Pub Date : 2024-09-07DOI: 10.1101/2024.09.04.611166
Rebecca Armstrong, Nikki J Marks, Timothy G Geary, John Harrington, Paul Selzer, Aaron G. Maule
Infection by the liver fluke, Fasciola hepatica, places a substantial burden on the global agri-food industry and poses a significant threat to human health in endemic regions. Widespread resistance to a limited arsenal of chemotherapeutics, including the frontline flukicide triclabendazole (TCBZ), renders F. hepatica control unsustainable and accentuates the need for novel therapeutic target discovery. A key facet of F. hepatica biology is a population of specialised stem cells which drive growth and development - their dysregulation is hypothesised to represent an appealing avenue for control. The exploitation of this system as a therapeutic target is impeded by a lack of understanding of the molecular mechanisms underpinning F. hepatica growth and development. Wnt signalling pathways govern a myriad of stem cell processes during embryogenesis and drive tumorigenesis in adult tissues. Here, we identify five putative Wnt ligands and five Frizzled receptors in liver fluke transcriptomic datasets and find that Wnt/β-catenin signalling is most active in juveniles, the most pathogenic life stage. FISH-mediated transcript localisation revealed partitioning of the five Wnt ligands, with each displaying a distinct expression pattern, consistent with each Wnt regulating the development of different cell/tissue types. The silencing of each individual Wnt or Frizzled gene yielded significant reductions in juvenile worm growth and, in select cases, blunted the proliferation of neoblast-like cells. Notably, silencing FhCTNNB1, the key effector of the Wnt/β-catenin signal cascade led to aberrant development of the neuromuscular system which ultimately proved fatal - the first report of a lethal RNAi-induced phenotype in F. hepatica. The absence of any discernible phenotypes following the silencing of the inhibitory Wnt/β-catenin destruction complex components is consistent with low destruction complex activity in rapidly developing juvenile worms, corroborates transcriptomic expression profiles and underscores the importance of Wnt signalling as a key molecular driver of growth and development in early stage juvenile fluke. The pharmacological inhibition of Wnt/β-catenin signalling using commercially available inhibitors phenocopied RNAi results and provides impetus for drug repurposing. Taken together, these data functionally and chemically validate the targeting of Wnt signalling as a novel strategy to undermine the pathogenicity of juvenile F. hepatica.
{"title":"Wnt/β-catenin signalling underpins juvenile Fasciola hepatica growth and development","authors":"Rebecca Armstrong, Nikki J Marks, Timothy G Geary, John Harrington, Paul Selzer, Aaron G. Maule","doi":"10.1101/2024.09.04.611166","DOIUrl":"https://doi.org/10.1101/2024.09.04.611166","url":null,"abstract":"Infection by the liver fluke, Fasciola hepatica, places a substantial burden on the global agri-food industry and poses a significant threat to human health in endemic regions. Widespread resistance to a limited arsenal of chemotherapeutics, including the frontline flukicide triclabendazole (TCBZ), renders F. hepatica control unsustainable and accentuates the need for novel therapeutic target discovery. A key facet of F. hepatica biology is a population of specialised stem cells which drive growth and development - their dysregulation is hypothesised to represent an appealing avenue for control. The exploitation of this system as a therapeutic target is impeded by a lack of understanding of the molecular mechanisms underpinning F. hepatica growth and development. Wnt signalling pathways govern a myriad of stem cell processes during embryogenesis and drive tumorigenesis in adult tissues. Here, we identify five putative Wnt ligands and five Frizzled receptors in liver fluke transcriptomic datasets and find that Wnt/β-catenin signalling is most active in juveniles, the most pathogenic life stage. FISH-mediated transcript localisation revealed partitioning of the five Wnt ligands, with each displaying a distinct expression pattern, consistent with each Wnt regulating the development of different cell/tissue types. The silencing of each individual Wnt or Frizzled gene yielded significant reductions in juvenile worm growth and, in select cases, blunted the proliferation of neoblast-like cells. Notably, silencing FhCTNNB1, the key effector of the Wnt/β-catenin signal cascade led to aberrant development of the neuromuscular system which ultimately proved fatal - the first report of a lethal RNAi-induced phenotype in F. hepatica. The absence of any discernible phenotypes following the silencing of the inhibitory Wnt/β-catenin destruction complex components is consistent with low destruction complex activity in rapidly developing juvenile worms, corroborates transcriptomic expression profiles and underscores the importance of Wnt signalling as a key molecular driver of growth and development in early stage juvenile fluke. The pharmacological inhibition of Wnt/β-catenin signalling using commercially available inhibitors phenocopied RNAi results and provides impetus for drug repurposing. Taken together, these data functionally and chemically validate the targeting of Wnt signalling as a novel strategy to undermine the pathogenicity of juvenile F. hepatica.","PeriodicalId":501269,"journal":{"name":"bioRxiv - Developmental Biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-09-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142211605","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}
Oocytes having meiotic defects are assumed to be eliminated by apoptosis in perinatal period. However, the oocyte apoptosis caused by meiotic defects has not been well analyzed, partly due to the great technical demands for tissue sectioning of perinatal ovaries. In the present study, we applied a squash method for immunohistochemical analysis of perinatal mouse ovaries as a substitute for tissue sectioning. As a result, we could show different kinetics of apoptosis caused by DMC1- and SPO11-deficiencies, indicating that DNA damage-induced apoptosis precedes asynapsis-induced apoptosis in mouse oocytes. Double mutant analysis revealed that only asynapsis-induced apoptosis was significantly dependent on HORMAD2. The present method is simple, easy, and able to analyze a sufficient number of oocytes for detecting infrequent events in a single specimen, accelerating detailed immunohistochemical analyses of mammalian ovaries during the fetal and perinatal periods.
{"title":"A simple immunohistochemical method for perinatal mammalian ovaries revealed different kinetics of oocyte apoptosis caused by DNA damage and asynapsis.","authors":"Hiroshi Kogo, Akiko Iizuka-Kogo, Hanako Yamamoto, Maiko Ikezawa, Yukiko Tajika, Toshiyuki Matsuzaki","doi":"10.1101/2024.09.05.611563","DOIUrl":"https://doi.org/10.1101/2024.09.05.611563","url":null,"abstract":"Oocytes having meiotic defects are assumed to be eliminated by apoptosis in perinatal period. However, the oocyte apoptosis caused by meiotic defects has not been well analyzed, partly due to the great technical demands for tissue sectioning of perinatal ovaries. In the present study, we applied a squash method for immunohistochemical analysis of perinatal mouse ovaries as a substitute for tissue sectioning. As a result, we could show different kinetics of apoptosis caused by DMC1- and SPO11-deficiencies, indicating that DNA damage-induced apoptosis precedes asynapsis-induced apoptosis in mouse oocytes. Double mutant analysis revealed that only asynapsis-induced apoptosis was significantly dependent on HORMAD2. The present method is simple, easy, and able to analyze a sufficient number of oocytes for detecting infrequent events in a single specimen, accelerating detailed immunohistochemical analyses of mammalian ovaries during the fetal and perinatal periods.","PeriodicalId":501269,"journal":{"name":"bioRxiv - Developmental Biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-09-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142226681","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 : 2024-09-07DOI: 10.1101/2024.09.05.611553
Ioannis Segos, Jens Van Eeckhoven, Simon Berger, Nikhil Mishra, Eric Lambie, Barbara Conradt
The non-random segregation of organelles has been proposed to be an intrinsic mechanism that contributes to cell fate divergence during asymmetric cell division; however, in vivo evidence is sparse. Using super-resolution microscopy, we analysed the segregation of organelles during the division of the neuroblast QL.p in C. elegans larvae. QL.p divides to generate a daughter that survives, QL.pa, and a daughter that dies, QL.pp. We found that mitochondria segregate unequally by density and morphology and that this is dependent on mitochondrial fission and fusion. Furthermore, we found that mitochondrial density in QL.pp correlates with the time it takes QL.pp to die. We propose that low mitochondrial density in QL.pp promotes the cell death fate and ensures that QL.pp dies in a highly reproducible and timely manner. Our results provide the first in vivo evidence that the non-random segregation of mitochondria can contribute to cell fate divergence during asymmetric cell division.
{"title":"Non-random segregation of mitochondria during asymmetric cell division contributes to cell fate divergence in daughter cells","authors":"Ioannis Segos, Jens Van Eeckhoven, Simon Berger, Nikhil Mishra, Eric Lambie, Barbara Conradt","doi":"10.1101/2024.09.05.611553","DOIUrl":"https://doi.org/10.1101/2024.09.05.611553","url":null,"abstract":"The non-random segregation of organelles has been proposed to be an intrinsic mechanism that contributes to cell fate divergence during asymmetric cell division; however, in vivo evidence is sparse. Using super-resolution microscopy, we analysed the segregation of organelles during the division of the neuroblast QL.p in C. elegans larvae. QL.p divides to generate a daughter that survives, QL.pa, and a daughter that dies, QL.pp. We found that mitochondria segregate unequally by density and morphology and that this is dependent on mitochondrial fission and fusion. Furthermore, we found that mitochondrial density in QL.pp correlates with the time it takes QL.pp to die. We propose that low mitochondrial density in QL.pp promotes the cell death fate and ensures that QL.pp dies in a highly reproducible and timely manner. Our results provide the first in vivo evidence that the non-random segregation of mitochondria can contribute to cell fate divergence during asymmetric cell division.","PeriodicalId":501269,"journal":{"name":"bioRxiv - Developmental Biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-09-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142211598","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 : 2024-09-07DOI: 10.1101/2024.09.06.611673
Devon E Harris, Jongmin J Kim, Sarah R Stern, Hannah M Vicars, Neuza R Matias, Lorenzo Gallicchio, Catherine C Baker, Margaret T Fuller
The switch from precursor cell proliferation to onset of differentiation in adult stem cell lineages must be carefully regulated to produce sufficient progeny to maintain and repair tissues, yet prevent overproliferation that may enable oncogenesis. In the Drosophila male germ cell lineage, spermatogonia produced by germ line stem cells undergo a limited number of transit amplifying mitotic divisions before switching to the spermatocyte program that sets up meiosis and eventual spermatid differentiation. The number of transit amplifying divisions is set by accumulation of the bag-of-marbles (Bam) protein to a critical threshold. In bam mutants, spermatogonia proliferate through several extra rounds of mitosis then die without becoming spermatocytes. Here we show that the key role of Bam for the mitosis to differentiation switch is repressing expression of Held Out Wings (how), homolog of mammalian Quaking. Knock down of how in germ cells was sufficient to allow spermatogonia mutant for bam or its partner benign gonial cell neoplasm (bgcn) to differentiate, while forced expression of nuclear-targeted How protein in spermatogonia wild-type for bam resulted in continued proliferation at the expense of differentiation. Our findings suggest that Bam targets how RNA for degradation by acting as an adapter to recruit the CCR4-NOT deadenylation complex via binding its subunit, Caf40. As How is itself an RNA binding protein with roles in RNA processing, our findings reveal that the switch from proliferation to meiosis and differentiation in the Drosophila male germ line adult stem cell lineage is regulated by a cascade of RNA-binding proteins.
成体干细胞系从前体细胞增殖到开始分化的转换必须经过仔细调节，以产生足够的后代来维持和修复组织，同时防止过度增殖，以免导致肿瘤发生。在果蝇雄性生殖细胞系中，由生殖系干细胞产生的精原细胞在转入精母细胞程序之前，要经历有限次数的有丝分裂中转放大分裂，以启动减数分裂和最终的精子分化。有丝分裂的次数由大理石袋（Bam）蛋白积累到临界阈值决定。在 Bam 突变体中，精原细胞通过几轮额外的有丝分裂增殖，然后在没有成为精母细胞的情况下死亡。在这里，我们发现 Bam 在有丝分裂到分化的转换过程中的关键作用是抑制哺乳动物 Quaking 的同源物 Held Out Wings（how）的表达。敲除生殖细胞中的how足以使bam突变的精原细胞或其伴侣良性绒毛膜细胞瘤（bgcn）分化，而在bam野生型精原细胞中强制表达核靶向How蛋白则会导致精原细胞继续增殖而牺牲分化。我们的研究结果表明，Bam通过结合CCR4-NOT的亚基Caf40，作为适配器招募CCR4-NOT去淀粉化复合物，从而靶向降解how RNA。由于How本身是一种RNA结合蛋白，在RNA加工中发挥作用，我们的研究结果揭示了果蝇雄性生殖系成体干细胞系中从增殖到减数分裂和分化的转换是由一连串RNA结合蛋白调控的。
{"title":"An RNA binding regulatory cascade controls the switch from proliferation to differentiation in the Drosophila male germ line stem cell lineage","authors":"Devon E Harris, Jongmin J Kim, Sarah R Stern, Hannah M Vicars, Neuza R Matias, Lorenzo Gallicchio, Catherine C Baker, Margaret T Fuller","doi":"10.1101/2024.09.06.611673","DOIUrl":"https://doi.org/10.1101/2024.09.06.611673","url":null,"abstract":"The switch from precursor cell proliferation to onset of differentiation in adult stem cell lineages must be carefully regulated to produce sufficient progeny to maintain and repair tissues, yet prevent overproliferation that may enable oncogenesis. In the Drosophila male germ cell lineage, spermatogonia produced by germ line stem cells undergo a limited number of transit amplifying mitotic divisions before switching to the spermatocyte program that sets up meiosis and eventual spermatid differentiation. The number of transit amplifying divisions is set by accumulation of the bag-of-marbles (Bam) protein to a critical threshold. In bam mutants, spermatogonia proliferate through several extra rounds of mitosis then die without becoming spermatocytes. Here we show that the key role of Bam for the mitosis to differentiation switch is repressing expression of Held Out Wings (how), homolog of mammalian Quaking. Knock down of how in germ cells was sufficient to allow spermatogonia mutant for bam or its partner benign gonial cell neoplasm (bgcn) to differentiate, while forced expression of nuclear-targeted How protein in spermatogonia wild-type for bam resulted in continued proliferation at the expense of differentiation. Our findings suggest that Bam targets how RNA for degradation by acting as an adapter to recruit the CCR4-NOT deadenylation complex via binding its subunit, Caf40. As How is itself an RNA binding protein with roles in RNA processing, our findings reveal that the switch from proliferation to meiosis and differentiation in the Drosophila male germ line adult stem cell lineage is regulated by a cascade of RNA-binding proteins.","PeriodicalId":501269,"journal":{"name":"bioRxiv - Developmental Biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-09-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142211612","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 : 2024-09-06DOI: 10.1101/2024.09.04.611313
Chris Siu Yeung Chow, Sumedha Negi, Shaine Chenxin Bao, Chen Fang, James Edward Hudson, Woo Jun Shim, Yuanzhao Cao, Nathan Palpant
Protocols for culturing cardiac cells derived from human-induced pluripotent stem cells (hiPSCs) are critical for studying mechanisms of development and disease. This study provides analysis of a community-oriented database to assess parameters used in published cardiac differentiation protocols, including a web-accessible portal for user-guided customisation of protocols. Validation studies support context-dependent roles for media composition in deriving functional maturation of hiPSC-derived cardiomyocytes.
{"title":"A community-oriented, data-driven resource to improve protocol design for cardiac modelling from human pluripotent stem cells","authors":"Chris Siu Yeung Chow, Sumedha Negi, Shaine Chenxin Bao, Chen Fang, James Edward Hudson, Woo Jun Shim, Yuanzhao Cao, Nathan Palpant","doi":"10.1101/2024.09.04.611313","DOIUrl":"https://doi.org/10.1101/2024.09.04.611313","url":null,"abstract":"Protocols for culturing cardiac cells derived from human-induced pluripotent stem cells (hiPSCs) are critical for studying mechanisms of development and disease. This study provides analysis of a community-oriented database to assess parameters used in published cardiac differentiation protocols, including a web-accessible portal for user-guided customisation of protocols. Validation studies support context-dependent roles for media composition in deriving functional maturation of hiPSC-derived cardiomyocytes.","PeriodicalId":501269,"journal":{"name":"bioRxiv - Developmental Biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-09-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142211613","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}
Neural development requires metabolic adaptations that coincide with a functional shift from differentiation to neurotransmission. Serine metabolism provides essential metabolites for cellular growth and proliferation, and also produces neurotransmitters. However, how serine metabolism coordinates functional development of neurons remains unclear. Here, we report that neurons undergo metabolic transitions through an enantiomeric shift of serine during functional maturation. Developmental alterations of neural transcriptional profiles and serine enantiomers indicated that L- to D-serine conversion is a signature of neural maturation. Metabolomic analysis of neural progenitors revealed that D-serine decreases glycine synthesis, thereby suppressing one-carbon metabolism, in which L-serine is a crucial carbon donor. D-serine inhibits one-carbon metabolism by competing with transport of cytosolic L-serine to mitochondria, which restrains proliferation and triggers apoptosis of neural progenitors as well as neural tumor cells, but not mature neurons, in vitro and ex vivo. Thus, our findings suggest that the metabolic transition from L- to D-serine during neural maturation inhibits one-carbon metabolism essential for proliferation of immature neural cells, leading to acquisition of characteristics tailored to functional development toward neurotransmission.
{"title":"Serine chirality guides metabolic flow between one-carbon metabolism and neuromodulator synthesis","authors":"Masataka Suzuki, Kenichiro Adachi, Pattama Wiriyasermukul, Mariko Fukumura, Ryota Tamura, Yoshinori Hirano, Yumi Aizawa, Tetsuya Miyamoto, Sakiko Taniguchi, Masahiro Toda, Hiroshi Homma, Kohsuke Kanekura, Kenji Yasuoka, Takanori Kanai, Masahiro Sugimoto, Shushi Nagamori, Masato Yasui, Jumpei Sasabe","doi":"10.1101/2024.09.03.610855","DOIUrl":"https://doi.org/10.1101/2024.09.03.610855","url":null,"abstract":"Neural development requires metabolic adaptations that coincide with a functional shift from differentiation to neurotransmission. Serine metabolism provides essential metabolites for cellular growth and proliferation, and also produces neurotransmitters. However, how serine metabolism coordinates functional development of neurons remains unclear. Here, we report that neurons undergo metabolic transitions through an enantiomeric shift of serine during functional maturation. Developmental alterations of neural transcriptional profiles and serine enantiomers indicated that L- to D-serine conversion is a signature of neural maturation. Metabolomic analysis of neural progenitors revealed that D-serine decreases glycine synthesis, thereby suppressing one-carbon metabolism, in which L-serine is a crucial carbon donor. D-serine inhibits one-carbon metabolism by competing with transport of cytosolic L-serine to mitochondria, which restrains proliferation and triggers apoptosis of neural progenitors as well as neural tumor cells, but not mature neurons, in vitro and ex vivo. Thus, our findings suggest that the metabolic transition from L- to D-serine during neural maturation inhibits one-carbon metabolism essential for proliferation of immature neural cells, leading to acquisition of characteristics tailored to functional development toward neurotransmission.","PeriodicalId":501269,"journal":{"name":"bioRxiv - Developmental Biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-09-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142211615","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 : 2024-09-06DOI: 10.1101/2024.09.05.610903
Aleena Patel, Vanessa Gonzalez, Triveni Menon, Stanislav Y Shvartsman, Rebecca D Burdine, Maria Avdeeva
Signaling pathways induce stereotyped transcriptional changes as stem cells progress into mature cell types during embryogenesis. Signaling perturbations are necessary to discover which genes are responsive or insensitive to pathway activity. However, gene regulation is additionally dependent on cell state-specific factors like chromatin modifications or transcription factor binding. Thus, transcriptional profiles need to be assayed in single cells to identify potentially multiple, distinct perturbation responses among heterogeneous cell states in an embryo. In perturbation studies, comparing heterogeneous transcriptional states among experimental conditions often requires samples to be collected over multiple independent experiments. Datasets produced in such complex experimental designs can be confounded by batch effects. We present Design-Aware Integration of Single Cell ExpEriments (DAISEE), a new algorithm that models perturbation responses in single-cell datasets with a complex experimental design. We demonstrate that DAISEE improves upon a previously available integrative non-negative matrix factorization framework, more efficiently separating perturbation responses from confounding variation. We use DAISEE to integrate newly collected single-cell RNA-sequencing datasets from 5-hour old zebrafish embryos expressing optimized photoswitchable MEK (psMEK), which globally activates the extracellular signal-regulated kinase (ERK), a signaling molecule involved in many cell specification events. psMEK drives some cells that are normally not exposed to ERK signals towards other wild type states and induces novel states expressing a mixture of transcriptional programs, including precociously activated endothelial genes. ERK signaling is therefore capable of introducing profoundly new gene expression states in developing embryos.
{"title":"Disrupted developmental signaling induces novel transcriptional states","authors":"Aleena Patel, Vanessa Gonzalez, Triveni Menon, Stanislav Y Shvartsman, Rebecca D Burdine, Maria Avdeeva","doi":"10.1101/2024.09.05.610903","DOIUrl":"https://doi.org/10.1101/2024.09.05.610903","url":null,"abstract":"Signaling pathways induce stereotyped transcriptional changes as stem cells progress into mature cell types during embryogenesis. Signaling perturbations are necessary to discover which genes are responsive or insensitive to pathway activity. However, gene regulation is additionally dependent on cell state-specific factors like chromatin modifications or transcription factor binding. Thus, transcriptional profiles need to be assayed in single cells to identify potentially multiple, distinct perturbation responses among heterogeneous cell states in an embryo. In perturbation studies, comparing heterogeneous transcriptional states among experimental conditions often requires samples to be collected over multiple independent experiments. Datasets produced in such complex experimental designs can be confounded by batch effects. We present Design-Aware Integration of Single Cell ExpEriments (DAISEE), a new algorithm that models perturbation responses in single-cell datasets with a complex experimental design. We demonstrate that DAISEE improves upon a previously available integrative non-negative matrix factorization framework, more efficiently separating perturbation responses from confounding variation. We use DAISEE to integrate newly collected single-cell RNA-sequencing datasets from 5-hour old zebrafish embryos expressing optimized photoswitchable MEK (psMEK), which globally activates the extracellular signal-regulated kinase (ERK), a signaling molecule involved in many cell specification events. psMEK drives some cells that are normally not exposed to ERK signals towards other wild type states and induces novel states expressing a mixture of transcriptional programs, including precociously activated endothelial genes. ERK signaling is therefore capable of introducing profoundly new gene expression states in developing embryos.","PeriodicalId":501269,"journal":{"name":"bioRxiv - Developmental Biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-09-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142211616","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 : 2024-09-06DOI: 10.1101/2024.09.03.610938
Alexandra de la Porte, Julia Schröder, Moritz Thomas, Johanna Geuder, Michael Sterr, Xavier Pastor, Leslie E. Sanderson, Tahsin Stefan Barakat, Wolfgang Enard, Carsten Marr, Christian Schröter, Micha Drukker
Embryos from different mammalian species develop at characteristic timescales. These timescales are recapitulated during the differentiation of pluripotent stem cells in vitro. Specific genes and molecular pathways that modulate cell differentiation speed between mammalian species remain to be determined. Here we use single-cell multi-omic analysis of neural differentiation of mouse, cynomolgus and human pluripotent cells to identify regulators for differentiation speed. We demonstrate that species-specific transcriptome dynamics are mirrored at the chromatin level, but that the speed of neural differentiation is insensitive to manipulations of cell growth and cycling. Exploiting the single-cell resolution of our data, we identify glycogen storage levels regulated by UDP-glucose pyrophosphorylase 2 (UGP2) as a species-dependent trait of pluripotent cells, and show that lowered glycogen storage in UGP2 mutant cells is associated with accelerated neural differentiation. The control of energy storage could be a general strategy for the regulation of cell differentiation speed.
{"title":"Single-cell multiome uncovers differences in glycogen metabolism underlying species-specific speed of development","authors":"Alexandra de la Porte, Julia Schröder, Moritz Thomas, Johanna Geuder, Michael Sterr, Xavier Pastor, Leslie E. Sanderson, Tahsin Stefan Barakat, Wolfgang Enard, Carsten Marr, Christian Schröter, Micha Drukker","doi":"10.1101/2024.09.03.610938","DOIUrl":"https://doi.org/10.1101/2024.09.03.610938","url":null,"abstract":"Embryos from different mammalian species develop at characteristic timescales. These timescales are recapitulated during the differentiation of pluripotent stem cells in vitro. Specific genes and molecular pathways that modulate cell differentiation speed between mammalian species remain to be determined. Here we use single-cell multi-omic analysis of neural differentiation of mouse, cynomolgus and human pluripotent cells to identify regulators for differentiation speed. We demonstrate that species-specific transcriptome dynamics are mirrored at the chromatin level, but that the speed of neural differentiation is insensitive to manipulations of cell growth and cycling. Exploiting the single-cell resolution of our data, we identify glycogen storage levels regulated by UDP-glucose pyrophosphorylase 2 (UGP2) as a species-dependent trait of pluripotent cells, and show that lowered glycogen storage in UGP2 mutant cells is associated with accelerated neural differentiation. The control of energy storage could be a general strategy for the regulation of cell differentiation speed.","PeriodicalId":501269,"journal":{"name":"bioRxiv - Developmental Biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-09-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142211444","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 : 2024-09-05DOI: 10.1101/2024.09.04.611161
RICHA ARYA, Papri Das, Smrithi Murthy, Eshan Abbas, Kristin White
The size of a cell is important for its function and physiology. Interestingly, size variation can be easily observed in clonally derived embryonic and hematopoietic stem cells. Here, we investigated the regulation of stem cell growth and its association with cell fate. We observed heterogeneous sizes of neuroblasts or neural stem cells (NSCs) in the Drosophila ventral nerve cord (VNC). Specifically, thoracic NSCs were larger than those in the abdominal region of the VNC. Our research uncovered a significant role of the Hox gene abdominal A (abdA) in the regulation of abdominal NSC growth. Developmental expression of AbdA retards their growth and delays mitotic entry compared to thoracic NSCs. The targeted loss of abdA enhanced their growth and caused an earlier entry into mitosis with a faster cycling rate. Furthermore, ectopic expression of abdA reduced the size of thoracic NSCs and delayed their entry into mitosis. We suggest that abdA plays an instructive role in regulating NSC size and exit from quiescence. This study demonstrates for the first time the involvement of abdA in NSC fate determination by regulating their growth, entry into mitosis and proliferation rate, and thus their potential to make appropriate number of progeny for CNS patterning.
{"title":"The Hox Gene, abdominal A controls timely mitotic entry of neural stem cell and their growth during CNS development in Drosophila","authors":"RICHA ARYA, Papri Das, Smrithi Murthy, Eshan Abbas, Kristin White","doi":"10.1101/2024.09.04.611161","DOIUrl":"https://doi.org/10.1101/2024.09.04.611161","url":null,"abstract":"The size of a cell is important for its function and physiology. Interestingly, size variation can be easily observed in clonally derived embryonic and hematopoietic\u0000stem cells. Here, we investigated the regulation of stem cell growth and its association with cell fate. We observed heterogeneous sizes of neuroblasts or neural stem cells (NSCs) in the Drosophila ventral nerve cord (VNC). Specifically, thoracic NSCs were larger than those in the abdominal region of the VNC. Our research uncovered a significant role of the Hox gene abdominal A (abdA) in the regulation of abdominal NSC growth. Developmental expression of AbdA retards their growth and delays mitotic entry compared to thoracic NSCs. The targeted loss of abdA enhanced their growth and caused an earlier entry into mitosis with a\u0000faster cycling rate. Furthermore, ectopic expression of abdA reduced the size of thoracic NSCs and delayed their entry into mitosis. We suggest that abdA plays an instructive role in regulating NSC size and exit from quiescence. This study demonstrates for the first time the involvement of abdA in NSC fate determination by regulating their growth, entry into mitosis and proliferation rate, and thus their potential to make appropriate number of progeny for CNS patterning.","PeriodicalId":501269,"journal":{"name":"bioRxiv - Developmental Biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-09-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142211618","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 : 2024-09-03DOI: 10.1101/2024.09.03.610492
Matthew French, Rosa Portero Migueles, J Kim Dale, Guillaume Blin, Valerie Wilson, Sally Lowell
Patterning of cell fates is central to embryonic development, tissue homeostasis, and disease. Quantitative analysis of patterning reveals the logic by which cell-cell interactions orchestrate changes in cell fate. However, it is challenging to quantify patterning when graded changes in identity occur over complex 4D trajectories, or where different cell states are intermingled. Furthermore, comparing patterns across multiple individual embryos, tissues, or organoids is difficult because these often vary in shape and size. Here we present a toolkit of computational approaches to tackle these problems. These strategies are based on measuring properties of each cell in relation to the properties of its neighbours to quantify patterning, and on using embryonic landmarks in order to compare these patterns between embryos. We use this toolkit to characterise patterning of cell identities within the caudal lateral epiblast of E8.5 embryos, revealing local patterning in emergence of early mesoderm cells that is sensitive to inhibition of Notch activity.
{"title":"A toolkit for mapping cell identities in relation to neighbours reveals Notch-dependent heterogeneity within neuromesodermal progenitor populations","authors":"Matthew French, Rosa Portero Migueles, J Kim Dale, Guillaume Blin, Valerie Wilson, Sally Lowell","doi":"10.1101/2024.09.03.610492","DOIUrl":"https://doi.org/10.1101/2024.09.03.610492","url":null,"abstract":"Patterning of cell fates is central to embryonic development, tissue homeostasis, and disease. Quantitative analysis of patterning reveals the logic by which cell-cell interactions orchestrate changes in cell fate. However, it is challenging to quantify patterning when graded changes in identity occur over complex 4D trajectories, or where different cell states are intermingled. Furthermore, comparing patterns across multiple individual embryos, tissues, or organoids is difficult because these often vary in shape and size.\u0000Here we present a toolkit of computational approaches to tackle these problems. These strategies are based on measuring properties of each cell in relation to the properties of its neighbours to quantify patterning, and on using embryonic landmarks in order to compare these patterns between embryos. We use this toolkit to characterise patterning of cell identities within the caudal lateral epiblast of E8.5 embryos, revealing local patterning in emergence of early mesoderm cells that is sensitive to inhibition of Notch activity.","PeriodicalId":501269,"journal":{"name":"bioRxiv - Developmental Biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-09-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142211620","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}
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