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Charles A. Ettensohn - One of the best experts on this subject based on the ideXlab platform.
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global analysis of primary Mesenchyme Cell cis regulatory modules by chromatin accessibility profiling
BMC Genomics, 2018Co-Authors: Tanvi Shashikant, Jian Ming Khor, Charles A. EttensohnAbstract: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). 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. 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.
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A fate map of the vegetal plate of the sea urchin (Lytechinus variegatus) Mesenchyme blastula.
Development (Cambridge England), 1996Co-Authors: S W Ruffins, Charles A. EttensohnAbstract:Previous lineage tracing experiments have shown that the vegetal blastomers of cleavage stage embryos give rise to all the mesoderm and endoderm of the sea urchin larva. In these studies, vegetal blastomers were labeled no later than the sixth cleavage division (60-64 Cell stage). In an earlier study we showed that single Cells in the vegetal plate of the blastula stage Lytechinus variegatus embryo could be labeled in situ with the fluorescent, lipophilic dye, DiI(C18), and that Cells labeled in the central region of the vegetal plate of the Mesenchyme blastula primarily gave rise to homogeneous clones consisting of a single secondary Mesenchyme Cell (SMC) type (Ruffins and Ettensohn (1993) Dev. Biol. 160, 285-288). Our clonal labeling showed that a detailed fate map could be generated using the DiI(C18) labeling technique. Such a fate map could provide information about the spatial relationships between the precursors of specific mesodermal and endodermal Cell types and information concerning the movements of these Cells during gastrulation and later embryogenesis. We have used this method to construct the first detailed fate map of the vegetal plate of the sea urchin embryo. Ours is a latitudinal map; mapping from the plate center, where the mesodermal precursors reside, through the region which contains the endodermal precursors and across the ectodermal boundary. We found that the precursors of certain SMC types are segregated in the Mesenchyme blastula stage vegetal plate and that prospective germ layers reside within specific boundaries. To determine whether the vegetal plate is radially symmetrical with respect to mesodermal Cell fates, single blastomeres of four Cell stage embryos were injected with lysyl-rhodamine dextran (LRD). The resulting ectodermal labeling patterns were classified and correlated with the SMC types labeled. This analysis indicates that the dorsal and ventral blastomers do not contribute equally to SMC derivatives in L. variegatus.
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primary Mesenchyme Cell migration in the sea urchin embryo distribution of directional cues
Developmental Biology, 1994Co-Authors: Katherine M Malinda, Charles A. EttensohnAbstract:The directional migration of the primary Mesenchyme Cells (PMCs) of the sea urchin embryo is a critical step in the process of gastrulation. Although interactions between the migrating Cells and the blastocoel environment are necessary for guiding the PMCs to their subequatorial target site, the nature of these interactions and the localization of guidance cues involved in directing the Cells are not yet known. Previous studies have suggested that PMC migration is the result of random exploration and selective trapping at the target site by a pattern of adhesiveness in the ectoderm or basal lamina. To better characterize the distribution of guidance cues in the blastocoel we used a combination of timelapse microscopy microsurgery and fluorescence photoablation to study the behavior of the migrating Cells. By using fluorescence time-lapse microscopy and a two-dimensional random-walk analysis of Cell trajectories we demonstrated that fluorescently labeled PMCs injected near the animal pole move in a directed fashion over a relatively long distance to reach the target site. This suggests that guidance cues are distributed globally throughout the embryo and are not restricted to the immediate ring area. To further test this hypothesis we investigated the migratory behavior of PMCs that were prevented from interacting directly with the target site. First we examined the behavior of PMCs injected into animal embryo fragments lacking the target site. We found that PMCs move to the vegetal-most area of such embryo fragments regardless of their size. Second we studied the effects of photoablating a stripe of ectoderm between PMCs injected at the animal pole region (APR) and the target site. PMCs were found to accumulate along the ablated stripe and were unable to cross it for up to 6 hr after ablation. We also examined the migratory behavior of endogenous PMCs in embryos treated with lithium a vegetalizing agent which shifts the position of the PMC ring toward the animal pole. We found that PMCs accumulated along an ablated stripe of ectoderm positioned below the shifted target site suggesting that endogenous PMCs follow a set of directional cues to the target site which may be similar to those used by PMCs injected into the APR. As a whole these results suggest that migrating PMCs follow a set of directional cues that are widely distributed throughout the blastocoel and that maybe arranged in a gradient.
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A clonal analysis of secondary Mesenchyme Cell fates in the sea urchin embryo.
Developmental Biology, 1993Co-Authors: S W Ruffins, Charles A. EttensohnAbstract:The secondary Mesenchyme Cells (SMCs) give rise to most of the mesoderm of the sea urchin embryo. Although the early embryonic lineage of these Cells has been described, the mechanisms that cause SMCs to become restricted to a particular mesodermal Cell fate are unknown. To begin to address this question, we performed a clonal analysis of the fates of SMC precursors in the vegetal plate by labeling single Cells with the fluorescent dye DiI (C18). Our data show that some presumptive SMCs remain pluripotent at the late blastula stage, since some Cells labeled at this stage gave rise to more than one mesodermal Cell type. Surprisingly, however, most labeled Cells gave rise to homogeneous clones composed of a single Cell type. This observation indicates that either many SMC precursors are restricted in their fate before the start of gastrulation or that all the progeny of a single vegetal plate Cell are influenced by the same instructional signals during gastrulation, despite the Cell divisions and extensive Cell movements that occur during this time. The percentage of clones composed of a single Cell type increased during the blastula stage, supporting the view that the process of SMC fate specification begins before the onset of gastrulation.
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The regulation of primary Mesenchyme Cell patterning
Developmental Biology, 1990Co-Authors: Charles A. EttensohnAbstract:Abstract The primary Mesenchyme Cells (PMCs) of the sea urchin embryo undergo a dramatic sequence of morphogenetic behaviors that includes migration, localization at specific sites within the embryo, and synthesis of the larval skeleton. To gain information about how these processes are regulated, PMC migration and patterning were analyzed in embryos with experimentally altered numbers of PMCs. PMC movements were followed by labeling the Cells with a fluorescent dye, rhodamine B isothiocyanate, or with the PMC-specific monoclonal antibody 6a9. These methods show that individual PMCs have the capacity to join any position in the pattern, and rule out the possibility that PMC morphogenesis involves a sorting out of discrete subpopulations of Cells to predetermined sites. All sites in the PMC pattern have the capacity to accept more Cells than they normally do, and PMCs do not appear to compete with one another for preferred sites in the pattern. Even in embryos with 2–3 times the normal complement of PMCs, all these Cells take part in spiculogenesis and the resultant skeleton is normal in size and configuration. Two special sites along the basal lamina (those corresponding to the positions of the PMC ventrolateral clusters) promote spicule elongation, an effect that is independent of the numbers of PMCs at these sites. These observations emphasize the role of the basal lamina, blastocoel matrix, and embryonic epithelium in regulating key aspects of PMC morphogenesis. The PMCs remain highly flexible in their ability to respond to patterning cues in the blastocoel, since postmigratory PMCs will repeat their patterning process if microinjected into the blastocoel of young recipient embryos.
Michael Solursh - One of the best experts on this subject based on the ideXlab platform.
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primary Mesenchyme Cell migration requires a chondroitin sulfate dermatan sulfate proteoglycan
Developmental Biology, 1991Co-Authors: Mary Constance Lane, Michael SolurshAbstract:Abstract Primary Mesenchyme Cell migration in the sea urchin embryo is inhibited by sulfate deprivation and exposure to exogenous β- d -xylosides, two treatments known to disrupt proteoglycan synthesis. We show that in the developing sea urchin, exogenous xyloside affects the synthesis by the primary Mesenchyme Cells of a very large, Cell surface chondroitin sulfate/dermatan sulfate proteoglycan. This proteoglycan is present in a partially purified fraction that restores migratory ability to defective Cells in vitro. The integrity of this chondroitin sulfate/dermatan sulfate proteoglycan appears essential for primary Mesenchyme Cell migration since treatment of actively migrating Cells with chondroitinase ABC reversibly inhibited their migration in vitro.
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Primary Mesenchyme Cell migration requires a chondroitin sulfate/dermatan sulfate proteoglycan.
Developmental Biology, 1991Co-Authors: Mary Constance Lane, Michael SolurshAbstract:Abstract Primary Mesenchyme Cell migration in the sea urchin embryo is inhibited by sulfate deprivation and exposure to exogenous β- d -xylosides, two treatments known to disrupt proteoglycan synthesis. We show that in the developing sea urchin, exogenous xyloside affects the synthesis by the primary Mesenchyme Cells of a very large, Cell surface chondroitin sulfate/dermatan sulfate proteoglycan. This proteoglycan is present in a partially purified fraction that restores migratory ability to defective Cells in vitro. The integrity of this chondroitin sulfate/dermatan sulfate proteoglycan appears essential for primary Mesenchyme Cell migration since treatment of actively migrating Cells with chondroitinase ABC reversibly inhibited their migration in vitro.
Mary Constance Lane - One of the best experts on this subject based on the ideXlab platform.
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primary Mesenchyme Cell migration requires a chondroitin sulfate dermatan sulfate proteoglycan
Developmental Biology, 1991Co-Authors: Mary Constance Lane, Michael SolurshAbstract:Abstract Primary Mesenchyme Cell migration in the sea urchin embryo is inhibited by sulfate deprivation and exposure to exogenous β- d -xylosides, two treatments known to disrupt proteoglycan synthesis. We show that in the developing sea urchin, exogenous xyloside affects the synthesis by the primary Mesenchyme Cells of a very large, Cell surface chondroitin sulfate/dermatan sulfate proteoglycan. This proteoglycan is present in a partially purified fraction that restores migratory ability to defective Cells in vitro. The integrity of this chondroitin sulfate/dermatan sulfate proteoglycan appears essential for primary Mesenchyme Cell migration since treatment of actively migrating Cells with chondroitinase ABC reversibly inhibited their migration in vitro.
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Primary Mesenchyme Cell migration requires a chondroitin sulfate/dermatan sulfate proteoglycan.
Developmental Biology, 1991Co-Authors: Mary Constance Lane, Michael SolurshAbstract:Abstract Primary Mesenchyme Cell migration in the sea urchin embryo is inhibited by sulfate deprivation and exposure to exogenous β- d -xylosides, two treatments known to disrupt proteoglycan synthesis. We show that in the developing sea urchin, exogenous xyloside affects the synthesis by the primary Mesenchyme Cells of a very large, Cell surface chondroitin sulfate/dermatan sulfate proteoglycan. This proteoglycan is present in a partially purified fraction that restores migratory ability to defective Cells in vitro. The integrity of this chondroitin sulfate/dermatan sulfate proteoglycan appears essential for primary Mesenchyme Cell migration since treatment of actively migrating Cells with chondroitinase ABC reversibly inhibited their migration in vitro.
David R. Mcclay - One of the best experts on this subject based on the ideXlab platform.
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The Snail repressor is required for PMC ingression in the sea urchin embryo
Development, 2007Co-Authors: Shu-yu Wu, David R. McclayAbstract:In metazoans, the epithelial-mesenchymal transition (EMT) is a crucial process for placing the mesoderm beneath the ectoderm. Primary Mesenchyme Cells (PMCs) at the vegetal pole of the sea urchin embryo ingress into the floor of the blastocoele from the blastula epithelium and later become the skeletogenic Mesenchyme. This ingression movement is a classic EMT during which the PMCs penetrate the basal lamina, lose adherens junctions and migrate into the blastocoele. Later, secondary Mesenchyme Cells (SMCs) also enter the blastocoele via an EMT, but they accompany the invagination of the archenteron initially, in much the same way vertebrate Mesenchyme enters the embryo along with endoderm. Here we identify a sea urchin ortholog of the Snail transcription factor, and focus on its roles regulating EMT during PMC ingression. Functional knockdown analyses of Snail in whole embryos and chimeras demonstrate that Snail is required in micromeres for PMC ingression. Snail represses the transcription of cadherin, a repression that appears evolutionarily conserved throughout the animal kingdom. Furthermore, Snail expression is required for endocytosis of cadherin, a Cellular activity that accompanies PMC ingression. Perturbation studies position Snail in the sea urchin micromere-PMC gene regulatory network (GRN), downstream of Pmar1 and Alx1, and upstream of several PMC-expressed proteins. Taken together, our findings indicate that Snail plays an essential role in PMCs to control the EMT process, in part through its repression of cadherin expression during PMC ingression, and in part through its role in the endocytosis that helps convert an epithelial Cell to a Mesenchyme Cell.
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activation of pmar1 controls specification of micromeres in the sea urchin embryo
Developmental Biology, 2003Co-Authors: Paola Oliveri, Eric H Davidson, David R. McclayAbstract:pmar1 is a transcription factor in the paired class homeodomain family that was identified and found to be transcribed in micromeres beginning at the fourth cleavage of sea urchin development [Dev. Biol. 246 (2002), 209]. Based on in situ data, molecular perturbation studies, and QPCR data, the recently published gene regulatory network (GRN) model for endomesoderm specification [Science 295 (2002) 1669; Dev. Biol. 246 (2002), 162] places pmar1 early in the micromere specification pathway, and upstream of two important micromere induction signals. The goal of this study was to test these three predictions of the network model. A series of embryo chimeras were produced in which pmar1 activity was perturbed in one Cell that was transplanted to control hosts. At the fourth cleavage, micromeres bearing altered pmar1 activity were combined with a normal micromereless host embryo. If β-catenin signaling is blocked, the micromeres remain unspecified and are unable to signal to the host Cells. When such β-catenin-blocked micromeres also express Pmar1, all observed micromere functions are rescued. The rescue includes expression of the primary Mesenchyme Cell (PMC) differentiation program, expression and execution of the Delta signal to induce secondary mesoderm Cell (SMC) specification in macromere progeny, and expression of the early endomesoderm induction signal necessary for full specification of the endoderm. Additionally, Pmar1 expressed mosaically from inserted DNA constructs causes induction of ectopic Endo 16 in adjacent Cells, demonstrating further that Pmar1 controls expression of the early endomesoderm induction signal. Based on these experiments, Pmar1 is an important transcription factor necessary for initiating the micromere specification program and for the expression of two inductive signals produced by micromeres. Each of the tests we describe supports the placement and function of Pmar1 in the endomesoderm GRN model.
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primary Mesenchyme Cell patterning during the early stages following ingression
Developmental Biology, 2003Co-Authors: Robert E Peterson, David R. McclayAbstract:Sea urchin primary Mesenchyme Cells (PMCs) ingress into the blastocoel during an epithelial-to-mesenchymal transition (EMT), migrate along the blastocoelar wall for a period of time, and then settle into a subequatorial ring to form the larval skeleton. Fluorescent-marked blastomeres alone, or in combination with blastomere recombination, were used to track the position of PMCs during the early phases of this movement. Micromeres expressing Golgi-tethered GFP (galtase-GFP) were transplanted onto TRITC-stained hosts (in place of the endogenous micromere) to observe the progeny of a single micromere. Galtase-GFP as a Golgi marker is not transferred between PMCs when the syncytium forms. Thus, the position of Cells can be followed relative to beginning position for longer periods than previously reported. The PMC progeny of a single micromere do not disperse upon ingression, but instead remain in a closely associated cluster. Generally, progeny of a single micromere remain in the quadrant of origin. In total, greater than approximately 94% of labeled PMCs remain within the local region of ingression. By contrast, when a transplanted micromere is placed at the vegetal plate after removing all 4 host micromeres, the resultant PMCs ingress and migrate into all 4 quadrants. Similarly, if 1 blastomere is injected at the 2-Cell stage, and later the 2 unlabeled micromeres are removed at the 16-Cell stage, the remaining PMCs ingress into all 4 quadrants of the vegetal plate. We conclude that the normal restriction of PMCs to a quadrant is due to mechanical constraint from other micromere-PMCs. If a labeled micromere is placed ectopically at the macromere/mesomere boundary, the PMC progeny ingress ectopically and migrate longitudinally along the animal-vegetal axis only. Injection of galtase-GFP into one blastomere at the 4-Cell stage shows a 2-step pattern of localization. At late Mesenchyme blastula and early gastrula stages, greater than 90% of GFP-expressing PMCs remain in the injected quadrant, while at mid- to late-gastrula stage and beyond, more PMCs are found outside the injected quadrant. The migration that sets up the asymmetry of the larval skeleton first occurs around mid- to late-gastrula stages, when some PMCs from an aboral quadrant migrate to the adjacent oral quadrant. In all, these data combined with previous data suggest that freshly ingressed PMCs migrate along a longitudinal path toward the animal pole and back toward the vegetal pole. Beginning at mid- to late-gastrula stage, PMCs utilize oral-aboral cues from the ectoderm for the first time. At this time, some aboral PMCs migrate into the adjacent oral quadrant to assist in the formation of the ventrolateral cluster.
S W Ruffins - One of the best experts on this subject based on the ideXlab platform.
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A fate map of the vegetal plate of the sea urchin (Lytechinus variegatus) Mesenchyme blastula.
Development (Cambridge England), 1996Co-Authors: S W Ruffins, Charles A. EttensohnAbstract:Previous lineage tracing experiments have shown that the vegetal blastomers of cleavage stage embryos give rise to all the mesoderm and endoderm of the sea urchin larva. In these studies, vegetal blastomers were labeled no later than the sixth cleavage division (60-64 Cell stage). In an earlier study we showed that single Cells in the vegetal plate of the blastula stage Lytechinus variegatus embryo could be labeled in situ with the fluorescent, lipophilic dye, DiI(C18), and that Cells labeled in the central region of the vegetal plate of the Mesenchyme blastula primarily gave rise to homogeneous clones consisting of a single secondary Mesenchyme Cell (SMC) type (Ruffins and Ettensohn (1993) Dev. Biol. 160, 285-288). Our clonal labeling showed that a detailed fate map could be generated using the DiI(C18) labeling technique. Such a fate map could provide information about the spatial relationships between the precursors of specific mesodermal and endodermal Cell types and information concerning the movements of these Cells during gastrulation and later embryogenesis. We have used this method to construct the first detailed fate map of the vegetal plate of the sea urchin embryo. Ours is a latitudinal map; mapping from the plate center, where the mesodermal precursors reside, through the region which contains the endodermal precursors and across the ectodermal boundary. We found that the precursors of certain SMC types are segregated in the Mesenchyme blastula stage vegetal plate and that prospective germ layers reside within specific boundaries. To determine whether the vegetal plate is radially symmetrical with respect to mesodermal Cell fates, single blastomeres of four Cell stage embryos were injected with lysyl-rhodamine dextran (LRD). The resulting ectodermal labeling patterns were classified and correlated with the SMC types labeled. This analysis indicates that the dorsal and ventral blastomers do not contribute equally to SMC derivatives in L. variegatus.
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A clonal analysis of secondary Mesenchyme Cell fates in the sea urchin embryo.
Developmental Biology, 1993Co-Authors: S W Ruffins, Charles A. EttensohnAbstract:The secondary Mesenchyme Cells (SMCs) give rise to most of the mesoderm of the sea urchin embryo. Although the early embryonic lineage of these Cells has been described, the mechanisms that cause SMCs to become restricted to a particular mesodermal Cell fate are unknown. To begin to address this question, we performed a clonal analysis of the fates of SMC precursors in the vegetal plate by labeling single Cells with the fluorescent dye DiI (C18). Our data show that some presumptive SMCs remain pluripotent at the late blastula stage, since some Cells labeled at this stage gave rise to more than one mesodermal Cell type. Surprisingly, however, most labeled Cells gave rise to homogeneous clones composed of a single Cell type. This observation indicates that either many SMC precursors are restricted in their fate before the start of gastrulation or that all the progeny of a single vegetal plate Cell are influenced by the same instructional signals during gastrulation, despite the Cell divisions and extensive Cell movements that occur during this time. The percentage of clones composed of a single Cell type increased during the blastula stage, supporting the view that the process of SMC fate specification begins before the onset of gastrulation.