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Jonathan P Rast - One of the best experts on this subject based on the ideXlab platform.
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analysis of immune response in the Sea Urchin Larva
Methods in Cell Biology, 2019Co-Authors: Katherine M Buckley, Nicholas W Schuh, Andreas Heyland, Jonathan P RastAbstract:Sea Urchin Larvae deploy a complex immune system in the context of relatively simple morphology. Several types of phagocytic or granular immune cells respond rapidly to microbes and microbial components within the body cavity. Many of these cells also respond to microbial disturbances in the gut lumen. In the course of immune response, hundreds of genes are up- and downregulated, many of which have homologs involved in immunity in other species. Thus, the Larval Sea Urchin provides an experimentally advantageous model for investigating the response to immune challenge at the level of cell behavior and gene regulatory networks. Importantly, the morphological simplicity and optical clarity of these Larvae allow studies to be carried out within the intact animal. Here, we outline techniques to probe and visualize the immune system of the feeding Sea Urchin Larva, particularly for quantifying gene expression and cell migration as the animal responds to both pathogens and symbionts. Techniques addressed in this chapter include (1) exposure of Larvae to microbes and microbial products in Sea water and by blastocoelar microinjection, (2) time-lapse imaging of immune response, (3) isolation of culturable bacteria associated with feeding Larvae, (4) quantification of Larval associations with isolated bacterial strains and (5) preparation of secreted products from isolated bacteria for testing in Larval culture.
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il17 factors are early regulators in the gut epithelium during inflammatory response to vibrio in the Sea Urchin Larva
eLife, 2017Co-Authors: Katherine M Buckley, Catherine S Schrankel, Nicholas W Schuh, Taku Hibino, Jonathan P Rast, Guizhi WangAbstract:IL17 cytokines are central mediators of mammalian immunity. In vertebrates, these factors derive from diverse cellular sources. Sea Urchins share a molecular heritage with chordates that includes the IL17 system. Here, we characterize the role of epithelial expression of IL17 in the Larval gut-associated immune response. The purple Sea Urchin genome encodes 10 IL17 subfamilies (35 genes) and 2 IL17 receptors. Most of these subfamilies are conserved throughout echinoderms. Two IL17 subfamilies are sequentially strongly upregulated and attenuated in the gut epithelium in response to bacterial disturbance. IL17R1 signal perturbation results in reduced expression of several response genes including an IL17 subtype, indicating a potential feedback. A third IL17 subfamily is activated in adult immune cells indicating that expression in immune cells and epithelia is divided among families. The Larva provides a tractable model to investigate the regulation and consequences of gut epithelial IL17 expression across the organism.
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An Organismal Model for Gene Regulatory Networks in the Gut-Associated Immune Response
Frontiers Media S.A., 2017Co-Authors: Katherine M Buckley, Jonathan P RastAbstract:The gut epithelium is an ancient site of complex communication between the animal immune system and the microbial world. While elements of self-non-self receptors and effector mechanisms differ greatly among animal phyla, some aspects of recognition, regulation, and response are broadly conserved. A gene regulatory network (GRN) approach provides a means to investigate the nature of this conservation and divergence even as more peripheral functional details remain incompletely understood. The Sea Urchin embryo is an unparalleled experimental model for detangling the GRNs that govern embryonic development. By applying this theoretical framework to the free swimming, feeding Larval stage of the purple Sea Urchin, it is possible to delineate the conserved regulatory circuitry that regulates the gut-associated immune response. This model provides a morphologically simple system in which to efficiently unravel regulatory connections that are phylogenetically relevant to immunity in vertebrates. Here, we review the organism-wide cellular and transcriptional immune response of the Sea Urchin Larva. A large set of transcription factors and signal systems, including epithelial expression of interleukin 17 (IL17), are important mediators in the activation of the early gut-associated response. Many of these have homologs that are active in vertebrate immunity, while others are ancient in animals but absent in vertebrates or specific to echinoderms. This Larval model provides a means to experimentally characterize immune function encoded in the Sea Urchin genome and the regulatory interconnections that control immune response and resolution across the tissues of the organism
Katherine M Buckley - One of the best experts on this subject based on the ideXlab platform.
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analysis of immune response in the Sea Urchin Larva
Methods in Cell Biology, 2019Co-Authors: Katherine M Buckley, Nicholas W Schuh, Andreas Heyland, Jonathan P RastAbstract:Sea Urchin Larvae deploy a complex immune system in the context of relatively simple morphology. Several types of phagocytic or granular immune cells respond rapidly to microbes and microbial components within the body cavity. Many of these cells also respond to microbial disturbances in the gut lumen. In the course of immune response, hundreds of genes are up- and downregulated, many of which have homologs involved in immunity in other species. Thus, the Larval Sea Urchin provides an experimentally advantageous model for investigating the response to immune challenge at the level of cell behavior and gene regulatory networks. Importantly, the morphological simplicity and optical clarity of these Larvae allow studies to be carried out within the intact animal. Here, we outline techniques to probe and visualize the immune system of the feeding Sea Urchin Larva, particularly for quantifying gene expression and cell migration as the animal responds to both pathogens and symbionts. Techniques addressed in this chapter include (1) exposure of Larvae to microbes and microbial products in Sea water and by blastocoelar microinjection, (2) time-lapse imaging of immune response, (3) isolation of culturable bacteria associated with feeding Larvae, (4) quantification of Larval associations with isolated bacterial strains and (5) preparation of secreted products from isolated bacteria for testing in Larval culture.
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il17 factors are early regulators in the gut epithelium during inflammatory response to vibrio in the Sea Urchin Larva
eLife, 2017Co-Authors: Katherine M Buckley, Catherine S Schrankel, Nicholas W Schuh, Taku Hibino, Jonathan P Rast, Guizhi WangAbstract:IL17 cytokines are central mediators of mammalian immunity. In vertebrates, these factors derive from diverse cellular sources. Sea Urchins share a molecular heritage with chordates that includes the IL17 system. Here, we characterize the role of epithelial expression of IL17 in the Larval gut-associated immune response. The purple Sea Urchin genome encodes 10 IL17 subfamilies (35 genes) and 2 IL17 receptors. Most of these subfamilies are conserved throughout echinoderms. Two IL17 subfamilies are sequentially strongly upregulated and attenuated in the gut epithelium in response to bacterial disturbance. IL17R1 signal perturbation results in reduced expression of several response genes including an IL17 subtype, indicating a potential feedback. A third IL17 subfamily is activated in adult immune cells indicating that expression in immune cells and epithelia is divided among families. The Larva provides a tractable model to investigate the regulation and consequences of gut epithelial IL17 expression across the organism.
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An Organismal Model for Gene Regulatory Networks in the Gut-Associated Immune Response
Frontiers Media S.A., 2017Co-Authors: Katherine M Buckley, Jonathan P RastAbstract:The gut epithelium is an ancient site of complex communication between the animal immune system and the microbial world. While elements of self-non-self receptors and effector mechanisms differ greatly among animal phyla, some aspects of recognition, regulation, and response are broadly conserved. A gene regulatory network (GRN) approach provides a means to investigate the nature of this conservation and divergence even as more peripheral functional details remain incompletely understood. The Sea Urchin embryo is an unparalleled experimental model for detangling the GRNs that govern embryonic development. By applying this theoretical framework to the free swimming, feeding Larval stage of the purple Sea Urchin, it is possible to delineate the conserved regulatory circuitry that regulates the gut-associated immune response. This model provides a morphologically simple system in which to efficiently unravel regulatory connections that are phylogenetically relevant to immunity in vertebrates. Here, we review the organism-wide cellular and transcriptional immune response of the Sea Urchin Larva. A large set of transcription factors and signal systems, including epithelial expression of interleukin 17 (IL17), are important mediators in the activation of the early gut-associated response. Many of these have homologs that are active in vertebrate immunity, while others are ancient in animals but absent in vertebrates or specific to echinoderms. This Larval model provides a means to experimentally characterize immune function encoded in the Sea Urchin genome and the regulatory interconnections that control immune response and resolution across the tissues of the organism
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perturbation of gut bacteria induces a coordinated cellular immune response in the purple Sea Urchin Larva
Immunology and Cell Biology, 2016Co-Authors: Katherine M Buckley, Catherine S Schrankel, Nicholas W Schuh, Taku Hibino, Cynthia M Solek, Koeun BaeAbstract:The purple Sea Urchin (Strongylocentrotus purpuratus) genome sequence contains a complex repertoire of genes encoding innate immune recognition proteins and homologs of important vertebrate immune regulatory factors. To characterize how this immune system is deployed within an experimentally tractable, intact animal, we investigate the immune capability of the Larval stage. Sea Urchin embryos and Larvae are morphologically simple and transparent, providing an organism-wide model to view immune response at cellular resolution. Here we present evidence for immune function in five mesenchymal cell types based on morphology, behavior and gene expression. Two cell types are phagocytic; the others interact at sites of microbial detection or injury. We characterize immune-associated gene markers for three cell types, including a perforin-like molecule, a scavenger receptor, a complement-like thioester-containing protein and the echinoderm-specific immune response factor 185/333. We elicit Larval immune responses by (1) bacterial injection into the blastocoel and (2) Seawater exposure to the marine bacterium Vibrio diazotrophicus to perturb immune state in the gut. Exposure at the epithelium induces a strong response in which pigment cells (one type of immune cell) migrate from the ectoderm to interact with the gut epithelium. Bacteria that accumulate in the gut later invade the blastocoel, where they are cleared by phagocytic and granular immune cells. The complexity of this coordinated, dynamic inflammatory program within the simple Larval morphology provides a system in which to characterize processes that direct both aspects of the echinoderm-specific immune response as well as those that are shared with other deuterostomes, including vertebrates.
Meike Stumpp - One of the best experts on this subject based on the ideXlab platform.
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cellular bicarbonate accumulation and vesicular proton transport promote calcification in the Sea Urchin Larva
Proceedings of The Royal Society B: Biological Sciences, 2020Co-Authors: Inga Petersen, William Weijen Chang, Christine Blurton, Meike StumppAbstract:The Sea Urchin embryo develops a calcitic endoskeleton through intracellular formation of amorphous calcium carbonate (ACC). Intracellular precipitation of ACC, requires [Formula: see text] concentrating as well as proton export mechanisms to promote calcification. These processes are of fundamental importance in biological mineralization, but remain largely unexplored. Here, we demonstrate that the calcifying primary mesenchyme cells (PMCs) use Na+/H+-exchange (NHE) mechanisms to control cellular pH homeostasis during maintenance of the skeleton. During skeleton re-calcification, pHi of PMCs is increased accompanied by substantial elevation in intracellular [Formula: see text] mediated by the [Formula: see text] cotransporter Sp_Slc4a10. However, PMCs lower their pHi regulatory capacities associated with a reduction in NHE activity. Live-cell imaging using green fluorescent protein reporter constructs in combination with intravesicular pH measurements demonstrated alkaline and acidic populations of vesicles in PMCs and extensive trafficking of large V-type H+-ATPase (VHA)-rich acidic vesicles in blastocoelar filopodial cells. Pharmacological and gene expression analyses underline a central role of the VHA isoforms Sp_ATP6V0a1, Sp_ATP6V01_1 and Sp_ATPa1-4 for the process of skeleton re-calcification. These results highlight novel pH regulatory strategies in calcifying cells of a marine species with important implications for our understanding of the mineralization process in times of rapid changes in oceanic pH.
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alkaline guts contribute to immunity during exposure to acidified Seawater in the Sea Urchin Larva
The Journal of Experimental Biology, 2020Co-Authors: Meike Stumpp, Inga Petersen, Femke Thoben, Matthias Leippe, Marian Y HuAbstract:ABSTRACT Larval stages of members of the Abulacraria superphylum including echinoderms and hemichordates have highly alkaline midguts. To date, the reason for the evolution of such extreme pH conditions in the gut of these organisms remains unknown. Here, we test the hypothesis that, analogous to the acidic stomachs of vertebrates, these alkaline conditions may represent a first defensive barrier to protect from environmental pathogens. pH-optimum curves for five different species of marine bacteria demonstrated a rapid decrease in proliferation rates by 50–60% between pH 8.5 and 9.5. Using the marine bacterium Vibrio diazotrophicus, which elicits a coordinated immune response in the Larvae of the Sea Urchin Strongylocentrotus purpuratus, we studied the physiological responses of the midgut pH regulatory machinery to this pathogen. Gastroscopic microelectrode measurements demonstrate a stimulation of midgut alkalization upon infection with V. diazotrophicus accompanied by an upregulation of acid–base transporter transcripts of the midgut. Pharmacological inhibition of midgut alkalization resulted in an increased mortality rate of Larvae during Vibrio infection. Reductions in Seawater pH resembling ocean acidification conditions lead to moderate reductions in midgut alkalization. However, these reductions in midgut pH do not affect the immune response or resilience of Sea Urchin Larvae to a Vibrio infection under ocean acidification conditions. Our study addressed the evolutionary benefits of the alkaline midgut of Ambulacraria Larval stages. The data indicate that alkaline conditions in the gut may serve as a first defensive barrier against environmental pathogens and that this mechanism can compensate for changes in Seawater pH.
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tipping points of gastric ph regulation and energetics in the Sea Urchin Larva exposed to co2 induced Seawater acidification
Comparative Biochemistry and Physiology A-molecular & Integrative Physiology, 2019Co-Authors: Haegyeong Lee, Meike Stumpp, Jiajiun Yan, Yungche Tseng, Sebastian HeinzelAbstract:Sea Urchin Larvae reduce developmental rates accompanied by changes in their energy budget when exposed to acidified conditions. The necessity to maintain highly alkaline conditions in their digestive systems led to the hypothesis that gastric pH homeostasis is a key trait affecting Larval energy budgets leading to distinct tipping points for growth and survival. To test this hypothesis, Sea Urchin (Strongylocentrotus purpuratus) Larvae were reared for 10 days in different pH conditions ranging from pH 7.0 to pH 8.2. Survival, development and growth rates were determined demonstrating severe impacts < pH 7.2. To test the effects of pH on midgut alkalization we measured midgut pH and monitored the expression of acid-base transporters. While Larvae were able to maintain their midgut pH at 8.9-9.1 up to an acidification level of pH 7.2, midgut pH was decreased in the lower pH treatments. The maintenance of midgut pH under low pH conditions was accompanied by dynamic changes in the expression level of midgut acid-base transporters. Metabolic rates of the Larvae increased with decreasing pH and reached a threshold between pH 7.0 and pH 7.3 where metabolic rates decreased again. Methylation analyses on promoter CpG islands were performed for midgut acid-base transporter genes to test for possible epigenetic modifications after 10-day exposure to different pH conditions. This analysis demonstrated no correlation between methylation level and pH treatments suggesting low potential for epigenetic modification of acid-base transporters upon short-term exposure. Since a clear tipping point was identified at pH 7.2, which is much lower than near-future ocean acidification (OA) scenarios, this study suggests that the early development of the purple Sea Urchin Larva has a comparatively high tolerance to Seawater acidification with substantial acclimation capacity and plasticity in a key physiological trait under near-future OA conditions.
Inga Petersen - One of the best experts on this subject based on the ideXlab platform.
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na h exchangers differentially contribute to midgut fluid sodium and proton concentration in the Sea Urchin Larva
The Journal of Experimental Biology, 2021Co-Authors: Inga Petersen, William Weijen ChangAbstract:Regulation of ionic composition and pH is a requisite of all digestive systems in the animal kingdom. Larval stages of the marine superphylum Ambulacraria, including echinoderms and hemichordates, were demonstrated to have highly alkaline conditions in their midgut with the underlying epithelial transport mechanisms being largely unknown. Using ion-selective microelectrodes, the present study demonstrated that pluteus Larvae of the purple Sea Urchin have highly alkaline pH (pH ∼9) and low [Na+] (∼120 mmol l-1) in their midgut fluids, compared with the ionic composition of the surrounding Seawater. We pharmacologically investigated the role of Na+/H+ exchangers (NHE) in intracellular pH regulation and midgut proton and sodium maintenance using the NHE inhibitor 5-(n-ethyl-n-isopropyl)amiloride (EIPA). Basolateral EIPA application decreased midgut pH while luminal application via micro-injections increased midgut [Na+], without affecting pH. Immunohistochemical analysis demonstrated a luminal localization of NHE-2 (SpSlc9a2) in the midgut epithelium. Specific knockdown of spslc9a2 using Vivo-Morpholinos led to an increase in midgut [Na+] without affecting pH. Acute acidification experiments in combination with quantitative PCR analysis and measurements of midgut pH and [Na+] identified two other NHE isoforms, Spslc9a7 and SpSlc9a8, which potentially contribute to the regulation of [Na+] and pH in midgut fluids. This work provides new insights into ion regulatory mechanisms in the midgut epithelium of Sea Urchin Larvae. The involvement of NHEs in regulating pH and Na+ balance in midgut fluids shows conserved features of insect and vertebrate digestive systems and may contribute to the ability of Sea Urchin Larvae to cope with changes in Seawater pH.
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cellular bicarbonate accumulation and vesicular proton transport promote calcification in the Sea Urchin Larva
Proceedings of The Royal Society B: Biological Sciences, 2020Co-Authors: Inga Petersen, William Weijen Chang, Christine Blurton, Meike StumppAbstract:The Sea Urchin embryo develops a calcitic endoskeleton through intracellular formation of amorphous calcium carbonate (ACC). Intracellular precipitation of ACC, requires [Formula: see text] concentrating as well as proton export mechanisms to promote calcification. These processes are of fundamental importance in biological mineralization, but remain largely unexplored. Here, we demonstrate that the calcifying primary mesenchyme cells (PMCs) use Na+/H+-exchange (NHE) mechanisms to control cellular pH homeostasis during maintenance of the skeleton. During skeleton re-calcification, pHi of PMCs is increased accompanied by substantial elevation in intracellular [Formula: see text] mediated by the [Formula: see text] cotransporter Sp_Slc4a10. However, PMCs lower their pHi regulatory capacities associated with a reduction in NHE activity. Live-cell imaging using green fluorescent protein reporter constructs in combination with intravesicular pH measurements demonstrated alkaline and acidic populations of vesicles in PMCs and extensive trafficking of large V-type H+-ATPase (VHA)-rich acidic vesicles in blastocoelar filopodial cells. Pharmacological and gene expression analyses underline a central role of the VHA isoforms Sp_ATP6V0a1, Sp_ATP6V01_1 and Sp_ATPa1-4 for the process of skeleton re-calcification. These results highlight novel pH regulatory strategies in calcifying cells of a marine species with important implications for our understanding of the mineralization process in times of rapid changes in oceanic pH.
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alkaline guts contribute to immunity during exposure to acidified Seawater in the Sea Urchin Larva
The Journal of Experimental Biology, 2020Co-Authors: Meike Stumpp, Inga Petersen, Femke Thoben, Matthias Leippe, Marian Y HuAbstract:ABSTRACT Larval stages of members of the Abulacraria superphylum including echinoderms and hemichordates have highly alkaline midguts. To date, the reason for the evolution of such extreme pH conditions in the gut of these organisms remains unknown. Here, we test the hypothesis that, analogous to the acidic stomachs of vertebrates, these alkaline conditions may represent a first defensive barrier to protect from environmental pathogens. pH-optimum curves for five different species of marine bacteria demonstrated a rapid decrease in proliferation rates by 50–60% between pH 8.5 and 9.5. Using the marine bacterium Vibrio diazotrophicus, which elicits a coordinated immune response in the Larvae of the Sea Urchin Strongylocentrotus purpuratus, we studied the physiological responses of the midgut pH regulatory machinery to this pathogen. Gastroscopic microelectrode measurements demonstrate a stimulation of midgut alkalization upon infection with V. diazotrophicus accompanied by an upregulation of acid–base transporter transcripts of the midgut. Pharmacological inhibition of midgut alkalization resulted in an increased mortality rate of Larvae during Vibrio infection. Reductions in Seawater pH resembling ocean acidification conditions lead to moderate reductions in midgut alkalization. However, these reductions in midgut pH do not affect the immune response or resilience of Sea Urchin Larvae to a Vibrio infection under ocean acidification conditions. Our study addressed the evolutionary benefits of the alkaline midgut of Ambulacraria Larval stages. The data indicate that alkaline conditions in the gut may serve as a first defensive barrier against environmental pathogens and that this mechanism can compensate for changes in Seawater pH.
Steve Weiner - One of the best experts on this subject based on the ideXlab platform.
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calcium transport into the cells of the Sea Urchin Larva in relation to spicule formation
Proceedings of the National Academy of Sciences of the United States of America, 2016Co-Authors: Netta Vidavsky, Sefi Addadi, Andreas Schertel, D Benezra, Muki Shpigel, Lia Addadi, Steve WeinerAbstract:We investigated the manner in which the Sea Urchin Larva takes up calcium from its body cavity into the primary mesenchymal cells (PMCs) that are responsible for spicule formation. We used the membrane-impermeable fluorescent dye calcein and alexa-dextran, with or without a calcium channel inhibitor, and imaged the Larvae in vivo with selective-plane illumination microscopy. Both fluorescent molecules are taken up from the body cavity into the PMCs and ectoderm cells, where the two labels are predominantly colocalized in particles, whereas the calcium-binding calcein label is mainly excluded from the endoderm and is concentrated in the spicules. The presence of vesicles and vacuoles inside the PMCs that have openings through the plasma membrane directly to the body cavity was documented using high-resolution cryo-focused ion beam-SEM serial imaging. Some of the vesicles and vacuoles are interconnected to form large networks. We suggest that these vacuolar networks are involved in direct Sea water uptake. We conclude that the calcium pathway from the body cavity into cells involves nonspecific endocytosis of Sea water with its calcium.