ScRNA-seq of roof and floor endothelial cells at 21 hpf and 28 hpf in zebrafish
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ABSTRACT:
ORGANISM(S): Danio rerio
PROVIDER: GSE197757 | GEO | 2022/03/26
REPOSITORIES: GEO
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Project description:scRNA-seq of roof and floor endothelial cells at 21 hpf and 28 hpf in zebrafish
| PRJNA812001 | ENA
Project description:How cells acquire their fate is a fundamental question in both developmental and regenerative biology. Multipotent progenitors undergo gradual cell fate restriction in response to temporal and positional cues from the microenvironment, the nature of which is far from being clear. In the case of the lymphatic system, venous endothelial cells are thought to give rise to lymphatic vessels, through a process of trans-differentiation. Upon expression of a set of transcription factors, venous cells acquire a lymphatic fate, and bud out to generate the lymphatic vasculature. In this work we challenge this view and show that while lymphatic endothelial cells (LECs) do arise in the Cardinal Vein (CV), they do so from a previously uncharacterized pool of multipotent angioblasts. Using lymphatic-specific transgenic zebrafish, in combination with endothelial photoconvertible reporters, and long-term live imaging, we demonstrate that these multipotent angioblasts can generate not only lymphatic, but also arterious, and venous fates. We further reveal that the underlying endoderm serves as a source of Wnt5b, which acts as a lymphatic inductive signal, promoting the angioblast-to-lymphatic transition. Moreover, Wnt5b induced lymphatic specification in human embryonic stem cells- derived vascular progenitors, suggesting that this process is evolutionary conserved. Our results uncover a novel mechanism of lymphatic vessel formation, whereby multipotent angioblasts and not venous endothelial cells give rise to the lymphatic endothelium, and provide the first characterization of their inductive niche. More broadly, our findings highlight the CV as a plastic and heterogeneous structure containing different cell populations, analogous to the hematopoietic niche in the aortic floor. Following Kaede photoconversion of dorsal or ventral halves of the PCV in Tg(fli1:gal4;uasKaede) embryos at 24 hpf, 6 embryos per group were used for FACS isolation of Kaede photconverted (red) ECs.
2015-05-21 | E-GEOD-65751 | biostudies-arrayexpress
Project description:Human pluripotent stem cells (hPSCs) are a promising source of cells for applications in regenerative medicine. Directed differentiation of hPSCs into specialized cells such as spinal motoneurons or midbrain dopamine (DA) neurons has been achieved. However the effective use of hPSCs for cell therapy has lagged far behind. While mouse PSC-derived DA neurons have shown efficacy in models of Parkinson’s disease, DA neurons derived from human PSCs generally display poor in vivo performance. There are also considerable safety concerns for hPSCs related to their potential for teratoma formation or neural overgrowth. Here we present a novel floor plate-based strategy for the derivation of human DA neurons that efficiently engraft, suggesting that past failures were due to incomplete specification rather than a specific vulnerability of the cells. Midbrain floor plate precursors are derived from hPSCs in days following exposure to small molecule activators of sonic hedgehog (SHH) and canonical WNT signaling. Engraftable midbrain DA neurons are obtained by day 25 and can be maintained in vitro for several months. Extensive in vitro molecular profiling, biochemical and electrophysiological data define developmental progression and confirm identity of hPSC-derived midbrain DA neurons. In vivo survival and function is demonstrated in PD animal models in three host species. Long-term engraftment in 6-OHDA-lesioned mouse and rats demonstrates robust survival of midbrain DA neurons, complete restoration of amphetamine-induced rotation behavior and improvements in tests of forelimb use and akinesia. Finally, scalability is demonstrated by transplantation into Parkinsonian monkeys. Excellent DA neuron survival, function and lack of neural overgrowth in the three animal models tested indicate considerable promise for the development of cell based therapies in PD.
2011-11-07 | GSE32658 | GEO
Project description:RNA-seq transcriptome analysis identified a functional requirement for zebrafish Rfx4 in the developing neural floor plate and roof plate.
2018-02-27 | GSE101390 | GEO
Project description:Human pluripotent stem cells (hPSCs) are a promising source of cells for applications in regenerative medicine. Directed differentiation of hPSCs into specialized cells such as spinal motoneurons or midbrain dopamine (DA) neurons has been achieved. However the effective use of hPSCs for cell therapy has lagged far behind. While mouse PSC-derived DA neurons have shown efficacy in models of Parkinson’s disease, DA neurons derived from human PSCs generally display poor in vivo performance. There are also considerable safety concerns for hPSCs related to their potential for teratoma formation or neural overgrowth. Here we present a novel floor plate-based strategy for the derivation of human DA neurons that efficiently engraft, suggesting that past failures were due to incomplete specification rather than a specific vulnerability of the cells. Midbrain floor plate precursors are derived from hPSCs in days following exposure to small molecule activators of sonic hedgehog (SHH) and canonical WNT signaling. Engraftable midbrain DA neurons are obtained by day 25 and can be maintained in vitro for several months. Extensive in vitro molecular profiling, biochemical and electrophysiological data define developmental progression and confirm identity of hPSC-derived midbrain DA neurons. In vivo survival and function is demonstrated in PD animal models in three host species. Long-term engraftment in 6-OHDA-lesioned mouse and rats demonstrates robust survival of midbrain DA neurons, complete restoration of amphetamine-induced rotation behavior and improvements in tests of forelimb use and akinesia. Finally, scalability is demonstrated by transplantation into Parkinsonian monkeys. Excellent DA neuron survival, function and lack of neural overgrowth in the three animal models tested indicate considerable promise for the development of cell based therapies in PD. Differentiated hESC with three conditions (LSB, LSB/S/F8, LSB/S/F8/CHIR) were subjected to RNA extraction in specific timepoint (day 0, 1, 3, 5, 7, 11, 13, 25) and hybridization on Illumina microarrays. Each sample has 3 or 4 biological repeats. Based on previous study* of dual SMAD inhibition neural induction, we developed new midbrain dopamine neuron protocol. It depends on time specific treatment of below factors (LSB/S/F8/CHIR): L (LDN193189 (BMP inhibitor) , day 0-11), SB (SB431542 (TGF-b signal inhibitor), day 0-5), S (SHH + Purmorphamine (Smo agonist), day 1-7), F8 (FGF8, day 1-7) and CHIR (CHIR99021 (GSK3b inhibitor), day 3-13) LSB and LSB/S/F8 are limited control conditions of dual SMAD only (LSB) or traditional patterning with Sonic and FGF (LSB/S/F8) *Chambers,S.M. et al. Highly efficient neural conversion of human ES and iPS cells by dual inhibition of SMAD signaling. Nat. Biotechnol. 27, 275-280 (2009).
2011-11-07 | E-GEOD-32658 | biostudies-arrayexpress
Project description:Floor plate-derived extracellular signaling molecules, including canonical axon guidance cues of the Netrin family, control neuronal circuit organization. Despite the importance of the floor plate as an essential signaling centre in the developing vertebrate central nervous system, no systematic approach to identify binding partners for floor plate-expressed cellsurface and secreted proteins has been carried out. Here, we used a high-throughput assay to discover extracellular protein-protein interactions, which likely take place in the zebrafish floor plate microenvironment. The assembled floor plate network contains 47 interactions including the hitherto not reported interaction between Netrin-1 and Draxin. We further characterized this interaction, narrowed down the binding interface, and demonstrated that Draxin competes with Netrin receptors for binding to Netrin-1. Our results suggest that Draxin functions as an extracellular Netrin signaling modulator in vertebrates. A reciprocal gradient of Draxin might shape or sharpen the active Netrin gradient, thereby critically modulating its effect.
2015-05-31 | E-MTAB-3609 | biostudies-arrayexpress
Project description:The data revealed differential expression between floor plate and ventral lateral region in E10.5 mouse embryo midbrain. Several differentially expressed genes in these regions have been reported in the literature, demonstrating reliability of tissue dissection. Midbrain floor plate and non-overlapping adjacent ventral lateral region of mouse E10.5 embryo midbrain was dissected. Each sample was a pool from 6 embryos. Three replicates for each region were used for the experiment.
2011-05-18 | E-GEOD-25178 | biostudies-arrayexpress
Project description:Transcription factors play diverse roles during embryonic development, combinatorially controlling multiple cellular states in a spatially and temporally defined manner. Resolving the dynamic transcriptional profiles that underlie these patterning processes is essential for understanding embryogenesis at the molecular level. Here we show how temporal, tissue-specific changes in embryonic transcription factor function can be discerned by integrating caged morpholinos (cMOs) with photoactivatable fluorophores, fluorescence-activated cell sorting (FACS), and microarray technologies. As a proof of principle, we have dynamically profiled No tail-a (Ntla)-dependent genes at different stages of axial mesoderm development in zebrafish, discovering and characterizing discrete sets of transcripts that are coincident with either notochord cell fate commitment or differentiation. Our studies demonstrate how optically controlled chemical tools can be use to probe developmental processes with spatiotemporal precision and reveal the sequential activation of distinct transcriptomes within a cell lineage by a single transcriptional factor. Zebrafish zygotes were injected with a mixture of ntla caged morpholino (cMO) and caged fluorescein dextran (cFD), and a 100 µm-diameter region of the shield was UV-irradiated at 6 hours post fertilization (hpf). The UV irradiation generated an active morpholino targeting the ntla 5'UTR and simultaneously labeled the cells with green fluorescence. By 36 hpf, the irradiated, green-fluorescent cells contributed to the medial floor plate rather than the notochord, consistent with earlier proposals that Ntla acts as a transcriptional switch between these two cell fates. Combining these caged reagents with microarray analysis identified transcriptional changes coincident with loss of ntla at 9 hpf and subsequent notochord fate respecification.
2012-03-01 | GSE31880 | GEO
Project description:Transcription factors play diverse roles during embryonic development, combinatorially controlling multiple cellular states in a spatially and temporally defined manner. Resolving the dynamic transcriptional profiles that underlie these patterning processes is essential for understanding embryogenesis at the molecular level. Here we show how temporal, tissue-specific changes in embryonic transcription factor function can be discerned by integrating caged morpholinos (cMOs) with photoactivatable fluorophores, fluorescence-activated cell sorting (FACS), and microarray technologies. As a proof of principle, we have dynamically profiled No tail-a (Ntla)-dependent genes at different stages of axial mesoderm development in zebrafish, discovering and characterizing discrete sets of transcripts that are coincident with either notochord cell fate commitment or differentiation. Our studies demonstrate how optically controlled chemical tools can be use to probe developmental processes with spatiotemporal precision and reveal the sequential activation of distinct transcriptomes within a cell lineage by a single transcriptional factor. Zebrafish zygotes were injected with a mixture of ntla caged morpholino (cMO) and caged fluorescein dextran (cFD), and a 100 µm-diameter region of the posterior chordamesoderm 125 μm anterior to the center of Kupffer’s vesicle was UV-irradiated at 12 hours post fertilization (hpf). The UV irradiation generated an active morpholino targeting the ntla 5'UTR and simultaneously labeled the cells with green fluorescence. By 36 hpf, the irradiated, green-fluorescent cells contributed to both notochord and floor plate, but the notochord cells were partially vacuolated and disorganized. Combining these caged reagents with microarray analysis identified transcriptional changes coincident with loss of ntla at 16 hpf and subsequent notochord differentiation.
2012-03-01 | GSE31881 | GEO