Pub Date : 2023-06-15DOI: 10.1101/2023.06.15.545059
Christopher B. Cooke, Christopher Barrington, Peter Baillie-Benson, J. Nichols, Naomi Moris
Primordial Germ Cells (PGCs) are the early embryonic precursors of gametes - sperm and egg cells. PGC-like cells (PGCLCs) can currently be derived in vitro from pluripotent cells exposed to signalling cocktails and aggregated into large embryonic bodies, but these do not recapitulate the native embryonic environment during PGC formation. Here we show that mouse gastruloids, a three-dimensional in vitro model of gastrulation, contain a population of Gastruloid-derived PGC-like cells (Gld-PGCLCs) that resemble early PGCs in vivo. Importantly, the conserved organisation of mouse gastruloids leads to coordinated spatial and temporal localisation of Gld-PGCLCs relative to surrounding somatic cells, even in the absence of specific exogenous PGC-specific signalling or extraembryonic tissues. In gastruloids, self-organised interactions between cells and tissues, including the endodermal epithelium, enables the specification and subsequent maturation of a pool of Gld-PGCLCs. As such, mouse gastruloids represent a new source of PGCLCs in vitro and, due to their inherent co-development, serve as a novel model to study the dynamics of PGC development within integrated tissue environments.
{"title":"Gastruloid-derived primordial germ cell-like cells develop dynamically within integrated tissues","authors":"Christopher B. Cooke, Christopher Barrington, Peter Baillie-Benson, J. Nichols, Naomi Moris","doi":"10.1101/2023.06.15.545059","DOIUrl":"https://doi.org/10.1101/2023.06.15.545059","url":null,"abstract":"Primordial Germ Cells (PGCs) are the early embryonic precursors of gametes - sperm and egg cells. PGC-like cells (PGCLCs) can currently be derived in vitro from pluripotent cells exposed to signalling cocktails and aggregated into large embryonic bodies, but these do not recapitulate the native embryonic environment during PGC formation. Here we show that mouse gastruloids, a three-dimensional in vitro model of gastrulation, contain a population of Gastruloid-derived PGC-like cells (Gld-PGCLCs) that resemble early PGCs in vivo. Importantly, the conserved organisation of mouse gastruloids leads to coordinated spatial and temporal localisation of Gld-PGCLCs relative to surrounding somatic cells, even in the absence of specific exogenous PGC-specific signalling or extraembryonic tissues. In gastruloids, self-organised interactions between cells and tissues, including the endodermal epithelium, enables the specification and subsequent maturation of a pool of Gld-PGCLCs. As such, mouse gastruloids represent a new source of PGCLCs in vitro and, due to their inherent co-development, serve as a novel model to study the dynamics of PGC development within integrated tissue environments.","PeriodicalId":77105,"journal":{"name":"Development (Cambridge, England). Supplement","volume":"330 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2023-06-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"80470756","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2023-06-14DOI: 10.1101/2023.06.14.544953
Yi-Chen Chen, Daisuke Saito, Takayuki Suzuki, T. Takemoto
Chicken embryos are a powerful and widely used animal model in developmental biology studies. After the development of CRISPR technology, gene-edited chickens have been generated by transferring primordial germ cells (PGCs) after genetic modifications. However, the low inheritance caused by the competition between host germ cells and the transferred ones is the most common complication and largely reduces the production efficiency in this way. Here, we generated a gene-edited chicken, in which germ cells can be ablated in a drug-dependent manner, as recipients for gene-edited PGC transfer. We used the nitroreductase/metronidazole (NTR/Mtz) system for cell ablation, in which NTR produces cytotoxic alkylating agents from administered Mtz, causing cell apoptosis. The chicken Vasa homolog (CVH) gene locus is used to drive the expression of the NTR gene in a germ cell-specific manner. In addition, a fluorescent protein gene, mCherry, was also placed in the CVH locus to visualize the PGCs. We named this system germ cell-Specific AutonoMoUs RemovAl Induction (gSAMURAI). gSAMURAI chickens will be an ideal recipient to produce offspring derived from transplanted exogenous germ cells.
{"title":"An inducible germ cell ablation chicken model for high-grade germline chimeras","authors":"Yi-Chen Chen, Daisuke Saito, Takayuki Suzuki, T. Takemoto","doi":"10.1101/2023.06.14.544953","DOIUrl":"https://doi.org/10.1101/2023.06.14.544953","url":null,"abstract":"Chicken embryos are a powerful and widely used animal model in developmental biology studies. After the development of CRISPR technology, gene-edited chickens have been generated by transferring primordial germ cells (PGCs) after genetic modifications. However, the low inheritance caused by the competition between host germ cells and the transferred ones is the most common complication and largely reduces the production efficiency in this way. Here, we generated a gene-edited chicken, in which germ cells can be ablated in a drug-dependent manner, as recipients for gene-edited PGC transfer. We used the nitroreductase/metronidazole (NTR/Mtz) system for cell ablation, in which NTR produces cytotoxic alkylating agents from administered Mtz, causing cell apoptosis. The chicken Vasa homolog (CVH) gene locus is used to drive the expression of the NTR gene in a germ cell-specific manner. In addition, a fluorescent protein gene, mCherry, was also placed in the CVH locus to visualize the PGCs. We named this system germ cell-Specific AutonoMoUs RemovAl Induction (gSAMURAI). gSAMURAI chickens will be an ideal recipient to produce offspring derived from transplanted exogenous germ cells.","PeriodicalId":77105,"journal":{"name":"Development (Cambridge, England). Supplement","volume":"82 298 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2023-06-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"83323524","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2023-03-30DOI: 10.1101/2022.08.15.502524
Anupama Rao, Baken Lyu, Ishrat Jahan, Anna Lubertozzi, Gao Zhou, Frank A. Tedeschi, E. Jankowsky, Junsu Kang, B. Carstens, K. Poss, Kedryn K. Baskin, J. A. Goldman
The eIF4E family of translation initiation factors bind 5’ methylated caps and act as the limiting-step for mRNA translation. The canonical eIF4E1A is required for cell viability, yet other related eIF4E families exist and are utilized in specific contexts or tissues. Here, we describe a family called Eif4e1c for which we find roles during heart development and regeneration in zebrafish. The Eif4e1c family is present in all aquatic vertebrates but is lost in all terrestrial species. A core group of amino acids shared over 500 million years of evolution forms an interface along the protein surface, suggesting Eif4e1c functions in a novel pathway. Deletion of eif4e1c in zebrafish caused growth deficits and impaired survival in juveniles. Mutants surviving to adulthood had fewer cardiomyocytes and reduced proliferative responses to cardiac injury. Ribosome profiling of mutant hearts demonstrated changes in translation efficiency of mRNA for genes known to regulate cardiomyocyte proliferation. Although eif4e1c is broadly expressed, its disruption had most notable impact on the heart and at juvenile stages. Our findings reveal context-dependent requirements for translation initiation regulators during heart regeneration.
{"title":"The translation initiation factor homolog eif4e1c regulates cardiomyocyte metabolism and proliferation during heart regeneration","authors":"Anupama Rao, Baken Lyu, Ishrat Jahan, Anna Lubertozzi, Gao Zhou, Frank A. Tedeschi, E. Jankowsky, Junsu Kang, B. Carstens, K. Poss, Kedryn K. Baskin, J. A. Goldman","doi":"10.1101/2022.08.15.502524","DOIUrl":"https://doi.org/10.1101/2022.08.15.502524","url":null,"abstract":"The eIF4E family of translation initiation factors bind 5’ methylated caps and act as the limiting-step for mRNA translation. The canonical eIF4E1A is required for cell viability, yet other related eIF4E families exist and are utilized in specific contexts or tissues. Here, we describe a family called Eif4e1c for which we find roles during heart development and regeneration in zebrafish. The Eif4e1c family is present in all aquatic vertebrates but is lost in all terrestrial species. A core group of amino acids shared over 500 million years of evolution forms an interface along the protein surface, suggesting Eif4e1c functions in a novel pathway. Deletion of eif4e1c in zebrafish caused growth deficits and impaired survival in juveniles. Mutants surviving to adulthood had fewer cardiomyocytes and reduced proliferative responses to cardiac injury. Ribosome profiling of mutant hearts demonstrated changes in translation efficiency of mRNA for genes known to regulate cardiomyocyte proliferation. Although eif4e1c is broadly expressed, its disruption had most notable impact on the heart and at juvenile stages. Our findings reveal context-dependent requirements for translation initiation regulators during heart regeneration.","PeriodicalId":77105,"journal":{"name":"Development (Cambridge, England). Supplement","volume":"29 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2023-03-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"76604804","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Epithelial cell death is highly prevalent during development and in adult tissues. It plays an essential role in the regulation of tissue size, shape, and turnover. Cell elimination relies on the concerted remodelling of cell junctions, so-called cell extrusion, which allows the seamless expulsion of dying cells. The dissection of the regulatory mechanism giving rise to a certain number and pattern of cell death was so far limited by our capacity to generate high-throughput quantitative data on cell death/extrusion number and distribution in various perturbed backgrounds. Indeed, quantitative studies of cell death rely so far on manual detection of cell extrusion events or through tedious systematic error-free segmentation and cell tracking. Recently, deep learning was used to automatically detect cell death and cell division in cell culture mostly using transmission light microscopy. However, so far, no method was developed for fluorescent images and confocal microscopy, which constitute most datasets in embryonic epithelia. Here, we devised DeXtrusion, a pipeline for automatic detection of cell extrusion/cell death events in larges movies of epithelia marked with cell contour and based on recurrent neural networks. The pipeline, initially trained on large movies of the Drosophila pupal notum marked with fluorescent E-cadherin, is easily trainable, provides fast and accurate extrusion/cell death predictions in a large range of imaging conditions, and can also detect other cellular events such as cell division or cell differentiation. It also performs well on other epithelial tissues with markers of cell junctions with reasonable retraining.
{"title":"DeXtrusion: automatic recognition of epithelial cell extrusion through machine learning in vivo","authors":"Alexis Villars, Gaelle Letort, Léo Valon, Romain Levayer","doi":"10.1101/2023.02.16.528845","DOIUrl":"https://doi.org/10.1101/2023.02.16.528845","url":null,"abstract":"Epithelial cell death is highly prevalent during development and in adult tissues. It plays an essential role in the regulation of tissue size, shape, and turnover. Cell elimination relies on the concerted remodelling of cell junctions, so-called cell extrusion, which allows the seamless expulsion of dying cells. The dissection of the regulatory mechanism giving rise to a certain number and pattern of cell death was so far limited by our capacity to generate high-throughput quantitative data on cell death/extrusion number and distribution in various perturbed backgrounds. Indeed, quantitative studies of cell death rely so far on manual detection of cell extrusion events or through tedious systematic error-free segmentation and cell tracking. Recently, deep learning was used to automatically detect cell death and cell division in cell culture mostly using transmission light microscopy. However, so far, no method was developed for fluorescent images and confocal microscopy, which constitute most datasets in embryonic epithelia. Here, we devised DeXtrusion, a pipeline for automatic detection of cell extrusion/cell death events in larges movies of epithelia marked with cell contour and based on recurrent neural networks. The pipeline, initially trained on large movies of the Drosophila pupal notum marked with fluorescent E-cadherin, is easily trainable, provides fast and accurate extrusion/cell death predictions in a large range of imaging conditions, and can also detect other cellular events such as cell division or cell differentiation. It also performs well on other epithelial tissues with markers of cell junctions with reasonable retraining.","PeriodicalId":77105,"journal":{"name":"Development (Cambridge, England). Supplement","volume":"16 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2023-02-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"79531380","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2023-02-13DOI: 10.1101/2022.06.20.496880
Chon U Chan, Fengzhu Xiong, Arthur Michaut, Joana M. N. Vidigueira, O. Pourquié, L. Mahadevan
Developmental morphogenesis is driven by tissue stresses acting on tissue rheology. Direct measurements of forces in small tissues (100μm-1mm) in situ such as in early embryos require high spatial precision and minimal invasiveness. Here we report tissue force microscopy (TiFM) integrating a vertical cantilever probe and live imaging to enable close-loop control of mechanical loading in early chicken embryos. By testing previously qualitatively characterized force-producing tissues in the elongating body axis, we show that TiFM quantitatively captures stress dynamics with high sensitivity. TiFM also provides the capacity of applying a stable, minimally-invasive and physiologically relevant load to drive tissue deformation, which alters morphogenetic progression and cell movements. Together, TiFM addresses a key technological gap in tissue force measurement and manipulation in small developing embryos, and promises to contribute to the quantitative understanding of complex multi-tissue mechanics during development.
{"title":"Direct force measurement and loading on developing tissues in intact avian embryos","authors":"Chon U Chan, Fengzhu Xiong, Arthur Michaut, Joana M. N. Vidigueira, O. Pourquié, L. Mahadevan","doi":"10.1101/2022.06.20.496880","DOIUrl":"https://doi.org/10.1101/2022.06.20.496880","url":null,"abstract":"Developmental morphogenesis is driven by tissue stresses acting on tissue rheology. Direct measurements of forces in small tissues (100μm-1mm) in situ such as in early embryos require high spatial precision and minimal invasiveness. Here we report tissue force microscopy (TiFM) integrating a vertical cantilever probe and live imaging to enable close-loop control of mechanical loading in early chicken embryos. By testing previously qualitatively characterized force-producing tissues in the elongating body axis, we show that TiFM quantitatively captures stress dynamics with high sensitivity. TiFM also provides the capacity of applying a stable, minimally-invasive and physiologically relevant load to drive tissue deformation, which alters morphogenetic progression and cell movements. Together, TiFM addresses a key technological gap in tissue force measurement and manipulation in small developing embryos, and promises to contribute to the quantitative understanding of complex multi-tissue mechanics during development.","PeriodicalId":77105,"journal":{"name":"Development (Cambridge, England). Supplement","volume":"35 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2023-02-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"77972210","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2023-02-02DOI: 10.1101/2023.02.01.526519
Bidushi Chandra, Matthew G. Voas, E. Davies, Rachel H. Roberts-Galbraith
Glia play multifaceted roles in nervous systems in response to injury. Depending on the species, extent of injury, and glial cell type in question, glia can help or hinder the regeneration of neurons. Studying glia in the context of successful regeneration could reveal key features of pro-regenerative glia that could be exploited for improvement of human therapies. Planarian flatworms completely regenerate their nervous systems after injury—including glia—and thus provide a strong model system with which to explore glia in the context of regeneration. Here, we report that planarian glia regenerate after neurons and that glia require neural structures to regenerate near the eyespot. We find that the planarian transcription factor-encoding gene ets-1 promotes glial cell maintenance and regeneration. We also find that ets-1(RNAi) impairs nervous system architecture, neuronal gene expression, and animal behavior. Taken together, the discovery of ets-1 as a regulator of glial persistence presents a critical first step in understanding glial regulation and potential roles of glia in planarian neurobiology. More importantly, we elucidate interrelationships between glia and neurons in the context of robust neural regeneration.
{"title":"Ets-1 transcription factor regulates glial cell regeneration and function in planarians","authors":"Bidushi Chandra, Matthew G. Voas, E. Davies, Rachel H. Roberts-Galbraith","doi":"10.1101/2023.02.01.526519","DOIUrl":"https://doi.org/10.1101/2023.02.01.526519","url":null,"abstract":"Glia play multifaceted roles in nervous systems in response to injury. Depending on the species, extent of injury, and glial cell type in question, glia can help or hinder the regeneration of neurons. Studying glia in the context of successful regeneration could reveal key features of pro-regenerative glia that could be exploited for improvement of human therapies. Planarian flatworms completely regenerate their nervous systems after injury—including glia—and thus provide a strong model system with which to explore glia in the context of regeneration. Here, we report that planarian glia regenerate after neurons and that glia require neural structures to regenerate near the eyespot. We find that the planarian transcription factor-encoding gene ets-1 promotes glial cell maintenance and regeneration. We also find that ets-1(RNAi) impairs nervous system architecture, neuronal gene expression, and animal behavior. Taken together, the discovery of ets-1 as a regulator of glial persistence presents a critical first step in understanding glial regulation and potential roles of glia in planarian neurobiology. More importantly, we elucidate interrelationships between glia and neurons in the context of robust neural regeneration.","PeriodicalId":77105,"journal":{"name":"Development (Cambridge, England). Supplement","volume":"31 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2023-02-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"91231889","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2022-12-23DOI: 10.1101/2022.12.22.521699
C. Yáñez-Domínguez, D. Lagunas-Gómez, D. M. Torres-Cifuentes, M. Bezanilla, O. Pantoja
Newly synthesized membrane proteins pass through the secretory pathway starting at the endoplasmic reticulum and packaged into COPII vesicles to continue to the Golgi apparatus before reaching their membrane of residence. It is known that cargo receptor proteins form part of the COPII complex and play a role in the recruitment of cargo proteins for their subsequent transport through the secretory pathway. The role of cornichon proteins is conserved from yeast to vertebrates, but it is poorly characterized in plants. To study the role of this protein in cellular traffic mechanisms in plants, the moss Physcomitrium patens has been selected since it can be studied at the single-cell level. Here, we studied the role of the two moss cornichon homologs in the secretory pathway. Mutant analyzes revealed that cornichon genes regulate different growth processes during the moss life cycle, by controlling auxin transport; with CNIH2 functioning as a specific cargo receptor for the auxin efflux carrier PINA, with the C-terminus of the receptor regulating the interaction and trafficking of PINA.
{"title":"A cornichon protein controls polar localization of the PINA auxin transporter in Physcomitrium patens","authors":"C. Yáñez-Domínguez, D. Lagunas-Gómez, D. M. Torres-Cifuentes, M. Bezanilla, O. Pantoja","doi":"10.1101/2022.12.22.521699","DOIUrl":"https://doi.org/10.1101/2022.12.22.521699","url":null,"abstract":"Newly synthesized membrane proteins pass through the secretory pathway starting at the endoplasmic reticulum and packaged into COPII vesicles to continue to the Golgi apparatus before reaching their membrane of residence. It is known that cargo receptor proteins form part of the COPII complex and play a role in the recruitment of cargo proteins for their subsequent transport through the secretory pathway. The role of cornichon proteins is conserved from yeast to vertebrates, but it is poorly characterized in plants. To study the role of this protein in cellular traffic mechanisms in plants, the moss Physcomitrium patens has been selected since it can be studied at the single-cell level. Here, we studied the role of the two moss cornichon homologs in the secretory pathway. Mutant analyzes revealed that cornichon genes regulate different growth processes during the moss life cycle, by controlling auxin transport; with CNIH2 functioning as a specific cargo receptor for the auxin efflux carrier PINA, with the C-terminus of the receptor regulating the interaction and trafficking of PINA.","PeriodicalId":77105,"journal":{"name":"Development (Cambridge, England). Supplement","volume":"2 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2022-12-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"88211492","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2022-12-07DOI: 10.1101/2022.12.07.519375
Micaela Lasser, Nawei Sun, Yuxiao Xu, Sheng Wang, Sam Drake, Karen Law, Silvano Gonzalez, Belinda Wang, Vanessa Drury, Octavio Castillo, Y. Zaltsman, Jeanselle Dea, Ethel Bader, Kate McCluskey, M. State, A. Willsey, H. Willsey
Gene ontology analyses of high confidence autism spectrum disorder (hcASD) risk genes have historically highlighted chromatin regulation and synaptic function as major contributors to pathobiology. Our recent functional work in vivo has additionally implicated microtubule biology and identified disrupted cellular proliferation as a convergent ASD phenotype. As many chromatin regulators, including ASD risk genes ADNP and CHD3, are known to directly regulate both tubulins and histones, we studied the five chromatin regulators most strongly associated with ASD (ADNP, CHD8, CHD2, POGZ, and SUV420H1/KMT5B) specifically with respect to microtubule biology. We observe that all five localize to microtubules of the mitotic spindle in vitro and in vivo. Further in-depth investigation of CHD2 provides evidence that patient-derived mutations lead to a range of microtubule-related phenotypes, including disrupted localization of the protein at the mitotic spindle, spindle defects, cell cycle stalling, DNA damage, and cell death. Lastly, we observe that ASD genetic risk is significantly enriched among microtubule-associated proteins, suggesting broader relevance. Together, these results provide further evidence that the role of tubulin biology and cellular proliferation in ASD warrant further investigation and highlight the pitfalls of relying solely on annotated gene functions in the search for pathological mechanisms.
{"title":"Pleiotropy of autism-associated chromatin regulators","authors":"Micaela Lasser, Nawei Sun, Yuxiao Xu, Sheng Wang, Sam Drake, Karen Law, Silvano Gonzalez, Belinda Wang, Vanessa Drury, Octavio Castillo, Y. Zaltsman, Jeanselle Dea, Ethel Bader, Kate McCluskey, M. State, A. Willsey, H. Willsey","doi":"10.1101/2022.12.07.519375","DOIUrl":"https://doi.org/10.1101/2022.12.07.519375","url":null,"abstract":"Gene ontology analyses of high confidence autism spectrum disorder (hcASD) risk genes have historically highlighted chromatin regulation and synaptic function as major contributors to pathobiology. Our recent functional work in vivo has additionally implicated microtubule biology and identified disrupted cellular proliferation as a convergent ASD phenotype. As many chromatin regulators, including ASD risk genes ADNP and CHD3, are known to directly regulate both tubulins and histones, we studied the five chromatin regulators most strongly associated with ASD (ADNP, CHD8, CHD2, POGZ, and SUV420H1/KMT5B) specifically with respect to microtubule biology. We observe that all five localize to microtubules of the mitotic spindle in vitro and in vivo. Further in-depth investigation of CHD2 provides evidence that patient-derived mutations lead to a range of microtubule-related phenotypes, including disrupted localization of the protein at the mitotic spindle, spindle defects, cell cycle stalling, DNA damage, and cell death. Lastly, we observe that ASD genetic risk is significantly enriched among microtubule-associated proteins, suggesting broader relevance. Together, these results provide further evidence that the role of tubulin biology and cellular proliferation in ASD warrant further investigation and highlight the pitfalls of relying solely on annotated gene functions in the search for pathological mechanisms.","PeriodicalId":77105,"journal":{"name":"Development (Cambridge, England). Supplement","volume":"44 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2022-12-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"80706292","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2022-12-02DOI: 10.1101/2022.11.30.518567
Damilola Olatunji, Natalie M. Clark, Dior R. Kelley
Myosins are evolutionarily conserved motor proteins that interact with actin filaments to regulate organelle transport, cytoplasmic streaming and cell growth. Plant-specific Class XI myosin proteins direct cell division and root organogenesis. However, the roles of plantspecific Class VIII myosin proteins in plant growth and development are less understood. Here, we investigated the function of an auxin-regulated Class VIII myosin, Arabidopsis thaliana Myosin 1 (ATM1), using genetics, transcriptomics, and live cell microscopy. ATM1 is expressed in the primary root, adventitious roots and throughout lateral root development. ATM1 is a plasma membrane localized protein that is enriched in actively dividing cells in the root apical meristem (RAM). Loss of ATM1 function results in impaired primary root growth due to decreased RAM size and reduced cell proliferation in a sugar-dependent manner. In ATM1 loss-of-function roots, columella reporter gene expression is diminished, and fewer columella stem cell divisions occur. In addition, atm1-1 roots displayed reduced auxin responses and auxin marker gene expression. Complementation of atm1-1 with a tagged ATM1 driven under the native ATM1 promoter restored root growth and cell cycle progression in the root meristem. Collectively, these results provide novel evidence that ATM1 functions to influence cell proliferation and columella differentiation in primary roots in response to auxin and sugar cues.
{"title":"The class VIII myosin ATM1 is required for root apical meristem function","authors":"Damilola Olatunji, Natalie M. Clark, Dior R. Kelley","doi":"10.1101/2022.11.30.518567","DOIUrl":"https://doi.org/10.1101/2022.11.30.518567","url":null,"abstract":"Myosins are evolutionarily conserved motor proteins that interact with actin filaments to regulate organelle transport, cytoplasmic streaming and cell growth. Plant-specific Class XI myosin proteins direct cell division and root organogenesis. However, the roles of plantspecific Class VIII myosin proteins in plant growth and development are less understood. Here, we investigated the function of an auxin-regulated Class VIII myosin, Arabidopsis thaliana Myosin 1 (ATM1), using genetics, transcriptomics, and live cell microscopy. ATM1 is expressed in the primary root, adventitious roots and throughout lateral root development. ATM1 is a plasma membrane localized protein that is enriched in actively dividing cells in the root apical meristem (RAM). Loss of ATM1 function results in impaired primary root growth due to decreased RAM size and reduced cell proliferation in a sugar-dependent manner. In ATM1 loss-of-function roots, columella reporter gene expression is diminished, and fewer columella stem cell divisions occur. In addition, atm1-1 roots displayed reduced auxin responses and auxin marker gene expression. Complementation of atm1-1 with a tagged ATM1 driven under the native ATM1 promoter restored root growth and cell cycle progression in the root meristem. Collectively, these results provide novel evidence that ATM1 functions to influence cell proliferation and columella differentiation in primary roots in response to auxin and sugar cues.","PeriodicalId":77105,"journal":{"name":"Development (Cambridge, England). Supplement","volume":"17 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2022-12-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"74785055","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2022-11-24DOI: 10.1101/2022.11.23.517632
Helen Thompson, Weiran Shen, Rodrigo Matus, Medhavi Kakkar, Carl M Jones, David Dolan, Sushma N. Grellscheid, Xiyan Yang, Na Zhang, S. Mozaffari-Jovin, Chunli Chen, Xianlong Zhang, J. Topping, K. Lindsey
Plants respond to environmental stresses through controlled stem cell maintenance and meristem activity. One level of transcriptional control is RNA alternative splicing. However the mechanistic link between stress, meristem function and RNA splicing is poorly understood. The MERISTEM-DEFECTIVE (MDF)/DEFECTIVELY ORGANIZED TRIBUTARIES (DOT2) gene of Arabidopsis encodes a SR-related family protein, required for meristem function and leaf vascularization, and is the likely orthologue of the human SART1 and yeast snu66 splicing factors. MDF is required for the correct splicing and expression of key transcripts associated with root meristem function. We identified RSZ33 and ACC1, both known to regulate cell patterning, as splicing targets required for MDF function in the meristem. MDF expression is modulated by osmotic and cold stress, associated with differential splicing and specific isoform accumulation and shuttling between nucleus and cytosol, and acts in part via a splicing target SR34. We propose a model in which MDF controls splicing in the root meristem to promote stemness and repress stress response and cell differentiation pathways. Summary statement The protein MERISTEM-DEFECTIVE regulates Arabidopsis meristem function through its role as a splicing factor, mediated through splicing targets RSZ33, ACC1 and SR34.
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