Biology subjects

Wang, D.

Publications and source records attributed to Wang, D..

At least 541 records · Page 30Linked to original sources

bioRxiv2021

LKB1 Is Physiologically Required for Sleep from Drosophila to the Mouse

LKB1 is known as a master kinase for 14 kinases related to the adenosine monophosphate (AMP)-activated protein kinase (AMPK). Two of them (SIK3 and AMPK) have previously been implicated in sleep regulation. We generated loss-of-function (LOF) mutants for Lkb1 in both Drosophila and mice. Sleep was reduced in Lkb1-mutant flies and in flies with neuronal deletion of Lkb1. Sleep was reduced in mice after virally mediated reduction of Lkb1 in the brain. Electroencephalography (EEG) analysis showed that non-rapid eye movement (NREM) sleep and sleep need were both reduced in Lkb1-mutant mice. These results indicate that LKB1 plays a physiological role in sleep regulation conserved from flies to mice.

neuroscience↗

bioRxiv2021

HBV enveloped particle secretion is positively regulated by GRP78 through direct interaction with preS1

Hepatitis B virus (HBV) infection is a common cause of liver diseases worldwide. Existing drugs do not effectively eliminate HBV from infected hepatocytes; thus, novel curative therapies are needed. Enveloped-particle secretion is a key but poorly studied aspect of the viral life cycle. Here, we report that GRP78 positively regulates HBV enveloped-particle secretion. GRP78 is the specific target of preS1 binding; HBV can upregulate GRP78 in liver cell lines and sera from chronic hepatitis B patients. GRP78 promoted intact HBV-particle secretion in liver cell lines and an HBV transgenic-mouse model. Some peptides screened from preS1 via phage display could inhibit viral-particle secretion by interacting with GRP78 via hydrogen bonds and hydrophobic interactions, thereby disturbing the interaction with HBV particles. These results provide insight into enveloped-particle secretion in the HBV life cycle. GRP78 might be a potential target for HBV-infection treatment via restricting GRP78-preS1 interactions to block viral-particle secretion. IMPORTANCEHBV is a major human pathogen. The virus More than 250 million n individuals globally are chronically infected with HBV and can result in the infected personsabout 800,000 die for HBV-related disease annually. The mature HBV enveloped particles containing HBs and the relaxed circular (RC) DNA genome could secreted extracellularly as virions. And these enveloped virions serves as the infectious HBV particles for initiating a complete life cycle of the virus. Here, we show the GRP78 is the specific target of preS1 binding, and it could positive regulate HBV enveloped particle secretion in cell and HBV transgenic mice model. Furthermore, several peptides screened from preS1 and Phage screening could inhibit viral particle secretion by interacting with GRP78 and disturbing its interaction with HBV, and then block the life cycle of the virus. This has important implications for our understanding of the mechanisms of antivirals that target intact HBV particles secretion.

microbiology↗

bioRxiv2021

CRISPR knockouts of pmela and pmelb engineered a golden tilapia by regulating relative pigment cell abundance

Premelanosome protein (pmel) is a key gene for melanogenesis in vertebrates. Mutations in this gene are responsible for white plumage in chicken, but its role in pigmentation of fish remains to be demonstrated. In this study we found that most fishes have two pmel genes arising from the teleost-specific whole genome duplication. Both pmela and pmelb were expressed at high levels in the eyes and skin of Nile tilapia. We mutated both genes in tilapia using CRISPR/Cas9 gene editing. Homozygous mutation of pmela resulted in yellowish body color with weak vertical bars and a hypo-pigmented retinal pigment epithelium (RPE) due to significantly reduced number and size of melanophores. In contrast, we observed an increased number and size of xanthophores in mutants compared to wild-type fish. Homozygous mutation of pmelb resulted in a similar, but milder phenotype than pmela-/- mutants, without effects on RPE pigmentation. Double mutation of pmela and pmelb resulted in loss of additional melanophores compared to the pmela-/- mutants, and also an increase in the number and size of xanthophores, producing a strong golden body color without bars in the trunk. The RPE pigmentation of pmela-/-;pmelb-/- was similar to pmela-/- mutants, with much less pigmentation than pmelb-/- mutants and wild-type fish. Taken together, our results indicate that, while both pmel genes are important for the formation of body color in tilapia, pmela plays a more important role than pmelb. To our knowledge, this is the first report on mutation of pmelb or both pmela;pmelb in fish. Studies on these mutants suggest new strategies for breeding golden tilapia, and also provide a new model for studies of pmel function in vertebrates. Author SummaryMelanophores, the most common pigment cell type, have been studied for nearly 150 years. Many genes are involved in melanoblast migration, melanophore differentiation, and melanin biosynthesis. Pmel is fundamental for melanosome development by directing melanin biosynthesis and melanosome phase transition. Specifically, PMEL can form a fibrillar structure within the melanosome upon which melanin is deposited. We identified two pmel genes in Nile tilapia arising from the teleost-specific whole genome duplication. Disruption of either pmela or pmelb in tilapia leads to significant hypo-pigmentation. PMEL disrupted fish showed not only a reduction in melanin and tiny melanophores, but also a significant increase in the number of xanthophores, and even guanine-filled melanophores, which led to a golden tilapia with hypo-pigmented RPE. Our study confirmed the role of pmel in melanin biosynthesis and maturation, and also highlighted its effects on melanophore number and size. These results provide new insights into pigment cell biology and will help us better understand the mechanisms of color patterning in teleosts. Knockout of both pmela and pmelb provide a new strategy for engineering a golden tilapia, which might provide a foundation for developing new strains in the tilapia industry.

genetics↗

bioRxiv2021

Interaction between theta-phase and spike-timing dependent plasticity simulates theta induced memory effects

Rodent studies suggest that spike timing relative to hippocampal theta activity determines whether potentiation or depression of synapses arise. Such changes also depend on spike timing between pre- and post-synaptic neurons, known as spike-timing-dependent plasticity (STDP). STDP, together with theta-phase-dependent learning, has inspired several computational models of learning and memory. However, evidence to elucidate how these mechanisms directly link to human episodic memory is lacking. In a computational model, we modulate long-term potentiation (LTP) and long-term depression (LTD) of STDP, by opposing phases of a simulated theta rhythm. We fit parameters to a hippocampal cell culture study in which LTP and LTD were observed to occur in opposing phases of a theta rhythm. Further, we modulated two inputs by cosine waves with synchronous and asynchronous phase offsets and replicate key findings in human episodic memory. Learning advantage was found for the synchronous condition, as compared to the asynchronous conditions, and was specific to theta modulated inputs. Importantly, simulations with and without each mechanism suggest that both STDP and theta-phase-dependent plasticity are necessary to replicate the findings. Together, the results indicate a role for circuit-level mechanisms, which bridges the gap between slice preparation studies and human memory. Author SummaryLong-lasting changes in synaptic connectivity between neurons have been suggested to support learning and memory processes at the cellular level in the brain. Such synaptic modifications depend on synchronous activation of neurons, which leads to generate brain oscillations. Human memory studies focus on the relationships between brain oscillations and memory processes. Direct evidence on how the cellular mechanism links to human memory behaviour is lacking. To investigate the direct link between synaptic plasticity mechanisms and human memory formation, we built a computational neural network that implements two synaptic plasticity mechanisms, which are well-established in the rodents hippocampus. One mechanism shows that strengthening or weakening in synaptic connectivity depends on the phases of ongoing brain oscillation at theta frequency (4 - 8 Hz), which is a dominant signal in the hippocampus. The other mechanism suggests that synaptic modification depends on the precise timing of action potentials between two neurons. Our model successfully reproduces results from rodents, as well as several human episodic memory studies which demonstrated that human associative memory performance depends on phase synchronisation in theta frequency. These findings suggest a link between specific learning mechanisms at cellular level and human memory behaviour.

neuroscience↗

bioRxiv2021

Biological network inference from single-cell multi-omics data using heterogeneous graph transformer

We present DeepMAPS (Deep learning-based Multi-omics Analysis Platform for Single-cell data) for biological network inference from single-cell multi-omics (scMulti-omics). DeepMAPS includes both cells and genes in a heterogeneous graph to simultaneously infer cell-cell, cell-gene, and gene-gene relations. The multi-head attention mechanism in a graph transformer considers the heterogeneous relation among cells and genes within both local and global context, making DeepMAPS robust to data noise and scale. We benchmarked DeepMAPS on 18 scMulti-omics datasets for cell clustering and biological network inference, and the results showed that our method outperformed various existing tools. We further applied DeepMAPS on lung tumor leukocyte CITE-seq data and matched diffuse small lymphocytic lymphoma scRNA-seq and scATAC-seq data. In both cases, DeepMAPS showed competitive performance in cell clustering and predicted biologically meaningful cell-cell communication pathways based on the inferred gene networks. Note that we deployed a webserver using DeepMAPS implementation equipped with multiple functions and visualizations to improve the feasibility and reproducibility of scMulti-omics data analysis. Overall, DeepMAPS represents a heterogeneous graph transformer for single-cell study and may benefit the use of scMulti-omics data in various biological systems.

bioinformatics↗

bioRxiv2021

DEAD-box RNA helicase 18 disrupts IRF3-binding to the interferon-β promoter

The production of type I interferons (IFN-/{beta}) requires strict control to avoid excessive activation during viral infections. The binding of interferon regulatory factor 3 (IRF3) to the IFN-{beta} promoter region in the nucleus is essential for IFN-{beta} transcription; however, whether nuclear factors have important negative-regulatory roles in this process is largely unknown. By screening for IRF3-interacting partners in the nucleus, we identified DEAD-box RNA helicase 18 (DDX18) as an important negative regulator of intranuclear IRF3. Overexpression of DDX18 suppressed virus- and IRF3-induced IFN-{beta} production, whereas knockdown of DDX18 expression or knockout of the DDX18 gene had opposite effects. Mechanistically, DDX18 interacts with IRF3 and decreases the binding of IRF3 to the IFN-{beta} promoter after viral infection. DDX18 knockdown mice (Ddx18+/-) further demonstrated that DDX18 suppressed antiviral innate immunity in vivo. Thus, despite many members of the DDX family act as important positive regulators in the cytoplasm, DDX18 plays a unique "braking" role in balancing virus-induced type I IFN production.

immunology↗

bioRxiv2021

Who takes care of the kids at when? Variation of sex-role patterns across different parental care forms in passerine birds

Parental care in birds consists of many elaborate forms, including nest building, incubation, and offspring provision, but we still do not know how much each parent contributes to the different forms. Furthermore, the variations, relationships, and potential drivers of sex differentiation in providing care across different care stages remain largely unknown. Here, we surveyed species in birds and uncovered remarkable differences in the sex role patterns across different care forms. This result implied that parental care should not be treated as a unitary trait but as a composite of integrated features with great variations. Further analyses revealed moderate correlations of the sex roles between care forms, indicating the existence of shared intrinsic drivers. We tested the effects of sexual selection, certainty of paternity, predation risk, and offsprings life history traits in driving sex role variations. Results showed that species with strong sexual selection on males or uncertainty of paternity tend to have female-biased care.

evolutionary biology↗

bioRxiv2021

OsMADS58 stabilizes gene regulatory circuits during rice stamen development

Rice (Oryza sativa) OsMADS58 is a C-class MADS box protein, and characterization of a transposon insertion mutant osmads58 suggested that OsMADS58 plays a role in stamen development. However, as no null mutation has been obtained, its role has remained unclear. Here, we report that the CRISPR knockout mutant osmads58 exhibits complex altered phenotypes, including anomalous diploid germ cell differentiation, aberrant meiosis, and delayed tapetum degeneration. This CRISPR mutant line exhibited stronger changes in expression of OsMADS58 target genes compared with the osmads58 dSpm (transposon insertion) line, along with changes in multiple pathways related to early stamen development. Notably, transcriptional regulatory circuits in young panicles covering the stamen at stages 4-6 were substantially altered in the CRISPR line compared to the dSpm line. These findings strongly suggest that the pleiotropic effects of OsMADS58 on stamen development derive from a potential role in stabilizing gene regulatory circuits during early stamen development. Thus, this work opens new avenues for viewing and deciphering the regulatory mechanisms of early stamen development from a network perspective.

plant biology↗

bioRxiv2021

Treatment with IFB-088 improves neuropathy in CMT1A and CMT1B mice

Charcot Marie Tooth diseases type 1A (CMT1A), caused by duplication of Peripheral Myelin Protein 22 (PMP22) gene, and CMT1B, caused by mutations in myelin protein zero (MPZ) gene are the two most common forms of demyelinating CMT (CMT1) and no treatments are available for either. Prior studies of the MpzSer63del mouse model of CMT1B have demonstrated that protein misfolding, endoplasmic reticulum (ER) retention and activation of the unfolded protein response (UPR) contributed to the neuropathy. Heterozygous patients with an arginine to cysteine mutation in MPZ (MPZR98C) develop a severe infantile form of CMT1B which is modeled by MpzR98C/+ mice that also show ER-stress and an activated UPR. C3-PMP22 mice are considered to effectively model CMT1A. Altered proteostasis, ER-stress and activation of the UPR have been demonstrated in mice carrying Pmp22 mutations. To determine whether enabling the ER-stress/UPR and readjusting protein homeostasis would effectively treat these models of CMT1B and CMT1A we administered Sephin1/IFB-088/icerguestat, a UPR modulator which showed efficacy in the MpzS63del model of CMT1B, to heterozygous MpzR98C and C3-PMP22 mice. Mice were analyzed by behavioral, neurophysiological, morphological and biochemical measures. Both MpzR98C/+ and C3-PMP22 mice improved in motor function and neurophysiology. Myelination, as demonstrated by g-ratios and myelin thickness, improved in CMT1B and CMT1A mice and markers of UPR activation returned towards wild type values. Taken together our results demonstrate the capability of IFB-088 to treat a second mouse model of CMT1B and a mouse model of CMT1A, the most common form of CMT. Given the recent benefits of IFB-088 treatment in Amyotrophic Lateral Sclerosis and Multiple Sclerosis animal models, these data demonstrate its potential in managing UPR and ER-stress for multiple mutations in CMT1 as well as in other neurodegenerative diseases.

neuroscience↗

bioRxiv2021

A unique genomic island-governed cannibalism in Bacillus enhanced biofilm formation through a novel regulation mechanism

Cannibalism is a differentiation strategy and social multicellular behavior in biofilms. The novel factors and mechanisms to trigger the bacterial cannibalism remain scarce. Here, we report a novel bacillunoic acids-mediated strategy for manipulating cannibalism in Bacillus velezensis SQR9 biofilm formation. A subfraction of cells differentiate into cannibals that secrete toxic bacillunoic acids to lyse a fraction of their sensitive siblings, and the released nutrients enhance biofilm formation. Meanwhile, the self-immunity of cannibal cells was induced by bacillunoic acids. A two-component system, the OmpS-OmpR signal-transduction pathway, controls the expression of the ABC transporter BnaAB for self-immunity. Specifically, bacillunoic acids activate the autophosphorylation of OmpS, a transmembrane histidine kinase, which then transfers a phosphoryl group to its response regulator OmpR. The phosphorylation of OmpR activates the transcription of the transporter gene bnaAB by binding its promoter. Thus, bacillunoic acids are pumped out of cells by the ABC transporter BnaAB. Moreover, we discovered that strain SQR9 could use the bacillunoic acids-mediated cannibalism to optimize its community to produce more bacillunoic acids for bacterial competition. This study revealed that bacillunoic acids play a previously undiscovered dual role in both cannibalism during biofilm formation and interspecies competition, which has an important biological significance.

microbiology↗

bioRxiv2021

Dalpiciclib and Pyrotinib Exert Synergistic Antitumor Effects in Triple Positive Breast Cancer

BackgroundThe therapeutic benefit of the standard combination of anti-HER2 and chemotherapy in triple-positive breast cancer (TPBC) is limited even after the addition of endocrine therapy to the regimen. Therefore, treatment optimization is required urgently. MethodsThrough the drug sensitivity test, the drug combination efficacy of anti-HER2 drug, endocrine drug and CDK4/6 inhibitor to BT474 cells were tested. The underlying molecular mechanisms were investigated using immunofluorescence, western blot analysis, immunohistochemical staining and cell cycle analysis. Potential biomarker which may indicate the responsiveness to drug treatment in triple positive breast cancer was selected out using RNA-sequence and tested using immunohistochemical staining. ResultsWe found that pyrotinib combined with dalpiciclib showed better efficacy than pyrotinib combined with tamoxifen in BT474 cells. Degradation of HER2 could enhance ER nuclear transportation, whereas cell cycle blockers could reverse this process. This may be the underlying mechanism by which the addition of dalpiciclib was more beneficial than the addition of pyrotinib plus tamoxifen. Furthermore, CALML5 was revealed to be a potential indicator of responsiveness to anti-HER2 therapy plus CDK4/6 inhibition in triple positive breast cancer. ConclusionOur study provided evidence for the introduction of CDK4/6 inhibitor in the treatment of TPBC and indicated that the combination of anti-HER2 therapy and cell cycle blockers may be a better strategy for TPBC treatment. FundingThis study was supported by the National Natural Science Foundation of China (#U20A20381, #81872159)

cell biology↗

bioRxiv2021

Mechanical loading and hyperosmolarity as a daily resetting cue for skeletal circadian clocks

Daily rhythms in mammalian behaviour and physiology are generated by a multi-oscillator circadian system entrained through environmental cues (e.g. light). Presence of niche-dependent physiological time cues has been proposed, allowing local tissues flexibility of phase adjustment. However, to date, such stimuli have remained elusive. Here we show that cycles of mechanical loading and osmotic stimuli within physiological range drive rhythmic expression of clock genes and reset clock phase and amplitude in cartilage and intervertebral disc tissues. Hyperosmolarity (not hypo-osmolarity) resets clocks in young and ageing skeletal tissues through mTORC2-AKT-GSK3{beta} pathway, leading to genome-wide induction of rhythmic genes. These results advocate diurnal patterns of mechanical loading and consequent daily surges in osmolarity as a bona fide tissue niche-specific time cue to maintain skeletal circadian rhythms in sync. One-Sentence SummaryCircadian clocks in aneural skeletal tissues sense the passage of time through rhythmic patterns of loading and osmolarity

physiology↗

bioRxiv2021

CAR T cell therapy drives endogenous locoregional T cell dynamics in a responding patient with glioblastoma

CAR T cell therapy has transformed clinical care and management of patients with certain hematological cancers. However, it remains unclear whether the success of CAR T cell therapy relies solely on CAR T cell engagement with tumor antigen, or if it also requires the stimulation of an individual patients endogenous T cell response. Here, we performed combined analysis of longitudinal, single cell RNA and T cell receptor sequencing on glioblastoma tumors, peripheral blood (PB), and cerebrospinal fluid (CSF) from a patient with recurrent multifocal glioblastoma that underwent a remarkable response followed by recurrence on IL13RA2-targeted CAR T cell therapy (Brown et al. 2016). Single cell analysis of a tumor resected prior to CAR T cell therapy revealed the existence of an inflamed tumor microenvironment including a CD8+ cytotoxic, clonally expanded and antigen specific T cell population that disappeared in the recurrent setting. Longitudinal tracking of T cell receptors uncovered distinct T cell dynamics classes in the CSF during CAR T cell therapy. These included T cell clones with transient dynamics, representing intraventricular CAR T cell delivery and endogenous T cell recruitment from the PB into the CSF; and a group of T cells in the cerebrospinal fluid, that tracked with clonally expanded tumor resident T cells and whose dynamics contracted concomitantly with tumor volume. Our results suggest the existence of an endogenous T cell population that was invigorated by intraventricular CAR T cell infusions, and combined with the therapy to produce a complete response.

immunology↗

bioRxiv2021

Necroptosis contributes to chronic inflammation and fibrosis in aging liver

Inflammaging, characterized by an increase in low-grade chronic inflammation with age, is a hallmark of aging and is strongly associated with various age-related diseases, including chronic liver disease (CLD) and hepatocellular carcinoma (HCC). Because necroptosis is a cell death pathway that induces inflammation through the release of DAMPs, we tested the hypothesis that age-associated increase in necroptosis contributes to chronic inflammation in aging liver. Phosphorylation of MLKL and MLKL-oligomers, markers of necroptosis, as well as phosphorylation of RIPK3 and RIPK1 were significantly upregulated in the livers of old mice relative to young mice and this increase occurred in the later half of life (i.e., after 18 months of age). Markers of M1 macrophages, expression of proinflammatory cytokines (TNF, IL6 and IL-1{beta}), and markers of fibrosis were significantly upregulated in the liver with age and the change in necroptosis paralleled the changes in inflammation and fibrosis. Hepatocytes and liver macrophages isolated from old mice showed elevated levels of necroptosis markers as well as increased expression of proinflammatory cytokines relative to young mice. Short term treatment with the necroptosis inhibitor, necrostatin-1s (Nec-1s), reduced necroptosis, markers of M1 macrophages, expression of proinflammatory cytokines, and markers of fibrosis in the livers of old mice. Thus, our data show for the first time that liver aging is associated with increased necroptosis and necroptosis contributes to chronic inflammation in the liver, which in turn appears to contribute to liver fibrosis and possibly CLD. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=158 SRC="FIGDIR/small/460953v1_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@15ab761org.highwire.dtl.DTLVardef@bd7336org.highwire.dtl.DTLVardef@1d6e3bcorg.highwire.dtl.DTLVardef@10159eb_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2021

m6A modifications regulate intestinal immunity and rotavirus infection

N6-methyladenosine (m6A) is an abundant mRNA modification and affects many biological processes. However, how m6A levels are regulated during physiological or pathological processes such as virus infections, and the in vivo function of m6A in the intestinal immune defense against virus infections are largely unknown. Here, we uncover a novel antiviral function of m6A modification during rotavirus (RV) infection in small bowel intestinal epithelial cells (IECs). We found that rotavirus infection induced global m6A modifications on mRNA transcripts by down-regulating the m6a eraser ALKBH5. Mice lacking the m6A writer enzymes METTL3 in IECs (Mettl3{Delta}IEC) were resistant to RV infection and showed increased expression of interferons (IFNs) and IFN-stimulated genes (ISGs). Using RNA-sequencing and m6A RNA immuno-precipitation (RIP)-sequencing, we identified IRF7, a master regulator of IFN responses, as one of the primary m6A targets during virus infection. In the absence of METTL3, IECs showed increased Irf7 mRNA stability and enhanced type I and III IFN expression. Deficiency in IRF7 attenuated the elevated expression of IFNs and ISGs and restored susceptibility to RV infection in Mettl3{Delta}IEC mice. Moreover, the global m6A modification on mRNA transcripts declined with age in mice, with a significant drop from 2 weeks to 3 weeks post birth, which likely has broad implications for the development of intestinal immune system against enteric viruses early in life. Collectively, we demonstrated a novel host m6A-IRF7-IFN antiviral signaling cascade that restricts rotavirus infection in vivo.

immunology↗

bioRxiv2021

Illuminating links between cis-regulators and trans-acting variants in the human prefrontal cortex

Psychiatric disorders exact immense human and economic tolls in societies globally. Underlying many of these disorders is a complex repertoire of genomic variants that influence the expression of genes involved in pathways and processes in the brain. Identifying such variants and their associated brain functions is thus essential for understanding the molecular underpinnings of psychiatric disorders. Genome-wide association studies (GWASes) have provided many variants associated with these disorders; however, our knowledge of the precise biological mechanisms by which these contribute to disease remains limited. In connection with this, expression quantitative trait loci (eQTLs) have provided useful information linking variants to genes and functions. However, most eQTL studies on human brain have focused exclusively on cis-eQTLs. A complete understanding of disease etiology should also include trans-regulatory mechanisms. Thus, we conduct one of the first genome-wide surveys of trans-eQTLs in the dorsolateral prefrontal cortex (DLPFC) by leveraging the large datasets from the PsychENCODE consortium. We identified [~]80,000 trans-eQTLs. We found that a significant number of these overlap with cis-eQTLs, thereby implicating cis-mediators as key players in trans-acting regulation. We show, furthermore, that trans-regulatory mechanisms provide novel insights into psychiatric disease. Particularly, colocalization analysis between trans-eQTLs and schizophrenia (SCZ) GWAS loci identified 90 novel SCZ risk genes and 23 GWAS loci previously uncharacterized by cis-eQTLs. Moreover, these 90 genes tend to be more central in transcriptome-wide co-expression networks and more susceptible to rare variants than SCZ-risk genes associated by cis-variation.

genomics↗

bioRxiv2021

RNASeq analysis of a Pax3-expressing myoblast clone in-vitro and effect of culture surface stiffness on differentiation.

Skeletal muscle satellite cells cultured on soft surfaces (12kPa) show improved differentiation than cells cultured on stiff surfaces (approximately 100kPa). To better understand the reasons for this, we performed an RNA-Seq analysis for a single satellite cell clone (C1F) derived from the H2kb-tsA58 immortomouse, which differentiates into myotubes under tightly regulated conditions (withdrawal of {gamma}-interferon, 37{degrees}C). As expected, the largest change in overall gene expression occurred at day 1, as cells switch from proliferation to differentiation. Surprisingly, further analysis showed that proliferating C1F cells express Pax3 and not Pax7, confirmed by immunostaining, yet their subsequent differentiation into myotubes is normal, and enhanced on softer surfaces, as evidenced by significantly higher expression levels of myogenic regulatory factors, sarcomeric genes, enhanced fusion and improved myofibrillogenesis. Levels of RNA encoding extracellular matrix structural constituents and related genes were consistently upregulated on hard surfaces, suggesting that a consequence of differentiating satellite cells on hard surfaces is that they attempt to manipulate their niche prior to differentiating. This comprehensive RNASeq dataset will be a useful resource for understanding Pax3 expressing cells.

cell biology↗

bioRxiv2021

Hematopoiesis at single cell resolution spanning human development and maturation

Hematopoiesis is a process of constitutive regeneration whereby hematopoietic stem and progenitor cells (HSPCs) replenish mature blood cells. During maturation and aging, HSPCs shift their output to support the demands of prenatal development and postnatal maturation both at homeostasis and in response to stress. How HSPC ontogeny changes throughout life is unknown; studies to date have largely focused on specific individual ages, particularly at single cell resolution. Here, we performed single cell RNA-seq of human HSPCs from early prenatal development into mature adulthood. We observed shifts in HSPC transcriptional states and differentiation trajectories over time. We identified age-specific gene expression patterns throughout human maturation and developed methods for identifying, prospectively purifying, and functionally validating age-specific HSC states. Together, our findings define the temporal maturation of human HSPCs and uncover principles applicable to age-biased blood diseases. SummarySingle cell RNA sequencing reveals that the mechanisms of human hematopoietic stem and progenitor cell (HSPC) fate commitment change over a lifetime from gestation to mature adulthood.

developmental biology↗