Embryo culture is crucial to achieve successful outcomes in assisted reproductive technology (ART) for cattle. This study explored the innovative use of dry incubators integrated with time-lapse monitoring systems for bovine embryo culture, building on their advantages in human medicine, such as reduced contamination risk, stable temperature control, and lower gas consumption. Our research demonstrates the feasibility of this approach, showing that although the osmotic pressure gradually increases over the culture period, it remains below the critical threshold for developmental impairment. Embryos cultured in dry incubators exhibited morphokinetics comparable to those cultured in conventional humidified time-lapse incubators. Furthermore, RNA-seq revealed that the transcriptomic profiles of blastocysts cultured in dry incubators closely matched those of blastocysts cultured in humidified incubators. These findings highlight the significant potential of dry incubators with time-lapse monitoring systems for the in vitro production of bovine embryos, marking a promising advancement in assisted reproductive technologies for the livestock industry and research setting.
{"title":"Compatibility of time-lapse dry incubator on in vitro production of bovine embryos","authors":"Haruhisa Tsuji, Hiroki Nagai, Sayaka Kobinata, Hinata Koyama, Atchalalt Khurchabilig, Noritaka Fukunaga, Yoshimasa Asada, Satoshi Sugimura","doi":"10.1101/2024.08.01.606272","DOIUrl":"https://doi.org/10.1101/2024.08.01.606272","url":null,"abstract":"Embryo culture is crucial to achieve successful outcomes in assisted reproductive technology (ART) for cattle. This study explored the innovative use of dry incubators integrated with time-lapse monitoring systems for bovine embryo culture, building on their advantages in human medicine, such as reduced contamination risk, stable temperature control, and lower gas consumption. Our research demonstrates the feasibility of this approach, showing that although the osmotic pressure gradually increases over the culture period, it remains below the critical threshold for developmental impairment. Embryos cultured in dry incubators exhibited morphokinetics comparable to those cultured in conventional humidified time-lapse incubators. Furthermore, RNA-seq revealed that the transcriptomic profiles of blastocysts cultured in dry incubators closely matched those of blastocysts cultured in humidified incubators. These findings highlight the significant potential of dry incubators with time-lapse monitoring systems for the in vitro production of bovine embryos, marking a promising advancement in assisted reproductive technologies for the livestock industry and research setting.","PeriodicalId":501269,"journal":{"name":"bioRxiv - Developmental Biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-08-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141943818","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 : 2024-08-06DOI: 10.1101/2024.08.02.606390
Tess A. Leathers, Raneesh Ramarapu, Crystal D. Rogers
Vertebrate development is regulated by several complex well-characterized morphogen signaling pathways, transcription factors, and structural proteins, but less is known about the enzymatic pathways that regulate early development. We have identified that factors from the inflammation-mediating cyclooxygenase (COX) signaling pathway are expressed at early stages of development in avian embryos. Using Gallus gallus (chicken) as a research model, we characterized the spatiotemporal expression of a subset of genes and proteins in the COX pathway during early neural development stages. Specifically, here we show expression patterns of COX1, COX2, and microsomal prostaglandin E synthase-2 (mPGES-2) as well as the genes encoding these enzymes. Unique expression patterns of individual players within the COX pathway suggest that they may play non-canonical/non-traditional roles in the embryo compared to their roles in the adult. Future work should examine the function of the COX pathway in tissue specification and morphogenesis and determine if these expression patterns are conserved across species.
{"title":"Spatiotemporal Characterization of Cyclooxygenase Pathway Enzymes During Vertebrate Embryonic Development","authors":"Tess A. Leathers, Raneesh Ramarapu, Crystal D. Rogers","doi":"10.1101/2024.08.02.606390","DOIUrl":"https://doi.org/10.1101/2024.08.02.606390","url":null,"abstract":"Vertebrate development is regulated by several complex well-characterized morphogen signaling pathways, transcription factors, and structural proteins, but less is known about the enzymatic pathways that regulate early development. We have identified that factors from the inflammation-mediating cyclooxygenase (COX) signaling pathway are expressed at early stages of development in avian embryos. Using <em>Gallus gallus</em> (chicken) as a research model, we characterized the spatiotemporal expression of a subset of genes and proteins in the COX pathway during early neural development stages. Specifically, here we show expression patterns of COX1, COX2, and microsomal prostaglandin E synthase-2 (mPGES-2) as well as the genes encoding these enzymes. Unique expression patterns of individual players within the COX pathway suggest that they may play non-canonical/non-traditional roles in the embryo compared to their roles in the adult. Future work should examine the function of the COX pathway in tissue specification and morphogenesis and determine if these expression patterns are conserved across species.","PeriodicalId":501269,"journal":{"name":"bioRxiv - Developmental Biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-08-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141943821","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 : 2024-08-06DOI: 10.1101/2024.08.05.606739
Emer Aisling King, Eleanor Jacobsen, Nicholas Woolner, Joaquin de Navascues, Owen J Marshall, Jerome Korzelius
Tissue-resident Adult Stem Cells (ASCs) need to continuously adapt their rate of division and differentiation based on their tissue environment. However, the gene regulatory networks that govern these decisions in ASCs and how they respond to challenges such as infection are often not fully understood. We use the Intestinal Stem Cells (ISCs) that maintain the adult Drosophila intestine or midgut as a model to study how transcriptional regulators govern ASC behaviour. We identify a novel role for the transcription factor (TF) Chronophage (Cph) in ISC proliferation and entero-endocrine (EE) cell differentiation. Cph is a Z2H2 zinc TF orthologous to mammalian BCL11A/B and recent work in Drosophila has demonstrated a role in regulating differentiation of neural stem cells (NSCs). We show here that Cph is expressed in ISCs and EEs in the Drosophila intestine. Increased levels of Cph correlates with increased ISC proliferation and EE differentiation. cph loss-of-function leads to impaired ISC proliferation. Cph levels are elevated during tumourigenesis as well as in ageing and infection conditions. Knockdown of Cph in a Notch-mutant tumour model reduces tumour size and incidence and extends lifespan. Mechanistically, Cph overexpression leads to an increase in enteroendocrine (EE) cells and DamID DNA-binding and qRT-PCR analysis reveals that Cph directly regulates the levels of key EE regulatory genes such as Prospero (pros) and Phyllopod (phyl). In addition, Cph directly regulates core cell cycle regulators such as E2F1 as well as the TF Nerfin-1 that controls ISC proliferation and maintenance. Together, these data support a role for Cph in finetuning the balance between differentiation and proliferation during entero-endocrine differentiation.
{"title":"The transcription factor Chronophage/BCL11A/B promotes intestinal stem cell proliferation and endocrine differentiation in the Drosophila intestine","authors":"Emer Aisling King, Eleanor Jacobsen, Nicholas Woolner, Joaquin de Navascues, Owen J Marshall, Jerome Korzelius","doi":"10.1101/2024.08.05.606739","DOIUrl":"https://doi.org/10.1101/2024.08.05.606739","url":null,"abstract":"Tissue-resident Adult Stem Cells (ASCs) need to continuously adapt their rate of division and differentiation based on their tissue environment. However, the gene regulatory networks that govern these decisions in ASCs and how they respond to challenges such as infection are often not fully understood. We use the Intestinal Stem Cells (ISCs) that maintain the adult Drosophila intestine or midgut as a model to study how transcriptional regulators govern ASC behaviour. We identify a novel role for the transcription factor (TF) Chronophage (Cph) in ISC proliferation and entero-endocrine (EE) cell differentiation. Cph is a Z2H2 zinc TF orthologous to mammalian BCL11A/B and recent work in Drosophila has demonstrated a role in regulating differentiation of neural stem cells (NSCs). We show here that Cph is expressed in ISCs and EEs in the Drosophila intestine. Increased levels of Cph correlates with increased ISC proliferation and EE differentiation. cph loss-of-function leads to impaired ISC proliferation. Cph levels are elevated during tumourigenesis as well as in ageing and infection conditions. Knockdown of Cph in a Notch-mutant tumour model reduces tumour size and incidence and extends lifespan. Mechanistically, Cph overexpression leads to an increase in enteroendocrine (EE) cells and DamID DNA-binding and qRT-PCR analysis reveals that Cph directly regulates the levels of key EE regulatory genes such as Prospero (pros) and Phyllopod (phyl). In addition, Cph directly regulates core cell cycle regulators such as E2F1 as well as the TF Nerfin-1 that controls ISC proliferation and maintenance. Together, these data support a role for Cph in finetuning the balance between differentiation and proliferation during entero-endocrine differentiation.","PeriodicalId":501269,"journal":{"name":"bioRxiv - Developmental Biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-08-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141943826","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 : 2024-08-06DOI: 10.1101/2024.08.05.606734
Liuliu Yang, Yuling Han, Tuo Zhang, Xue Dong, Jian Ge, Aadita Roy, Jiajun Zhu, Tiankun Lu, J. Jeya Vandana, Neranjan de Silva, Catherine C. Robertson, Jenny Z Xiang, Chendong Pan, Yanjie Sun, Jianwen Que, Todd Evans, Chengyang Liu, Wei Wang, Ali Naji, Stephen C.J. Parker, Robert E. Schwartz, Shuibing Chen
There is a paucity of human models to study immune-mediated host damage. Here, we utilized the GeoMx spatial multi-omics platform to analyze immune cell changes in COVID-19 pancreatic autopsy samples, revealing an accumulation of proinflammatory macrophages. Single cell RNA-seq analysis of human islets exposed to SARS-CoV-2 or Coxsackievirus B4 (CVB4) viruses identified activation of proinflammatory macrophages and β cell pyroptosis. To distinguish viral versus proinflammatory macrophage-mediated β cell pyroptosis, we developed human pluripotent stem cell (hPSC)-derived vascularized macrophage-islet (VMI) organoids. VMI organoids exhibited enhanced marker expression and function in both β cells and endothelial cells compared to separately cultured cells. Notably, proinflammatory macrophages within VMI organoids induced β cell pyroptosis. Mechanistic investigations highlighted TNFSF12-TNFRSF12A involvement in proinflammatory macrophage-mediated β cell pyroptosis. This study established hPSC- derived VMI organoids as a valuable tool for studying immune cell-mediated host damage and uncovered mechanism of β cell damage during viral exposure.
{"title":"Human Vascularized Macrophage-Islet Organoids to Model Immune-Mediated Pancreatic β cell Pyroptosis upon Viral Infection","authors":"Liuliu Yang, Yuling Han, Tuo Zhang, Xue Dong, Jian Ge, Aadita Roy, Jiajun Zhu, Tiankun Lu, J. Jeya Vandana, Neranjan de Silva, Catherine C. Robertson, Jenny Z Xiang, Chendong Pan, Yanjie Sun, Jianwen Que, Todd Evans, Chengyang Liu, Wei Wang, Ali Naji, Stephen C.J. Parker, Robert E. Schwartz, Shuibing Chen","doi":"10.1101/2024.08.05.606734","DOIUrl":"https://doi.org/10.1101/2024.08.05.606734","url":null,"abstract":"There is a paucity of human models to study immune-mediated host damage. Here, we utilized the GeoMx spatial multi-omics platform to analyze immune cell changes in COVID-19 pancreatic autopsy samples, revealing an accumulation of proinflammatory macrophages. Single cell RNA-seq analysis of human islets exposed to SARS-CoV-2 or Coxsackievirus B4 (CVB4) viruses identified activation of proinflammatory macrophages and β cell pyroptosis. To distinguish viral versus proinflammatory macrophage-mediated β cell pyroptosis, we developed human pluripotent stem cell (hPSC)-derived vascularized macrophage-islet (VMI) organoids. VMI organoids exhibited enhanced marker expression and function in both β cells and endothelial cells compared to separately cultured cells. Notably, proinflammatory macrophages within VMI organoids induced β cell pyroptosis. Mechanistic investigations highlighted TNFSF12-TNFRSF12A involvement in proinflammatory macrophage-mediated β cell pyroptosis. This study established hPSC- derived VMI organoids as a valuable tool for studying immune cell-mediated host damage and uncovered mechanism of β cell damage during viral exposure.","PeriodicalId":501269,"journal":{"name":"bioRxiv - Developmental Biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-08-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141943824","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 : 2024-08-06DOI: 10.1101/2024.08.06.606766
Alessa R. Ringel, Andreas Magg, Natalia Benetti, Robert Schöpflin, Mira Kühnlein, Asita Carola Stiege, Ute Fischer, Lars Wittler, Stephan Lorenz, Stefan Mundlos, Lila Allou
The precise spatiotemporal expression of developmental genes is required for proper embryonic development. EN1 plays a key role in dorsal-ventral patterning in mouse limb development from embryonic day (E) 9.5 to E11.5. Previously, we identified the lncRNA locus Maenli which drives En1 expression at E9.5, specifically in the limb. Here we addressed how En1 expression is maintained at later developmental stages when Maenli transcriptional activity is absent. With a series of in vivo CRISPR editing, we demonstrate that at later stages E10.5 and E11.5, En1 expression is driven by two intergenic enhancer elements, LSEE1 and LSEE2. Upon simultaneous loss of these two enhancers, mice only exhibit a subset of the En1 mutant and Maenli-/- limb malformations. We show that the timing of En1 misexpression during limb development causes distinct phenotypes. These findings demonstrate that the temporally restricted activities of cis-regulatory elements, including lncRNA loci and enhancers, may underlie subtle differences in complex disease phenotypes.
{"title":"Temporally restricted activities of En1 regulatory elements underlie distinct limb malformations","authors":"Alessa R. Ringel, Andreas Magg, Natalia Benetti, Robert Schöpflin, Mira Kühnlein, Asita Carola Stiege, Ute Fischer, Lars Wittler, Stephan Lorenz, Stefan Mundlos, Lila Allou","doi":"10.1101/2024.08.06.606766","DOIUrl":"https://doi.org/10.1101/2024.08.06.606766","url":null,"abstract":"The precise spatiotemporal expression of developmental genes is required for proper embryonic development. EN1 plays a key role in dorsal-ventral patterning in mouse limb development from embryonic day (E) 9.5 to E11.5. Previously, we identified the lncRNA locus <em>Maenli</em> which drives <em>En1</em> expression at E9.5, specifically in the limb. Here we addressed how <em>En1</em> expression is maintained at later developmental stages when <em>Maenli</em> transcriptional activity is absent. With a series of <em>in vivo</em> CRISPR editing, we demonstrate that at later stages E10.5 and E11.5, <em>En1</em> expression is driven by two intergenic enhancer elements, LSEE1 and LSEE2. Upon simultaneous loss of these two enhancers, mice only exhibit a subset of the <em>En1</em> mutant and <em>Maenli</em><sup>-/-</sup> limb malformations. We show that the timing of <em>En1</em> misexpression during limb development causes distinct phenotypes. These findings demonstrate that the temporally restricted activities of <em>cis</em>-regulatory elements, including lncRNA loci and enhancers, may underlie subtle differences in complex disease phenotypes.","PeriodicalId":501269,"journal":{"name":"bioRxiv - Developmental Biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-08-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141943820","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}
The declining rates of male fertility pose a significant clinical challenge, primarily due to our limited understanding of the testicular interstitium, which is crucial for male reproductive health. Here, we conducted a comprehensive analysis of the single-cell transcriptomic landscape of the murine testicular interstitium across the postnatal lifespan. Our investigation unveiled a previously unrecognized population of Cd34+/Sox4+ mesenchymal cells nestled within the interstitium, hinting at their potential as Leydig cell progenitors. During the aging process of Cd34+/Sox4+ mesenchymal cells, we observed a decline in glutathione levels within the testicular interstitium. Remarkably, these Cd34+/Sox4+ mesenchymal cells exhibited clonogenic self-renewal capacity and an impressive propensity to differentiate into Leydig cells. Intriguingly, when transplanted into Leydig cell-disrupted or failure models, Cd34+/Sox4+ cells efficiently colonized the testicular interstitium, resulting in a notable increase in testosterone production. Exploring the epigenetic landscape, we identified critical transcription factors, most notably Sox4, governing the stem cell fate of Cd34+/Sox4+ mesenchymal cells. Overall, this comprehensive reference atlas of lifespan testicular Leydig cells presents significant findings that may guide the development of cell-based strategies for treating testicular hypogonadism in elderly individuals.
{"title":"A comprehensive atlas of testicular interstitium reveals Cd34+/Sox4+ mesenchymal cells as potential Leydig cell progenitors","authors":"Xiaojia Huang, Kai Xiao Xia, Meiling Yang, Mengzhi Xiao Hong, Meihua Xiao Jiang, Weiqiang Li, Zhenmin Lei, Andy Peng Xiang, Wei Zhao","doi":"10.1101/2024.08.02.606288","DOIUrl":"https://doi.org/10.1101/2024.08.02.606288","url":null,"abstract":"The declining rates of male fertility pose a significant clinical challenge, primarily due to our limited understanding of the testicular interstitium, which is crucial for male reproductive health. Here, we conducted a comprehensive analysis of the single-cell transcriptomic landscape of the murine testicular interstitium across the postnatal lifespan. Our investigation unveiled a previously unrecognized population of Cd34<sup>+</sup>/Sox4<sup>+</sup> mesenchymal cells nestled within the interstitium, hinting at their potential as Leydig cell progenitors. During the aging process of Cd34<sup>+</sup>/Sox4<sup>+</sup> mesenchymal cells, we observed a decline in glutathione levels within the testicular interstitium. Remarkably, these Cd34<sup>+</sup>/Sox4<sup>+</sup> mesenchymal cells exhibited clonogenic self-renewal capacity and an impressive propensity to differentiate into Leydig cells. Intriguingly, when transplanted into Leydig cell-disrupted or failure models, Cd34<sup>+</sup>/Sox4<sup>+</sup> cells efficiently colonized the testicular interstitium, resulting in a notable increase in testosterone production. Exploring the epigenetic landscape, we identified critical transcription factors, most notably Sox4, governing the stem cell fate of Cd34<sup>+</sup>/Sox4<sup>+</sup> mesenchymal cells. Overall, this comprehensive reference atlas of lifespan testicular Leydig cells presents significant findings that may guide the development of cell-based strategies for treating testicular hypogonadism in elderly individuals.","PeriodicalId":501269,"journal":{"name":"bioRxiv - Developmental Biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-08-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141969237","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}
Tissue damage and inflammation trigger systemic signals that induce catabolic breakdown and nutrient release in distant organs, a process well-characterized in the context of tumor cachexia. While mechanisms allowing tumors to circumvent these systemic growth restrictions are known, the physiological processes that overcome inflammation-induced growth restrictions to support tissue repair and regeneration remain largely unexplored. In our study, we use a model of tissue inflammation and regeneration in developing Drosophila imaginal discs to dissect the key metabolic and signaling adaptations that help tissue overcome systemic growth restrictions. Our findings reveal a unique metabolic strategy used by rapidly proliferating cells in the regenerating domain. Instead of relying on the conventional Insulin-PI3K-Akt signaling pathway, these cells utilize a JAK/STAT-PDK1-S6K axis. This adaptation facilitates sustained protein synthesis and cellular growth despite the systemic catabolism associated with low insulin signaling. Specifically, we find that catabolic breakdown of the fat body is driven by the insulin-binding factor Impl2, which is expressed at the site of inflammatory damage. Notably, regenerative proliferation is also supported by mTORC1 activity and is associated with the upregulation of amino acid transporters in proliferating cells of the regenerating domain. These amino acid transporters align with a specific amino acid metabolite signature in the hemolymph, revealing a specialized metabolic program that meets the demands of fast-proliferating cells. Our work provides insight into how regenerating tissues rewire signaling pathways and adapt their metabolic growth to coordinate tissue repair with a conserved systemic nutrient provision response. These findings have important implications for understanding human diseases such as chronic wounds and cancer.
{"title":"PDK-1/S6K and mTORC1 bypass systemic growth restrictions to promote regeneration","authors":"Ananthakrishnan Vijayakumar Maya, Liyne Nogay, Lara Heckmann, Isabelle Grass, Katrin Kierdorf, Joerg Buescher, Anne-Kathrin Classen","doi":"10.1101/2024.08.05.606658","DOIUrl":"https://doi.org/10.1101/2024.08.05.606658","url":null,"abstract":"Tissue damage and inflammation trigger systemic signals that induce catabolic breakdown and nutrient release in distant organs, a process well-characterized in the context of tumor cachexia. While mechanisms allowing tumors to circumvent these systemic growth restrictions are known, the physiological processes that overcome inflammation-induced growth restrictions to support tissue repair and regeneration remain largely unexplored. In our study, we use a model of tissue inflammation and regeneration in developing Drosophila imaginal discs to dissect the key metabolic and signaling adaptations that help tissue overcome systemic growth restrictions. Our findings reveal a unique metabolic strategy used by rapidly proliferating cells in the regenerating domain. Instead of relying on the conventional Insulin-PI3K-Akt signaling pathway, these cells utilize a JAK/STAT-PDK1-S6K axis. This adaptation facilitates sustained protein synthesis and cellular growth despite the systemic catabolism associated with low insulin signaling. Specifically, we find that catabolic breakdown of the fat body is driven by the insulin-binding factor Impl2, which is expressed at the site of inflammatory damage. Notably, regenerative proliferation is also supported by mTORC1 activity and is associated with the upregulation of amino acid transporters in proliferating cells of the regenerating domain. These amino acid transporters align with a specific amino acid metabolite signature in the hemolymph, revealing a specialized metabolic program that meets the demands of fast-proliferating cells. Our work provides insight into how regenerating tissues rewire signaling pathways and adapt their metabolic growth to coordinate tissue repair with a conserved systemic nutrient provision response. These findings have important implications for understanding human diseases such as chronic wounds and cancer.","PeriodicalId":501269,"journal":{"name":"bioRxiv - Developmental Biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-08-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141943773","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 : 2024-08-04DOI: 10.1101/2024.08.01.606161
Jing Lu, Hao Xu, Dongyue Wang, Yanlu Chen, Takeshi Inoue, Liang Gao, Kai Lei
The intricate coordination of the neural network in planarian growth and regeneration has remained largely unrevealed, partly due to the challenges of imaging the central nervous system (CNS) in three dimensions (3D) with high resolution and within a reasonable timeframe. To address this gap in systematic imaging of the CNS in planarians, we adopted high-resolution, nanoscale imaging by combining tissue expansion and tiling light-sheet microscopy, achieving up to 4-fold linear expansion. Using a semi-automatic 3D cell segmentation pipeline, we quantitatively profiled neurons and muscle fibers at the single-cell level in over 400 wild-type planarians during homeostasis and regeneration. We validated previous observations of neuronal cell number changes and muscle fiber distribution. We found that the rate of neuron cell proliferation tends to lag behind the rapid expansion of somatic cells during the later phase of homeostasis. By imaging the planarian with up to 120 nm resolution, we also observed distinct muscle distribution patterns at the anterior and posterior poles. Furthermore, we investigated the effects of beta-catenin RNAi on muscle fiber distribution at the posterior pole, consistent with changes in anterior-posterior polarity. The glial cells were observed to be close in contact with dorsal-ventral muscle fibers. Finally, we observed disruptions in neural-muscular networks in inr-1 RNAi planarians. These findings provide insights into the detailed structure and potential functions of the neural-muscular system in planarians and highlight the accessibility of our imaging tool in unraveling the biological functions underlying their diverse phenotypes and behaviors.
{"title":"3D Reconstruction of Neuronal Allometry and Neuromuscular Projections in Asexual Planarians Using Expansion Tiling Light Sheet Microscopy","authors":"Jing Lu, Hao Xu, Dongyue Wang, Yanlu Chen, Takeshi Inoue, Liang Gao, Kai Lei","doi":"10.1101/2024.08.01.606161","DOIUrl":"https://doi.org/10.1101/2024.08.01.606161","url":null,"abstract":"The intricate coordination of the neural network in planarian growth and regeneration has remained largely unrevealed, partly due to the challenges of imaging the central nervous system (CNS) in three dimensions (3D) with high resolution and within a reasonable timeframe. To address this gap in systematic imaging of the CNS in planarians, we adopted high-resolution, nanoscale imaging by combining tissue expansion and tiling light-sheet microscopy, achieving up to 4-fold linear expansion. Using a semi-automatic 3D cell segmentation pipeline, we quantitatively profiled neurons and muscle fibers at the single-cell level in over 400 wild-type planarians during homeostasis and regeneration. We validated previous observations of neuronal cell number changes and muscle fiber distribution. We found that the rate of neuron cell proliferation tends to lag behind the rapid expansion of somatic cells during the later phase of homeostasis. By imaging the planarian with up to 120 nm resolution, we also observed distinct muscle distribution patterns at the anterior and posterior poles. Furthermore, we investigated the effects of beta-catenin RNAi on muscle fiber distribution at the posterior pole, consistent with changes in anterior-posterior polarity. The glial cells were observed to be close in contact with dorsal-ventral muscle fibers. Finally, we observed disruptions in neural-muscular networks in inr-1 RNAi planarians. These findings provide insights into the detailed structure and potential functions of the neural-muscular system in planarians and highlight the accessibility of our imaging tool in unraveling the biological functions underlying their diverse phenotypes and behaviors.","PeriodicalId":501269,"journal":{"name":"bioRxiv - Developmental Biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-08-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141943756","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 : 2024-08-04DOI: 10.1101/2024.08.02.606305
Mathieu Reynaud, Stefano Vianello, Shu-Hua Lee, Pauline Salis, Mélanie Dusseune, Kai Wu, Bruno Frederich, David Lecchini, Laurence Besseau, Natacha Roux, Vincent Laudet
Chemical pollution in coastal waters, particularly from agricultural runoff organophosphates, poses a significant threat to marine ecosystems, including coral reefs. Pollutants such as chlorpyrifos (CPF) are widely used in agriculture and have adverse effects on marine life and humans. In this paper, we investigate the impact of CPF on the metamorphosis of a coral reef fish model, the clownfish Amphiprion ocellaris, focusing on the disruption of thyroid hormone (TH) signalling pathways. Our findings reveal that by reducing TH levels, CPF exposure impairs the formation of characteristic white bands in clownfish larvae, indicative of metamorphosis progression. Interestingly these effects can be rescued by TH treatment, establishing a direct causal link between CPF effect and TH disruption. Moreover, transcriptomic analysis elucidates CPF’s effects on all components of the TH signalling pathway. Additionally, CPF induces systemic effects on cholesterol and vitamin D metabolism, DNA repair, and immunity, highlighting its broader TH-independent impacts. These results enhance understanding of the intricate interplay between CPF exposure, TH signalling and metamorphosis, emphasising the urgent need for mitigating the detrimental consequences of chemical pollutants on marine ecosystems.
{"title":"The multi-level effect of chlorpyrifos during clownfish metamorphosis","authors":"Mathieu Reynaud, Stefano Vianello, Shu-Hua Lee, Pauline Salis, Mélanie Dusseune, Kai Wu, Bruno Frederich, David Lecchini, Laurence Besseau, Natacha Roux, Vincent Laudet","doi":"10.1101/2024.08.02.606305","DOIUrl":"https://doi.org/10.1101/2024.08.02.606305","url":null,"abstract":"Chemical pollution in coastal waters, particularly from agricultural runoff organophosphates, poses a significant threat to marine ecosystems, including coral reefs. Pollutants such as chlorpyrifos (CPF) are widely used in agriculture and have adverse effects on marine life and humans. In this paper, we investigate the impact of CPF on the metamorphosis of a coral reef fish model, the clownfish <em>Amphiprion ocellaris</em>, focusing on the disruption of thyroid hormone (TH) signalling pathways. Our findings reveal that by reducing TH levels, CPF exposure impairs the formation of characteristic white bands in clownfish larvae, indicative of metamorphosis progression. Interestingly these effects can be rescued by TH treatment, establishing a direct causal link between CPF effect and TH disruption. Moreover, transcriptomic analysis elucidates CPF’s effects on all components of the TH signalling pathway. Additionally, CPF induces systemic effects on cholesterol and vitamin D metabolism, DNA repair, and immunity, highlighting its broader TH-independent impacts. These results enhance understanding of the intricate interplay between CPF exposure, TH signalling and metamorphosis, emphasising the urgent need for mitigating the detrimental consequences of chemical pollutants on marine ecosystems.","PeriodicalId":501269,"journal":{"name":"bioRxiv - Developmental Biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-08-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141943817","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 : 2024-08-03DOI: 10.1101/2024.08.03.606480
Shreeta Chakraborty, Nina Wenzlitschke, Matthew J Anderson, Ariel Eraso, Manon Baudic, Joyce J Thompson, Alicia A Evans, Lilly M Shatford Adams, Raj Chari, Parirokh Awasthi, Ryan K Dale, Mark Lewandoski, Timothy J Petros, Pedro P Rocha
Chromatin domain boundaries delimited by CTCF motifs can restrict the range of enhancer action. However, disruption of domain structure often results in mild gene dysregulation and thus predicting the impact of boundary rearrangements on animal development remains challenging. Here, we tested whether structural perturbation of a chromatin domain with multiple developmental regulators can result in more acute gene dysregulation and severe developmental phenotypes. We targeted clusters of CTCF motifs in a domain of the mouse genome containing three FGF ligand genes - Fgf3, Fgf4, and Fgf15 - that regulate several developmental processes. Deletion of the 23.9kb cluster that defines the centromeric boundary of this domain resulted in ectopic interactions of the FGF genes with enhancers located across the deleted boundary that are active in the developing brain. This caused strong induction of FGF expression and perinatal lethality with encephalocele and orofacial cleft phenotypes. Heterozygous boundary deletion was sufficient to cause these fully penetrant phenotypes, and strikingly, loss of a single CTCF motif within the cluster also recapitulated ectopic FGF expression and caused encephalocele. However, such phenotypic sensitivity to perturbation of domain structure did not extend to all CTCF clusters of this domain, nor to all developmental processes controlled by these three FGF genes - for example, the ability to undergo lineage specification in the blastocyst and pre-implantation development were not affected. By tracing the impact of different chromosomal rearrangements throughout mouse development, we start to uncover the determinants of phenotypic robustness and sensitivity to perturbation of chromatin boundaries. Our data show how small sequence variants at certain domain boundaries can have a surprisingly outsized effect and must be considered as potential sources of gene dysregulation during development and disease.
{"title":"Structural perturbation of chromatin domains with multiple developmental regulators can severely impact gene regulation and development","authors":"Shreeta Chakraborty, Nina Wenzlitschke, Matthew J Anderson, Ariel Eraso, Manon Baudic, Joyce J Thompson, Alicia A Evans, Lilly M Shatford Adams, Raj Chari, Parirokh Awasthi, Ryan K Dale, Mark Lewandoski, Timothy J Petros, Pedro P Rocha","doi":"10.1101/2024.08.03.606480","DOIUrl":"https://doi.org/10.1101/2024.08.03.606480","url":null,"abstract":"Chromatin domain boundaries delimited by CTCF motifs can restrict the range of enhancer action. However, disruption of domain structure often results in mild gene dysregulation and thus predicting the impact of boundary rearrangements on animal development remains challenging. Here, we tested whether structural perturbation of a chromatin domain with multiple developmental regulators can result in more acute gene dysregulation and severe developmental phenotypes. We targeted clusters of CTCF motifs in a domain of the mouse genome containing three FGF ligand genes - Fgf3, Fgf4, and Fgf15 - that regulate several developmental processes. Deletion of the 23.9kb cluster that defines the centromeric boundary of this domain resulted in ectopic interactions of the FGF genes with enhancers located across the deleted boundary that are active in the developing brain. This caused strong induction of FGF expression and perinatal lethality with encephalocele and orofacial cleft phenotypes. Heterozygous boundary deletion was sufficient to cause these fully penetrant phenotypes, and strikingly, loss of a single CTCF motif within the cluster also recapitulated ectopic FGF expression and caused encephalocele. However, such phenotypic sensitivity to perturbation of domain structure did not extend to all CTCF clusters of this domain, nor to all developmental processes controlled by these three FGF genes - for example, the ability to undergo lineage specification in the blastocyst and pre-implantation development were not affected. By tracing the impact of different chromosomal rearrangements throughout mouse development, we start to uncover the determinants of phenotypic robustness and sensitivity to perturbation of chromatin boundaries. Our data show how small sequence variants at certain domain boundaries can have a surprisingly outsized effect and must be considered as potential sources of gene dysregulation during development and disease.","PeriodicalId":501269,"journal":{"name":"bioRxiv - Developmental Biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-08-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141943822","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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