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      Rab5c-mediated endocytic trafficking regulates hematopoietic stem and progenitor cell development via Notch and AKT signaling

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          Abstract

          It is well known that various developmental signals play diverse roles in hematopoietic stem and progenitor cell (HSPC) production; however, how these signaling pathways are orchestrated remains incompletely understood. Here, we report that Rab5c is essential for HSPC specification by endocytic trafficking of Notch and AKT signaling in zebrafish embryos. Rab5c deficiency leads to defects in HSPC production. Mechanistically, Rab5c regulates hemogenic endothelium (HE) specification by endocytic trafficking of Notch ligands and receptor. We further show that the interaction between Rab5c and Appl1 in the endosome is required for the survival of HE in the ventral wall of the dorsal aorta through AKT signaling. Interestingly, Rab5c overactivation can also lead to defects in HSPC production, which is attributed to excessive endolysosomal trafficking inducing Notch signaling defect. Taken together, our findings establish a previously unrecognized role of Rab5c-mediated endocytic trafficking in HSPC development and provide new insights into how spatiotemporal signals are orchestrated to accurately execute cell fate transition.

          Abstract

          Cell-autonomous Notch signaling regulated by the membrane trafficking protein Rab5c plays an instructive role in hematopoietic stem and progenitor cell specification, while the AKT signaling seems to provide a permissive signal to maintain hemogenic endothelium survival.

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          Most cited references74

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          The Tol2kit: a multisite gateway-based construction kit for Tol2 transposon transgenesis constructs.

          Transgenesis is an important tool for assessing gene function. In zebrafish, transgenesis has suffered from three problems: the labor of building complex expression constructs using conventional subcloning; low transgenesis efficiency, leading to mosaicism in transient transgenics and infrequent germline incorporation; and difficulty in identifying germline integrations unless using a fluorescent marker transgene. The Tol2kit system uses site-specific recombination-based cloning (multisite Gateway technology) to allow quick, modular assembly of [promoter]-[coding sequence]-[3' tag] constructs in a Tol2 transposon backbone. It includes a destination vector with a cmlc2:EGFP (enhanced green fluorescent protein) transgenesis marker and a variety of widely useful entry clones, including hsp70 and beta-actin promoters; cytoplasmic, nuclear, and membrane-localized fluorescent proteins; and internal ribosome entry sequence-driven EGFP cassettes for bicistronic expression. The Tol2kit greatly facilitates zebrafish transgenesis, simplifies the sharing of clones, and enables large-scale projects testing the functions of libraries of regulatory or coding sequences. Copyright 2007 Wiley-Liss, Inc.
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            Genetic compensation triggered by mutant mRNA degradation

            Genetic robustness, or the ability of an organism to maintain fitness in the presence of mutations, can be achieved via protein feedback loops. Recent evidence suggests that organisms may also respond to mutations by upregulating related gene(s) independently of protein feedback loops, a phenomenon called transcriptional adaptation. However, the prevalence of transcriptional adaptation and its underlying molecular mechanisms are unknown. Here, by analyzing several models of transcriptional adaptation in zebrafish and mouse, we show a requirement for mRNA degradation. Alleles that fail to transcribe the mutated gene do not display transcriptional adaptation and exhibit more severe phenotypes than alleles displaying mutant mRNA decay. Transcriptome analysis reveals the upregulation of a substantial proportion of the genes that exhibit sequence similarity with the mutated gene’s mRNA, suggesting a sequence dependent mechanism. Besides implications for our understanding of disease-causing mutations, these findings will help design mutant alleles with minimal transcriptional adaptation-derived compensation.
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              Endocytosis and signalling: intertwining molecular networks.

              Cell signalling and endocytic membrane trafficking have traditionally been viewed as distinct processes. Although our present understanding is incomplete and there are still great controversies, it is now recognized that these processes are intimately and bidirectionally linked in animal cells. Indeed, many recent examples illustrate how endocytosis regulates receptor signalling (including signalling from receptor tyrosine kinases and G protein-coupled receptors) and, conversely, how signalling regulates the endocytic pathway. The mechanistic and functional principles that underlie the relationship between signalling and endocytosis in cell biology are becoming increasingly evident across many systems.
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                Author and article information

                Contributors
                Role: ConceptualizationRole: InvestigationRole: Writing – original draft
                Role: Investigation
                Role: Investigation
                Role: Investigation
                Role: Investigation
                Role: Investigation
                Role: ConceptualizationRole: Funding acquisitionRole: Project administrationRole: SupervisionRole: Writing – original draftRole: Writing – review & editing
                Role: Academic Editor
                Journal
                PLoS Biol
                PLoS Biol
                plos
                plosbiol
                PLoS Biology
                Public Library of Science (San Francisco, CA USA )
                1544-9173
                1545-7885
                10 April 2020
                April 2020
                10 April 2020
                : 18
                : 4
                : e3000696
                Affiliations
                [1 ] State Key Laboratory of Membrane Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China
                [2 ] Institute for Stem Cell and Regeneration, Chinese Academy of Sciences, Beijing, China
                [3 ] University of Chinese Academy of Sciences, Beijing, China
                [4 ] State Key Laboratory of Experimental Hematology, Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin, China
                National Cancer Institute, UNITED STATES
                Author notes

                The authors have declared that no competing interests exist.

                Author information
                http://orcid.org/0000-0002-1208-9757
                http://orcid.org/0000-0003-4788-3237
                http://orcid.org/0000-0002-9710-5970
                http://orcid.org/0000-0003-3228-0943
                Article
                PBIOLOGY-D-19-03269
                10.1371/journal.pbio.3000696
                7176290
                32275659
                3ae0d2c6-184e-44b7-af88-5a0eae87d43f
                © 2020 Heng et al

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 6 November 2019
                : 24 March 2020
                Page count
                Figures: 7, Tables: 0, Pages: 28
                Funding
                Funded by: funder-id http://dx.doi.org/10.13039/501100001809, National Natural Science Foundation of China;
                Award ID: 31830061
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/501100001809, National Natural Science Foundation of China;
                Award ID: 81530004
                Award Recipient :
                The research was funded by grants from the National Key Research and Development Program of China (2018YFA0800200, and 2016YFA0100500, FL; 2018YFA0801000, DM), the Strategic Priority Research Program of the Chinese Academy of Sciences, China (XDA16010207, FL), the National Natural Science Foundation of China (31830061, 81530004, and 31425016, FL), and Youth Innovation Promotion Association, CAS (2016083, DM). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Biology and Life Sciences
                Cell Biology
                Signal Transduction
                Cell Signaling
                Notch Signaling
                Biology and Life Sciences
                Developmental Biology
                Embryology
                Embryos
                Research and analysis methods
                Biological cultures
                Cell lines
                293T cells
                Research and Analysis Methods
                Animal Studies
                Experimental Organism Systems
                Model Organisms
                Zebrafish
                Research and Analysis Methods
                Model Organisms
                Zebrafish
                Research and Analysis Methods
                Animal Studies
                Experimental Organism Systems
                Animal Models
                Zebrafish
                Biology and Life Sciences
                Organisms
                Eukaryota
                Animals
                Vertebrates
                Fish
                Osteichthyes
                Zebrafish
                Biology and Life Sciences
                Biochemistry
                Proteins
                Luminescent Proteins
                Green Fluorescent Protein
                Biology and Life Sciences
                Cell Biology
                Cellular Structures and Organelles
                Vesicles
                Endosomes
                Research and Analysis Methods
                Specimen Preparation and Treatment
                Staining
                Immunostaining
                Biology and Life Sciences
                Developmental Biology
                Fertilization
                Custom metadata
                vor-update-to-uncorrected-proof
                2020-04-22
                The relevant data are within the paper and its Supporting Information files. The original RNA-Seq data of fli1a+ cells in control and rab5c morphants have been deposited in the SRA database under accession number PRJNA542436.

                Life sciences
                Life sciences

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