Pub Date : 2024-09-19DOI: 10.1101/2024.09.18.613729
Justin A Avila, Joseph T Benthal, Jenny C Schafer, E. Michelle Southard-Smith
Background & Aims Enteric nervous system (ENS) development requires migration, proliferation, and appropriate neuronal diversification from progenitors to enable normal gastrointestinal (GI) motility. Sox10 deficit causes aganglionosis, modeling Hirschsprung disease, and disrupts ratios of postnatal enteric neurons in proximal ganglionated bowel. How Sox10 deficiency alters ratios of enteric neuron subtypes is unclear. Sox10's prominent expression in enteric neural crest-derived progenitors (ENCP) and lack of this gene in enteric neurons led us to examine Sox10Dom effects on ENS progenitors and early differentiating enteric neurons. Methods ENS progenitors, developing neurons, and enteric glia were isolated from Sox10+/+ and Sox10Dom/+ littermates for single-cell RNA sequencing (scRNA-seq). scRNA-seq data was processed to identify cell type-specific markers, differentially expressed genes, cell fate trajectories, and gene regulatory network activity between genotypes. Hybridization chain reaction (HCR) validated expression changes detected in scRNA-seq. Results scRNA-seq profiles revealed three neuronal lineages emerging from cycling progenitors via two transition pathways accompanied by elevated activity of Hox gene regulatory networks (GRN) as progenitors transition to neuronal fates. Sox10Dom/+ scRNA-seq profiles exhibited a novel progenitor cluster, decreased abundance of cells in transitional states, and shifts in cell distributions between two neuronal trajectories. Hoxa6 was differentially expressed in the neuronal lineages impacted in Sox10Dom/+ mutants and HCR identified altered Hoxa6 expression in early developing neurons of Sox10Dom/+ ENS. ConclusionsSox10Dom/+ mutation shifts enteric neuron types by altering neuronal trajectories during early ENS lineage segregation. Multiple neurogenic transcription factors are reduced in Sox10Dom/+ scRNA-seq profiles including multiple Hox genes. This is the first report that implicates Hox genes in lineage diversification of enteric neurons.
{"title":"Single Cell Profiling in the Sox10Dom/+ Hirschsprung Mouse Implicates Hoxa6 in Enteric Neuron Lineage Allocation","authors":"Justin A Avila, Joseph T Benthal, Jenny C Schafer, E. Michelle Southard-Smith","doi":"10.1101/2024.09.18.613729","DOIUrl":"https://doi.org/10.1101/2024.09.18.613729","url":null,"abstract":"<strong>Background & Aims</strong> Enteric nervous system (ENS) development requires migration, proliferation, and appropriate neuronal diversification from progenitors to enable normal gastrointestinal (GI) motility. <em>Sox10</em> deficit causes aganglionosis, modeling Hirschsprung disease, and disrupts ratios of postnatal enteric neurons in proximal ganglionated bowel. How <em>Sox10</em> deficiency alters ratios of enteric neuron subtypes is unclear. <em>Sox10</em>'s prominent expression in enteric neural crest-derived progenitors (ENCP) and lack of this gene in enteric neurons led us to examine <em>Sox10</em><sup>Dom</sup> effects on ENS progenitors and early differentiating enteric neurons.\u0000<strong>Methods</strong>\u0000ENS progenitors, developing neurons, and enteric glia were isolated from <em>Sox10+/+</em> and <em>Sox10</em><sup>Dom/+</sup> littermates for single-cell RNA sequencing (scRNA-seq). scRNA-seq data was processed to identify cell type-specific markers, differentially expressed genes, cell fate trajectories, and gene regulatory network activity between genotypes. Hybridization chain reaction (HCR) validated expression changes detected in scRNA-seq. <strong>Results</strong>\u0000scRNA-seq profiles revealed three neuronal lineages emerging from cycling progenitors via two transition pathways accompanied by elevated activity of <em>Hox</em> gene regulatory networks (GRN) as progenitors transition to neuronal fates. <em>Sox10</em><sup>Dom/+</sup> scRNA-seq profiles exhibited a novel progenitor cluster, decreased abundance of cells in transitional states, and shifts in cell distributions between two neuronal trajectories. <em>Hoxa6</em> was differentially expressed in the neuronal lineages impacted in <em>Sox10</em><sup>Dom/+</sup> mutants and HCR identified altered <em>Hoxa6</em> expression in early developing neurons of <em>Sox10</em><sup>Dom/+</sup> ENS.\u0000<strong>Conclusions</strong>\u0000<em>Sox10</em><sup>Dom/+</sup> mutation shifts enteric neuron types by altering neuronal trajectories during early ENS lineage segregation. Multiple neurogenic transcription factors are reduced in <em>Sox10</em><sup>Dom/+</sup> scRNA-seq profiles including multiple <em>Hox</em> genes. This is the first report that implicates <em>Hox</em> genes in lineage diversification of enteric neurons.","PeriodicalId":501269,"journal":{"name":"bioRxiv - Developmental Biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-09-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142252545","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 : 2024-09-19DOI: 10.1101/2024.09.18.613761
Tatsuya Kato, Olga Skorobogata, Christian E Rocheleau
Caenorhabditis elegans vulval development is a relatively simple model of organ development whereby a signal from the overlying gonad induces three epithelial cells to undergo three rounds of cell division to generate 22 cells that make up the vulva. Specification of the vulva cell fates requires coordination between cell division and cell signaling via LIN-12/Notch and LET-23/EGFR pathways in the somatic gonad and the underlying epithelium. Here we characterize the positive regulation of vulval development by the centralspindlin complex, a conserved cytokinesis regulator. Centralspindlin, a heterotetramer of ZEN-4/KIF23 and CYK-4/RacGAP1, is essential for completion of cytokinesis during early embryonic cell divisions. We found that centralspindlin is required in the somatic gonad for division of somatic gonad precursor cells and hence specification of the LIN-3/EGF-secreting anchor cell critical for LET-23/EGFR-mediated vulval induction. However, the requirements for centralspindlin for cytokinesis during postembryonic development are incomplete as a binucleate anchor cell is frequently specified. The presence of the binucleate anchor cell correlates with vulva induction and demonstrates that LIN-12/Notch signaling, required for anchor cell specification, and LET-23/EGFR signaling, required for vulva induction, is largely functional in these cells. Centralspindlin is also partially required for cytokinesis of the vulval cells where it regulates vulva morphogenesis rather than induction. We also found that the GAP domain of CYK-4/RacGAP1 required for contractile ring assembly during embryonic division is not essential for vulval development. Thus, there appears to be different requirements for centralspindlin during postembryonic development of the somatic gonad and vulva as compared to early embryogenesis.
{"title":"Centralspindlin promotes C. elegans anchor cell specification, vulva induction and morphogenesis","authors":"Tatsuya Kato, Olga Skorobogata, Christian E Rocheleau","doi":"10.1101/2024.09.18.613761","DOIUrl":"https://doi.org/10.1101/2024.09.18.613761","url":null,"abstract":"<em>Caenorhabditis elegans</em> vulval development is a relatively simple model of organ development whereby a signal from the overlying gonad induces three epithelial cells to undergo three rounds of cell division to generate 22 cells that make up the vulva. Specification of the vulva cell fates requires coordination between cell division and cell signaling via LIN-12/Notch and LET-23/EGFR pathways in the somatic gonad and the underlying epithelium. Here we characterize the positive regulation of vulval development by the centralspindlin complex, a conserved cytokinesis regulator. Centralspindlin, a heterotetramer of ZEN-4/KIF23 and CYK-4/RacGAP1, is essential for completion of cytokinesis during early embryonic cell divisions. We found that centralspindlin is required in the somatic gonad for division of somatic gonad precursor cells and hence specification of the LIN-3/EGF-secreting anchor cell critical for LET-23/EGFR-mediated vulval induction. However, the requirements for centralspindlin for cytokinesis during postembryonic development are incomplete as a binucleate anchor cell is frequently specified. The presence of the binucleate anchor cell correlates with vulva induction and demonstrates that LIN-12/Notch signaling, required for anchor cell specification, and LET-23/EGFR signaling, required for vulva induction, is largely functional in these cells. Centralspindlin is also partially required for cytokinesis of the vulval cells where it regulates vulva morphogenesis rather than induction. We also found that the GAP domain of CYK-4/RacGAP1 required for contractile ring assembly during embryonic division is not essential for vulval development. Thus, there appears to be different requirements for centralspindlin during postembryonic development of the somatic gonad and vulva as compared to early embryogenesis.","PeriodicalId":501269,"journal":{"name":"bioRxiv - Developmental Biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-09-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142252505","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 : 2024-09-19DOI: 10.1101/2024.09.18.613721
Tayo E Adekeye, Emily M Teets, Emily A Tomak, Sadie L Waterman, Kailee A Sprague, Angelina White, Maddison L Coffin, Sabrina M Varga, Teresa E Easterbrooks, Sarah J Shepherd, Jared D Austin, Dmitrii Krivorotko, Troy E Hupper, Joshua B Kelley, Sharon L Amacher, Jared C Talbot
Muscle cells become stronger by expanding myofibrils, the chains of sarcomeres that produce contraction. Here we investigate how Mylpf (Myosin Light Chain Phosphorylatable Fast) abundance impacts myofibril assembly in fast-twitch muscle. The two zebrafish Mylpf genes (mylpfa and mylpfb) are exclusively expressed in fast-twitch muscle. We show that these cells initially produce six times more mylpfa mRNA and protein than mylpfb. The combined Mylpf protein dosage is necessary for and proportionate to fast-twitch myofibril growth in the embryo. Fast-twitch myofibrils are severely reduced in the mylpfa-/- mutant, leading to loss of high-speed movement; however, by persistent slow movement this mutant swims as far through time as its wild-type sibling. Although the mylpfb-/- mutant has normal myofibrils, myofibril formation fails entirely in the mylpfa-/-;mylpfb-/- double mutant, indicating that the two genes are collectively essential to myofibril formation. Fast-twitch myofibril width is restored in the mylpfa-/- mutant by transgenic expression of mylpfa-GFP, mylpfb-GFP, and by human MYLPF-GFP to a degree corresponding linearly with GFP brightness. This correlate is inverted by expression of MYLPF alleles that cause Distal Arthrogryposis, which reduce myofibril size in proportion to protein abundance. These effects indicate that Mylpf dosage controls myofibril growth, impacting embryonic development and lifelong health.
{"title":"Mylpf dosage is proportionate to fast-twitch myofibril size in the zebrafish embryo","authors":"Tayo E Adekeye, Emily M Teets, Emily A Tomak, Sadie L Waterman, Kailee A Sprague, Angelina White, Maddison L Coffin, Sabrina M Varga, Teresa E Easterbrooks, Sarah J Shepherd, Jared D Austin, Dmitrii Krivorotko, Troy E Hupper, Joshua B Kelley, Sharon L Amacher, Jared C Talbot","doi":"10.1101/2024.09.18.613721","DOIUrl":"https://doi.org/10.1101/2024.09.18.613721","url":null,"abstract":"Muscle cells become stronger by expanding myofibrils, the chains of sarcomeres that produce contraction. Here we investigate how Mylpf (Myosin Light Chain Phosphorylatable Fast) abundance impacts myofibril assembly in fast-twitch muscle. The two zebrafish Mylpf genes (<em>mylpfa</em> and <em>mylpfb</em>) are exclusively expressed in fast-twitch muscle. We show that these cells initially produce six times more <em>mylpfa</em> mRNA and protein than <em>mylpfb</em>. The combined Mylpf protein dosage is necessary for and proportionate to fast-twitch myofibril growth in the embryo. Fast-twitch myofibrils are severely reduced in the <em>mylpfa<sup>-/-</sup></em> mutant, leading to loss of high-speed movement; however, by persistent slow movement this mutant swims as far through time as its wild-type sibling. Although the <em>mylpfb<sup>-/-</sup></em> mutant has normal myofibrils, myofibril formation fails entirely in the <em>mylpfa<sup>-/-</sup>;mylpfb<sup>-/-</sup></em> double mutant, indicating that the two genes are collectively essential to myofibril formation. Fast-twitch myofibril width is restored in the <em>mylpfa<sup>-/-</sup></em> mutant by transgenic expression of <em>mylpfa-GFP</em>, <em>mylpfb-GFP</em>, and by human <em>MYLPF-GFP</em> to a degree corresponding linearly with GFP brightness. This correlate is inverted by expression of <em>MYLPF</em> alleles that cause Distal Arthrogryposis, which reduce myofibril size in proportion to protein abundance. These effects indicate that Mylpf dosage controls myofibril growth, impacting embryonic development and lifelong health.","PeriodicalId":501269,"journal":{"name":"bioRxiv - Developmental Biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-09-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142252546","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 : 2024-09-19DOI: 10.1101/2024.09.18.613197
Philippa Harding, Maja Wojtynska, Alexander J Smith, Robin Ali, Rachael A Pearson
The human macula is a specialized, M/L cone-rich region of the eye, critical for high acuity vision, but little is known about the pathways regulating its development. Transcriptional regulation by Retinoic Acid (RA) is essential for many aspects of human eye development. Here, we report a striking biphasic expression of the RA-catabolizing enzyme, CYP26A1, in early human macular development between post-conception weeks 6-17. Early inhibition of RA signaling in human retinal organoids (hROs) prompts early cell cycle exit, and an increase in cone photoreceptors, while late inhibition alters cone subtype specification. Conversely, FGF8, which is negatively regulated by RA and vital for High Acuity Area specification in chick, is not expressed in the nascent human macula and had no effect on hRO photoreceptor fate.
{"title":"Human macula formation involves two waves of retinoic acid signaling suppression via CYP26A1 regulating cell cycle exit and cone specification","authors":"Philippa Harding, Maja Wojtynska, Alexander J Smith, Robin Ali, Rachael A Pearson","doi":"10.1101/2024.09.18.613197","DOIUrl":"https://doi.org/10.1101/2024.09.18.613197","url":null,"abstract":"The human macula is a specialized, M/L cone-rich region of the eye, critical for high acuity vision, but little is known about the pathways regulating its development. Transcriptional regulation by Retinoic Acid (RA) is essential for many aspects of human eye development. Here, we report a striking biphasic expression of the RA-catabolizing enzyme, CYP26A1, in early human macular development between post-conception weeks 6-17. Early inhibition of RA signaling in human retinal organoids (hROs) prompts early cell cycle exit, and an increase in cone photoreceptors, while late inhibition alters cone subtype specification. Conversely, FGF8, which is negatively regulated by RA and vital for High Acuity Area specification in chick, is not expressed in the nascent human macula and had no effect on hRO photoreceptor fate.","PeriodicalId":501269,"journal":{"name":"bioRxiv - Developmental Biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-09-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142252544","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 : 2024-09-19DOI: 10.1101/2024.09.18.613484
Hannah R. Moran, Obed O. Nyarko, Rebecca O'Rourke, Ryenne-Christine R. Ching, Brisa Peña, Fréderike W. Riemslagh, Alexa Burger, Carmen C. Sucharov, Christian Mosimann
The heart integrates diverse cell lineages into a functional unit, including the pericardium, a mesothelial sac that supports heart movement, homeostasis, and immune responses. However, despite its critical roles, the developmental origins of the pericardium remain uncertain due to disparate models. Here, using live imaging, lineage tracking, and single-cell transcriptomics in zebrafish, we find the pericardium forms within the lateral plate mesoderm from dedicated anterior mesothelial progenitors and distinct from the classic heart field. Machine learning-based tracking and transcriptional trajectories uncover how pericardial precursors emerge among a bilateral mesothelial progenitor band to enclose the embryonic heart. This process occurs independently of heart tube formation, with canonical Wnt/β-catenin signaling modulating pericardial cell number and tissue rigidity. We link Wnt antagonist expression found in pediatric dilated cardiomyopathy to increased pericardial stiffness in a neonatal rat model. Our findings conceptually advance our models of heart formation with pericardium formation as a distinct process.
{"title":"The pericardium forms as a distinct structure during heart formation","authors":"Hannah R. Moran, Obed O. Nyarko, Rebecca O'Rourke, Ryenne-Christine R. Ching, Brisa Peña, Fréderike W. Riemslagh, Alexa Burger, Carmen C. Sucharov, Christian Mosimann","doi":"10.1101/2024.09.18.613484","DOIUrl":"https://doi.org/10.1101/2024.09.18.613484","url":null,"abstract":"The heart integrates diverse cell lineages into a functional unit, including the pericardium, a mesothelial sac that supports heart movement, homeostasis, and immune responses. However, despite its critical roles, the developmental origins of the pericardium remain uncertain due to disparate models. Here, using live imaging, lineage tracking, and single-cell transcriptomics in zebrafish, we find the pericardium forms within the lateral plate mesoderm from dedicated anterior mesothelial progenitors and distinct from the classic heart field. Machine learning-based tracking and transcriptional trajectories uncover how pericardial precursors emerge among a bilateral mesothelial progenitor band to enclose the embryonic heart. This process occurs independently of heart tube formation, with canonical Wnt/β-catenin signaling modulating pericardial cell number and tissue rigidity. We link Wnt antagonist expression found in pediatric dilated cardiomyopathy to increased pericardial stiffness in a neonatal rat model. Our findings conceptually advance our models of heart formation with pericardium formation as a distinct process.","PeriodicalId":501269,"journal":{"name":"bioRxiv - Developmental Biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-09-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142252504","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 : 2024-09-19DOI: 10.1101/2024.09.17.611698
Stanley M. Kanai, Chloe R. Garcia, MaCalia R. Augustus, Shujan A. Sharafeldeen, Elliott P. Brooks, Juliana Sucharov, Ezra S. Lencer, James T. Nichols, David E. Clouthier
Vertebrate jaw development is coordinated by highly conserved ligand-receptor systems such as the peptide ligand Endothelin 1 (Edn1) and Endothelin receptor type A (Ednra), which are required for patterning of lower jaw structures. The Edn1/Ednra signaling pathway establishes the identity of lower jaw progenitor cells by regulating expression of numerous patterning genes, but the intracellular signaling mechanisms linking receptor activation to gene regulation remain poorly understood. As a first step towards elucidating this mechanism, we examined the function of the Gq/11 family of Gα subunits in zebrafish using pharmacological inhibition and genetic ablation of Gq/11 activity and transgenic induction of a constitutively active Gq protein in edn1-/- embryos. Genetic loss of Gq/11 activity fully recapitulated the edn1-/- phenotype, with genes encoding G11 being most essential. Furthermore, inducing Gq activity in edn1-/- embryos not only restored Edn1/Ednra-dependent jaw structures and gene expression signatures but also caused homeosis of the upper jaw structure into a lower jaw-like structure. These results indicate that Gq/11 is necessary and sufficient to mediate the lower jaw patterning mechanism for Ednra in zebrafish.
{"title":"The Gq/11 family of Gα subunits is necessary and sufficient for lower jaw development","authors":"Stanley M. Kanai, Chloe R. Garcia, MaCalia R. Augustus, Shujan A. Sharafeldeen, Elliott P. Brooks, Juliana Sucharov, Ezra S. Lencer, James T. Nichols, David E. Clouthier","doi":"10.1101/2024.09.17.611698","DOIUrl":"https://doi.org/10.1101/2024.09.17.611698","url":null,"abstract":"Vertebrate jaw development is coordinated by highly conserved ligand-receptor systems such as the peptide ligand Endothelin 1 (Edn1) and Endothelin receptor type A (Ednra), which are required for patterning of lower jaw structures. The Edn1/Ednra signaling pathway establishes the identity of lower jaw progenitor cells by regulating expression of numerous patterning genes, but the intracellular signaling mechanisms linking receptor activation to gene regulation remain poorly understood. As a first step towards elucidating this mechanism, we examined the function of the Gq/11 family of Gα subunits in zebrafish using pharmacological inhibition and genetic ablation of Gq/11 activity and transgenic induction of a constitutively active Gq protein in edn1-/- embryos. Genetic loss of Gq/11 activity fully recapitulated the edn1-/- phenotype, with genes encoding G11 being most essential. Furthermore, inducing Gq activity in edn1-/- embryos not only restored Edn1/Ednra-dependent jaw structures and gene expression signatures but also caused homeosis of the upper jaw structure into a lower jaw-like structure. These results indicate that Gq/11 is necessary and sufficient to mediate the lower jaw patterning mechanism for Ednra in zebrafish.","PeriodicalId":501269,"journal":{"name":"bioRxiv - Developmental Biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-09-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142252547","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 : 2024-09-17DOI: 10.1101/2024.09.17.613495
Kelli D. Fenelon, Priyanshi Borad, Biraaj Rout, Parisa Boodaghimalidarreh, Mohammad S. Nasr, Jacob Luber, Theodora Koromila
Transcriptional regulation, orchestrated by the interplay between transcription factors (TFs) and enhancers, governs gene expression dynamics crucial for cellular processes. While gross, qualitative fluctuations in transcription factor-dependent gene expression patterning have a long history of characterization, the roles of these factors in the nuclei retaining expression in the presence or absence of these factors are now observable using modern techniques. Our study investigates the impact of Suppressor of Hairless (Su(H)), a broadly expressed transcription factor, on enhancer-driven transcriptional modulation using Drosophila early embryos as a model system. Building upon previous findings, we employ super-resolution microscopy to dissect Su(H)'s influence on sog-Distal (sogD) enhancer activity specifically in nuclei with preserved sogD-driven expression in the absence of Su(H) binding. We demonstrate that Su(H) occupancy perturbations alter expression levels and bursting dynamics. Notably, Su(H) absence during embryonic development exhibits region-specific effects, inhibiting expression dorsally and enhancing expression ventrally, implying a nuanced role in enhancer regulation. Our findings shed light on the intricate mechanisms that govern transcriptional dynamics and suggest a critical patterning role for Notch/Hairless signaling in sog expression as embryos transition to gastrulation.
{"title":"Notch/Hairless Pathway Modulation of sog Transcriptional Bursting in Prelude to Gastrulation","authors":"Kelli D. Fenelon, Priyanshi Borad, Biraaj Rout, Parisa Boodaghimalidarreh, Mohammad S. Nasr, Jacob Luber, Theodora Koromila","doi":"10.1101/2024.09.17.613495","DOIUrl":"https://doi.org/10.1101/2024.09.17.613495","url":null,"abstract":"Transcriptional regulation, orchestrated by the interplay between transcription factors (TFs) and enhancers, governs gene expression dynamics crucial for cellular processes. While gross, qualitative fluctuations in transcription factor-dependent gene expression patterning have a long history of characterization, the roles of these factors in the nuclei retaining expression in the presence or absence of these factors are now observable using modern techniques. Our study investigates the impact of Suppressor of Hairless (Su(H)), a broadly expressed transcription factor, on enhancer-driven transcriptional modulation using <em>Drosophila</em> early embryos as a model system. Building upon previous findings, we employ super-resolution microscopy to dissect Su(H)'s influence on <em>sog-Distal</em> (<em>sogD</em>) enhancer activity specifically in nuclei with preserved <em>sogD</em>-driven expression in the absence of Su(H) binding. We demonstrate that Su(H) occupancy perturbations alter expression levels and bursting dynamics. Notably, Su(H) absence during embryonic development exhibits region-specific effects, inhibiting expression dorsally and enhancing expression ventrally, implying a nuanced role in enhancer regulation. Our findings shed light on the intricate mechanisms that govern transcriptional dynamics and suggest a critical patterning role for Notch/Hairless signaling in <em>sog</em> expression as embryos transition to gastrulation.","PeriodicalId":501269,"journal":{"name":"bioRxiv - Developmental Biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-09-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142252549","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 : 2024-09-17DOI: 10.1101/2024.09.16.613227
Kutay Karatepe, Bruna Mafra de Faria, jian zhang, Xinyue Chen, Hugo Pinto, Dmitry Fyodorov, Esen Sefik, Michael Willcockson, Richard Flavell, Arthur I Skoultchi, Shangqin Guo
Myeloid-biased differentiation of multipotent hematopoietic stem and progenitor cells (HSPCs) occurs with aging or exhaustion. The molecular mechanism(s) responsible for this fate bias remain unclear. Here we report that linker histone regulates HSPC fate choice at the lymphoid versus myeloid bifurcation. HSPCs expressing H1.0 from a doxycycline (dox) inducible transgene favor the lymphoid fate, display strengthened nucleosome organization and reduced chromatin accessibility at genomic regions hosting key myeloid fate drivers. The transcription factor Hlf is located in one of such regions, where chromatin accessibility and gene expression is reduced in H1.0high HSPCs. Furthermore, H1.0 protein in HSPCs can decreases in an aspartyl protease dependent manner, a process enhanced in response to interferon alpha signaling. Aspartyl protease inhibitors preserve endogenous H1.0 levels and promote the lymphoid fate of wild type HSPCs. Thus, our work uncovers a point of intervention to mitigate myeloid skewed hematopoiesis.
{"title":"Linker histone regulates the myeloid versus lymphoid bifurcation of multipotent hematopoietic stem and progenitors","authors":"Kutay Karatepe, Bruna Mafra de Faria, jian zhang, Xinyue Chen, Hugo Pinto, Dmitry Fyodorov, Esen Sefik, Michael Willcockson, Richard Flavell, Arthur I Skoultchi, Shangqin Guo","doi":"10.1101/2024.09.16.613227","DOIUrl":"https://doi.org/10.1101/2024.09.16.613227","url":null,"abstract":"Myeloid-biased differentiation of multipotent hematopoietic stem and progenitor cells (HSPCs) occurs with aging or exhaustion. The molecular mechanism(s) responsible for this fate bias remain unclear. Here we report that linker histone regulates HSPC fate choice at the lymphoid versus myeloid bifurcation. HSPCs expressing H1.0 from a doxycycline (dox) inducible transgene favor the lymphoid fate, display strengthened nucleosome organization and reduced chromatin accessibility at genomic regions hosting key myeloid fate drivers. The transcription factor Hlf is located in one of such regions, where chromatin accessibility and gene expression is reduced in H1.0high HSPCs. Furthermore, H1.0 protein in HSPCs can decreases in an aspartyl protease dependent manner, a process enhanced in response to interferon alpha signaling. Aspartyl protease inhibitors preserve endogenous H1.0 levels and promote the lymphoid fate of wild type HSPCs. Thus, our work uncovers a point of intervention to mitigate myeloid skewed hematopoiesis.","PeriodicalId":501269,"journal":{"name":"bioRxiv - Developmental Biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-09-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142252556","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 : 2024-09-17DOI: 10.1101/2024.09.17.613303
Zhiyuan Hu, Sarah Mayes, Weixu Wang, Jose M. Santos-Pereira, Fabian Theis, Tatjana Sauka-Spengler
Cranial neural crest (NC) cells, which can migrate, adopt multiple fates, and form most of the craniofacial skeleton, are an excellent model for studying cell fate decisions. Using time-resolved single-cell multi-omics, spatial transcriptomics, and systematic Perturb-seq, we fully deciphered zebrafish cranial NC programs, including 23 cell states and three spatial trajectories, reconstructed and tested the complete gene regulatory network (GRN). Our GRN model, combined with a novel velocity-embedded simulation method, accurately predicted functions of all major regulons, with over a 3-fold increase in correlation between in vivo and in silico perturbations. Using our new approach based on regulatory synchronization, we discovered a post-epithelial-mesenchymal-transition endothelial-like program crucial for migration, identified motif coordinators for dual-fate priming, and quantified lineage-specific cooperative transcription factor functions. This study provides a comprehensive and validated NC regulatory landscape with unprecedented resolution, offering general regulatory models for cell fate decisions in vertebrates.
{"title":"Single-cell multi-omics, spatial transcriptomics and systematic perturbation decode circuitry of neural crest fate decisions","authors":"Zhiyuan Hu, Sarah Mayes, Weixu Wang, Jose M. Santos-Pereira, Fabian Theis, Tatjana Sauka-Spengler","doi":"10.1101/2024.09.17.613303","DOIUrl":"https://doi.org/10.1101/2024.09.17.613303","url":null,"abstract":"Cranial neural crest (NC) cells, which can migrate, adopt multiple fates, and form most of the craniofacial skeleton, are an excellent model for studying cell fate decisions. Using time-resolved single-cell multi-omics, spatial transcriptomics, and systematic Perturb-seq, we fully deciphered zebrafish cranial NC programs, including 23 cell states and three spatial trajectories, reconstructed and tested the complete gene regulatory network (GRN). Our GRN model, combined with a novel velocity-embedded simulation method, accurately predicted functions of all major regulons, with over a 3-fold increase in correlation between in vivo and in silico perturbations. Using our new approach based on regulatory synchronization, we discovered a post-epithelial-mesenchymal-transition endothelial-like program crucial for migration, identified motif coordinators for dual-fate priming, and quantified lineage-specific cooperative transcription factor functions. This study provides a comprehensive and validated NC regulatory landscape with unprecedented resolution, offering general regulatory models for cell fate decisions in vertebrates.","PeriodicalId":501269,"journal":{"name":"bioRxiv - Developmental Biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-09-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142252552","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 : 2024-09-17DOI: 10.1101/2024.09.17.613448
Dan J Hayman, Lola M Morrin, Sudipta Halder, Eleanor J Phillips, Mirre J P Simons, Iwan Robert Evans
Macrophages are responsible for diverse and fundamental functions in vertebrates. Fruit flies harbour an innate immune system of which the most populous blood cell (haemocyte) type bears striking homology to the vertebrate macrophage. The importance of these cells has been demonstrated previously, where immune and developmental phenotypes have been observed upon haemocyte ablation using pro-apoptotic transgenes driven by the Hml promoter. Here we show that, as well as ablating Hml-positive cells in vivo using the pro-apoptotic transgene bax, we can also increase Hml-positive cell numbers using a constitutively-active form of ras. However, in adults, compared to larvae, total blood cell numbers were not significantly affected by experimental expansion or ablation. This therefore implies the existence of feedback mechanisms which regulate the number of haemocytes. No effect on lifespan was observed from driving ras and bax in Hml-positive cells using a conditional genetic system (Hml-GeneSwitch). Using a constitutive driver system, we did observe differences in lifespan, however we attribute this to differences in genetic background that could have led to spurious conclusions. Additionally, no effect of either transgene was observed upon infection with two different bacterial species, although a striking pupal lethality phenotype was observed upon expansion of Hml-positive cells in the context of a self-encapsulation mutant genetic background. The latter confirms that the change in Hml-positive cell number does result in a phenotype. The lack of adult phenotypes could be due to the strength of our experimental manipulation or due to compensation via feedback mechanisms that operate to maintain total blood cell numbers. Our study demonstrates the importance of a conditional approach to modulate haemocyte cell numbers in vivo which allows for more precise study of innate immune system function. This approach could be especially fruitful to uncover the mechanisms that regulate total blood cell numbers across development and ageing.
{"title":"Expansion of Drosophila haemocytes using a conditional GeneSwitch driver affects larval haemocyte function, but does not modulate adult lifespan or survival from infection","authors":"Dan J Hayman, Lola M Morrin, Sudipta Halder, Eleanor J Phillips, Mirre J P Simons, Iwan Robert Evans","doi":"10.1101/2024.09.17.613448","DOIUrl":"https://doi.org/10.1101/2024.09.17.613448","url":null,"abstract":"Macrophages are responsible for diverse and fundamental functions in vertebrates. Fruit flies harbour an innate immune system of which the most populous blood cell (haemocyte) type bears striking homology to the vertebrate macrophage. The importance of these cells has been demonstrated previously, where immune and developmental phenotypes have been observed upon haemocyte ablation using pro-apoptotic transgenes driven by the Hml promoter.\u0000Here we show that, as well as ablating Hml-positive cells in vivo using the pro-apoptotic transgene bax, we can also increase Hml-positive cell numbers using a constitutively-active form of ras. However, in adults, compared to larvae, total blood cell numbers were not significantly affected by experimental expansion or ablation. This therefore implies the existence of feedback mechanisms which regulate the number of haemocytes.\u0000No effect on lifespan was observed from driving ras and bax in Hml-positive cells using a conditional genetic system (Hml-GeneSwitch). Using a constitutive driver system, we did observe differences in lifespan, however we attribute this to differences in genetic background that could have led to spurious conclusions. Additionally, no effect of either transgene was observed upon infection with two different bacterial species, although a striking pupal lethality phenotype was observed upon expansion of Hml-positive cells in the context of a self-encapsulation mutant genetic background. The latter confirms that the change in Hml-positive cell number does result in a phenotype. The lack of adult phenotypes could be due to the strength of our experimental manipulation or due to compensation via feedback mechanisms that operate to maintain total blood cell numbers. Our study demonstrates the importance of a conditional approach to modulate haemocyte cell numbers in vivo which allows for more precise study of innate immune system function. This approach could be especially fruitful to uncover the mechanisms that regulate total blood cell numbers across development and ageing.","PeriodicalId":501269,"journal":{"name":"bioRxiv - Developmental Biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-09-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142252550","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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