potenciadoras de Unión a CCAAT/genética , Factores de Transcripción de Tipo Kruppel/genética , Strongylocentrotus purpuratus/genética , Animales , ADN Complementario , Femenino , Regulación de la Expresión Génica , Células HEK293 , Humanos , Lipogénesis/genética , Masculino , Regiones Promotoras Genéticas , Strongylocentrotus purpuratus/crecimiento & desarrolloRESUMEN
The sea urchin larval embryo elaborates two calcitic endoskeletal elements called spicules. Spicules are synthesized by the primary mesenchyme cells (PMCs) and begin to form at early gastrula stage. It is known that the calcium comprising the spicules comes from the seawater and we wish to further consider the mode of calcium transport from the extracellular seawater to the PMCs and then onto the forming spicules. We used PMC in vitro cultures, calcein, fluorescently labeled dextran, and fluorescently labeled Wheat Germ Agglutinin (WGA) to track calcium transport from the seawater into PMCs and spicules and to determine how molecules from the surface of PMCs interact with the incoming calcium. Labeling of PMC endocytic vesicles and forming spicules by both calcein and fluorescently tagged dextran indicate that calcium is taken up from the seawater by endocytosis and directly incorporated into spicules. Calcein labeling studies also indicate that calcium from the extracellular seawater begins to be incorporated into spicules within 30min of uptake. In addition, we demonstrate that fluorescently labeled WGA and calcein are taken up by many of the same endocytic vesicles and are incorporated into growing spicules. These findings suggest that PMC specific surface molecules accompany calcium ions as they enter PMCs via endocytosis and are incorporated together in the growing spicule. Using anti-spicule matrix protein antibodies, we pinpoint a subset of spicule matrix proteins that may accompany calcium ions from the surface of the PMCs until they are incorporated into spicules. Msp130 is identified as one of these spicule matrix proteins.
Asunto(s)
Endocitosis , Mesodermo/citología , Osteogénesis , Strongylocentrotus purpuratus/citología , Strongylocentrotus purpuratus/crecimiento & desarrollo , Animales , Calcio/metabolismo , Células Cultivadas , Dextranos/metabolismo , Proteínas de la Matriz Extracelular/metabolismo , Espacio Extracelular/metabolismo , Fluoresceínas/metabolismo , Cinética , Larva/citología , Larva/metabolismo , Mesodermo/metabolismo , Strongylocentrotus purpuratus/metabolismo , Aglutininas del Germen de Trigo/metabolismoRESUMEN
BACKGROUND: Digestive cells are present in all metazoans and provide the energy necessary for the whole organism. Pancreatic exocrine cells are a unique vertebrate cell type involved in extracellular digestion of a wide range of nutrients. Although the organization and regulation of this cell type is intensively studied in vertebrates, its evolutionary history is still unknown. In order to understand which are the elements that define the pancreatic exocrine phenotype, we have analyzed the expression of genes that contribute to specification and function of this cell-type in an early branching deuterostome, the sea urchin Strongylocentrotus purpuratus. RESULTS: We defined the spatial and temporal expression of sea urchin orthologs of pancreatic exocrine genes and described a unique population of cells clustered in the upper stomach of the sea urchin embryo where exocrine markers are co-expressed. We used a combination of perturbation analysis, drug and feeding experiments and found that in these cells of the sea urchin embryo gene expression and gene regulatory interactions resemble that of bona fide pancreatic exocrine cells. We show that the sea urchin Ptf1a, a key transcriptional activator of digestive enzymes in pancreatic exocrine cells, can substitute for its vertebrate ortholog in activating downstream genes. CONCLUSIONS: Collectively, our study is the first to show with molecular tools that defining features of a vertebrate cell-type, the pancreatic exocrine cell, are shared by a non-vertebrate deuterostome. Our results indicate that the functional cell-type unit of the vertebrate pancreas may evolutionarily predate the emergence of the pancreas as a discrete organ. From an evolutionary perspective, these results encourage to further explore the homologs of other vertebrate cell-types in traditional or newly emerging deuterostome systems.
Asunto(s)
Evolución Biológica , Estómago/citología , Strongylocentrotus purpuratus/citología , Animales , Diferenciación Celular , Linaje de la Célula , Digestión/genética , Digestión/fisiología , Regulación del Desarrollo de la Expresión Génica , Genes Reguladores , Células HEK293 , Células HeLa , Humanos , Larva/citología , Larva/metabolismo , Páncreas/citología , Ratas , Strongylocentrotus purpuratus/crecimiento & desarrollo , Strongylocentrotus purpuratus/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Factores de Transcripción/fisiología , Vertebrados/anatomía & histología , Vertebrados/metabolismoRESUMEN
Energy is required to maintain physiological homeostasis in response to environmental change. Although responses to environmental stressors frequently are assumed to involve high metabolic costs, the biochemical bases of actual energy demands are rarely quantified. We studied the impact of a near-future scenario of ocean acidification [800 µatm partial pressure of CO2 (pCO2)] during the development and growth of an important model organism in developmental and environmental biology, the sea urchin Strongylocentrotus purpuratus. Size, metabolic rate, biochemical content, and gene expression were not different in larvae growing under control and seawater acidification treatments. Measurements limited to those levels of biological analysis did not reveal the biochemical mechanisms of response to ocean acidification that occurred at the cellular level. In vivo rates of protein synthesis and ion transport increased â¼50% under acidification. Importantly, the in vivo physiological increases in ion transport were not predicted from total enzyme activity or gene expression. Under acidification, the increased rates of protein synthesis and ion transport that were sustained in growing larvae collectively accounted for the majority of available ATP (84%). In contrast, embryos and prefeeding and unfed larvae in control treatments allocated on average only 40% of ATP to these same two processes. Understanding the biochemical strategies for accommodating increases in metabolic energy demand and their biological limitations can serve as a quantitative basis for assessing sublethal effects of global change. Variation in the ability to allocate ATP differentially among essential functions may be a key basis of resilience to ocean acidification and other compounding environmental stressors.
Asunto(s)
Metabolismo Energético , Expresión Génica , Strongylocentrotus purpuratus/genética , Strongylocentrotus purpuratus/metabolismo , Ácidos/química , Factores de Edad , Análisis de Varianza , Animales , Dióxido de Carbono/metabolismo , Femenino , Concentración de Iones de Hidrógeno , Larva/genética , Larva/crecimiento & desarrollo , Larva/metabolismo , Masculino , Océanos y Mares , Proteínas/genética , Proteínas/metabolismo , Agua de Mar/química , ATPasa Intercambiadora de Sodio-Potasio/genética , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Strongylocentrotus purpuratus/crecimiento & desarrollo , Factores de TiempoRESUMEN
Sea urchins have been used as experimental organisms for developmental biology for over a century. Yet, as is the case for many other marine invertebrates, understanding the development of the juveniles and adults has lagged far behind that of their embryos and larvae. The reasons for this are, in large part, due to the difficulty of experimentally manipulating juvenile development. Here we develop and validate a technique for injecting compounds into juvenile rudiments of the purple sea urchin, Strongylocentrotus purpuratus. We first document the distribution of rhodaminated dextran injected into different compartments of the juvenile rudiment of sea urchin larvae. Then, to test the potential of this technique to manipulate development, we injected Vivo-Morpholinos (vMOs) designed to knock down p58b and p16, two proteins involved in the elongation of S. purpuratus larval skeleton. Rudiments injected with these vMOs showed a delay in the growth of some juvenile skeletal elements relative to controls. These data provide the first evidence that vMOs, which are designed to cross cell membranes, can be used to transiently manipulate gene function in later developmental stages in sea urchins. We therefore propose that injection of vMOs into juvenile rudiments, as shown here, is a viable approach to testing hypotheses about gene function during development, including metamorphosis.
Asunto(s)
Técnicas de Silenciamiento del Gen/métodos , Morfolinos/farmacología , Strongylocentrotus purpuratus/crecimiento & desarrollo , Animales , Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , Dextranos/farmacología , Femenino , Colorantes Fluorescentes/farmacología , Larva/crecimiento & desarrollo , Masculino , Modelos Biológicos , Rodaminas/farmacología , Strongylocentrotus purpuratus/efectos de los fármacos , Strongylocentrotus purpuratus/genéticaRESUMEN
BACKGROUND: The purple sea urchin, Strongylocentrotus purpuratus, has long been the focus of developmental and ecological studies, and its recently-sequenced genome has spawned a diversity of functional genomics approaches. S. purpuratus has an indirect developmental mode with a pluteus larva that transforms after 1-3 months in the plankton into a juvenile urchin. Compared to insects and frogs, mechanisms underlying the correspondingly dramatic metamorphosis in sea urchins remain poorly understood. In order to take advantage of modern techniques to further our understanding of juvenile morphogenesis, organ formation, metamorphosis and the evolution of the pentameral sea urchin body plan, it is critical to assess developmental progression and rate during the late larval phase. This requires a staging scheme that describes developmental landmarks that can quickly and consistently be used to identify the stage of individual living larvae, and can be tracked during the final two weeks of larval development, as the juvenile is forming. RESULTS: Notable structures that are easily observable in developing urchin larvae are the developing spines, test and tube feet within the juvenile rudiment that constitute much of the oral portion of the adult body plan. Here we present a detailed staging scheme of rudiment development in the purple urchin using soft structures of the rudiment and the primordia of these juvenile skeletal elements. We provide evidence that this scheme is robust and applicable across a range of temperature and feeding regimes. CONCLUSIONS: Our proposed staging scheme provides both a useful method to study late larval development in the purple urchin, and a framework for developing similar staging schemes across echinoderms. Such efforts will have a high impact on evolutionary developmental studies and larval ecology, and facilitate research on this important deuterostome group.
Asunto(s)
Estadios del Ciclo de Vida , Metamorfosis Biológica , Strongylocentrotus purpuratus/crecimiento & desarrollo , Animales , Biología Evolutiva/métodos , Femenino , Larva/crecimiento & desarrollo , Masculino , Strongylocentrotus purpuratus/embriología , Factores de TiempoRESUMEN
Regulatory interactions buffer development against genetic and environmental perturbations, but adaptation requires phenotypes to change. We investigated the relationship between robustness and evolvability within the gene regulatory network underlying development of the larval skeleton in the sea urchin Strongylocentrotus purpuratus. We find extensive variation in gene expression in this network throughout development in a natural population, some of which has a heritable genetic basis. Switch-like regulatory interactions predominate during early development, buffer expression variation, and may promote the accumulation of cryptic genetic variation affecting early stages. Regulatory interactions during later development are typically more sensitive (linear), allowing variation in expression to affect downstream target genes. Variation in skeletal morphology is associated primarily with expression variation of a few, primarily structural, genes at terminal positions within the network. These results indicate that the position and properties of gene interactions within a network can have important evolutionary consequences independent of their immediate regulatory role.
Asunto(s)
Evolución Biológica , Regulación del Desarrollo de la Expresión Génica , Redes Reguladoras de Genes , Strongylocentrotus purpuratus/genética , Animales , Huesos/anatomía & histología , Perfilación de la Expresión Génica , Larva/anatomía & histología , Larva/genética , Strongylocentrotus purpuratus/crecimiento & desarrolloRESUMEN
Across heterogeneous landscapes, populations may have adaptive differences in gene regulation that adjust their physiologies to match local environments. Such differences could have origins in acclimation or in genetically fixed variation between habitats. Here we use common-garden experiments to evaluate differences in gene expression between populations of the purple sea urchin, Strongylocentrotus purpuratus, spanning 1700 km and average temperature differences of 5°C to 8°C. Across expression profiles from 18,883 genes after 3 years of common conditions, we find highly correlated expression patterns (Pearson's r = 0.992) among most genes. However, 66 genes were differentially expressed, including many ribosomal protein and biomineralization genes, which had higher expression in urchins originally from the southern population. Gene function analyses revealed slight but pervasive expression differences in genes related to ribosomal function, metabolism, transport, "bone" development, and response to stimuli. In accord with gene expression patterns, a post-hoc spine regrowth experiment revealed that urchins of southern origin regrew spines at a faster rate than northern urchins. These results suggest that there may be genetically controlled, potentially adaptive differences in gene regulation across habitats and that gene expression differences may be under strong enough selection to overcome high, dispersal-mediated gene flow in this marine species.