Biology subjects

Fu, D.

Publications and source records attributed to Fu, D..

48 records · Page 3Linked to original sources

bioRxiv2021

Large field of view-spatially resolved transcriptomics at nanoscale resolution

Spatially resolved transcriptomic technologies are promising tools to study cell fate decisions in a physical microenvironment, which is fundamental for enhancing our knowledge of mammalian development. However, the imbalance between resolution, transcript capture and field of view of current methodologies precludes their systematic application to analyze relatively large and three-dimensional mid- and late-gestation mammalian embryos. Here, we combined DNA nanoball (DNB) patterned arrays and tissue RNA capture to create SpaTial Enhanced REsolution Omics-sequencing (Stereo-seq). This approach allows transcriptomic profiling of large histological sections with high resolution and sensitivity. We have applied Stereo-seq to study the kinetics and directionality of transcriptional variation in a time course of mouse organogenesis. We used this information to gain insight into the molecular basis of regional specification, neuronal migration and differentiation in the developing brain. Furthermore, we mapped the expression of a panel of developmental disease-related loci on our global transcriptomic maps to define the spatiotemporal windows of tissue vulnerability. Our panoramic atlas constitutes an essential resource to investigate longstanding questions concerning normal and abnormal mammalian development.

cell biology↗

bioRxiv2020

CD73 maintains hepatocyte metabolic integrity and mouse liver homeostasis in a sex-dependentmanner

Background & AimsMetabolic imbalance and inflammation are common features of chronic liver diseases. Molecular factors controlling these mechanisms represent potential therapeutic targets. One promising target is CD73, the major enzyme that dephosphorylates extracellular adenosine monophosphate (AMP) to form the anti-inflammatory adenosine. In normal liver, CD73 is expressed on pericentral hepatocytes, which are important for long-term liver homeostasis. The aim of this study was to determine if CD73 has non-redundant hepatoprotective functions. Approach & ResultsWe generated mice with a targeted deletion of the CD73-encoding gene (Nt5e) in hepatocytes (CD73-LKO). Deletion of hepatocyte Nt5e resulted in approximately 70% reduction in total liver CD73 protein (p<0.0001). Male and female CD73-LKO mice developed normally during the first 21 weeks, without significant liver phenotypes. Between 21-42 weeks, the CD73-LKO mice developed spontaneous onset liver disease with significant severity in male mice. Notably, middle-aged male CD73-LKO mice displayed hepatocyte swelling and ballooning (p<0.05), inflammation (p<0.01) and variable steatosis. Female CD73-LKO mice had lower serum albumin (p<0.05) and elevated inflammatory markers (p<0.01), but did not exhibit the spectrum of histopathologic changes characteristic of the male mice, potentially due to compensatory induction of adenosine receptors. Serum analysis and proteomic profiling of hepatocytes from male CD73-LKO mice revealed significant metabolic imbalance, with elevated blood urea nitrogen (p<0.0001) and impairments in major metabolic pathways, including oxidative phosphorylation and AMP-activated protein kinase (AMPK) signaling. There was significant hypo-phosphorylation in AMPK substrate in CD73-LKO livers (p<0.0001), while in isolated hepatocytes treated with AMP, soluble CD73 induced AMPK activation (p<0.001). ConclusionsHepatocyte CD73 supports long-term metabolic liver homeostasis through AMPK in a sex-dependent manner. These findings have implications for human liver diseases marked by CD73 dysregulation.

physiology↗

bioRxiv2020

SMAD2 promotes myogenin expression and terminal myogenic differentiation.

SMAD2 is a transcription factor whose activity is regulated by members of the Transforming Growth Factor beta (TGF{beta}) superfamily. While activation of SMAD2 and SMAD3 downstream of TGF{beta} or myostatin signaling is known to inhibit myogenesis, we find that SMAD2 in the absence of TGF{beta} signaling promotes terminal myogenic differentiation. We find that during myogenic differentiation, SMAD2 expression is induced. Knockout of SMAD2 expression in primary myoblasts did not affect the efficiency of myogenic differentiation but produced smaller myotubes with reduced expression of the terminal differentiation marker myogenin. Conversely, overexpression of SMAD2 stimulated myogenin expression, and enhanced both differentiation and fusion, and these effects were independent of classical activation by the TGF{beta} receptor complex. Loss of Smad2 in muscle satellite cells in vivo resulted in decreased muscle fiber caliber and impaired regeneration after acute injury. Taken together, we demonstrate that SMAD2 is an important positive regulator of myogenic differentiation, in part through the regulation of Myog. SUMMARY STATEMENTTGF{beta} and SMAD2 are normally associated with inhibition of myogenesis. Our work identifies a pro-myogenic role for SMAD2 during terminal myogenic differentiation, through regulation of Myog and Klf4 expression.

cell biology↗

bioRxiv2020

ALKBH7 mediates necrosis via rewiring of glyoxal metabolism

Alkb homolog 7 (ALKBH7) is a mitochondrial -ketoglutarate dioxygenase required for necrotic cell death in response to DNA alkylating agents, but its physiologic role within tissues remains unclear. Herein, we show that ALKBH7 plays a key role in the regulation of dialdehyde metabolism, which impacts cardiac survival in response to ischemia-reperfusion (IR) injury. Using a multi-omics approach, we do not find evidence that ALKBH7 functions as a prolyl-hydroxylase. However, we do find that mice lacking ALKBH7 exhibit a significant increase in glyoxalase I (GLO-1), a dialdehyde detoxifying enzyme. Consistent with increased dialdehyde production, metabolomics analysis reveals rewiring of metabolic pathways related to the toxic glycolytic by-product methylglyoxal (MGO), as well as accelerated glycolysis and elevated levels of MGO protein adducts, in mice lacking ALKBH7. Consistent with roles for both necrosis and glycative stress in cardiac IR injury, hearts from male but not female Alkbh7-/- mice are protected against IR, although somewhat unexpectedly this protection does not appear to involve modulation of the mitochondrial permeability transition pore. Highlighting the importance of MGO metabolism for the observed protection, removal of glucose as a metabolic substrate or pharmacologic inhibition of GLO-1 both abrogate cardioprotection in ALKBH7 deficient mice. Integrating these observations, we propose that ALKBH7 plays a role in the regulation of glyoxal metabolism, and that protection against necrosis and IR injury bought on by ALKBH7 deficiency originates from hormetic signaling in response to elevated MGO stress.

cell biology↗

bioRxiv2019

A missense variant impairing TRMT1 function in tRNA modification is linked to intellectual disability

The human TRMT1 gene encodes a tRNA methyltransferase enzyme responsible for the formation of the dimethylguanosine (m2,2G) modification in cytoplasmic and mitochondrial tRNAs. Frameshift mutations in the TRMT1 gene have been shown to cause autosomal-recessive intellectual disability (ID) in the human population but additional TRMT1 variants remain to be characterized. Moreover, the impact of ID-associated TRMT1 mutations on m2,2G levels in ID-affected patients is unknown. Here, we describe a homozygous missense variant in TRMT1 in a patient displaying developmental delay, ID, and epilepsy. The missense variant changes a conserved arginine residue to a cysteine (R323C) within the methyltransferase domain of TRMT1 and is expected to perturb protein folding. Patient cells expressing the TRMT1-R323C variant exhibit a severe deficiency in m2,2G modifications within tRNAs, indicating that the mutation causes loss-of-function. Notably, the TRMT1 R323C mutant retains the ability to bind tRNA but is unable to rescue m2,2G formation in TRMT1-deficient human cells. Our results identify a pathogenic point mutation in TRMT1 that severely perturbs tRNA modification activity, and provide the first demonstration that m2,2G modifications are disrupted in patients with TRMT1-associated ID disorders.

molecular biology↗

bioRxiv2019

METTL2 forms a complex with the DALRD3 anticodon-domain binding protein to catalyze formation of 3-methylcytosine in specific arginine tRNA isoacceptors

In mammals, a subset of arginine tRNA isoacceptors are methylated in the anticodon loop by the METTL2 methyltransferase to form the 3-methylcytosine (m3C) modification. However, the mechanism by which METTL2 identifies specific arginine tRNAs for m3C formation as well as the biological role of m3C in mammals is unknown. Here, we show that human METTL2 forms a complex with DALR anticodon binding domain containing 3 (DALRD3) protein in order to recognize particular arginine tRNAs destined for m3C modification. Using biochemical reconstitution, we find that METTL2-DALDR3 complexes catalyze m3C formation in vitro that is dependent upon sequence elements specific to certain arginine tRNAs. Notably, DALRD3-deficient human cells exhibit nearly complete loss of the m3C modification in arginine tRNAs. These findings uncover an unexpected function for the DALRD3 protein in the targeting of distinct arginine tRNAs for m3C modification.

molecular biology↗

bioRxiv2019

In situ measurement of absolute concentrations by Normalized Raman Imaging

Cell mass and its chemical composition are important aggregate cellular variables for physiological processes including growth control and tissue homeostasis. Despite their central importance, it has been difficult to quantitatively measure these quantities from single cells in intact tissue. Here, we introduce Normalized Raman Imaging (NoRI), a Stimulated Raman Scattering (SRS) microscopy method that provides the local concentrations of protein, lipid and water from live or fixed tissue samples with high spatial resolution. Using NoRI, we demonstrate that single cell protein, lipid and water concentrations are maintained in a tight range in cells under same physiological conditions and are altered in different physiological states such as cell cycle stages, attachment to substrates of different stiffness, or by entering senescence. In animal tissues, protein and lipid concentration varies with cell types, yet an unexpected cell-to-cell heterogeneity was found in cerebellar Purkinje cells. Protein and lipid concentration profile provides a new means to quantitatively compare disease-related pathology as demonstrated using models of Alzheimers disease. Our demonstration shows that NoRI is a broadly applicable tool for probing the biological regulation of protein mass, lipid mass and water in cellular and tissue growth, homeostasis, and disease.

bioengineering↗

bioRxiv2019

Sequence based mapping identifies AWNS1, a candidate transcription repressor underlying awn suppression at the B1 locus in wheat

Awns are stiff, hair-like structures that grow from the spikelets of wheat (Triticum aestivum L.) and other grasses. In wild wheat relatives, awns play a role in seed dispersal. Awn suppression in domesticated wheat, where awns are shortened or even eliminated entirely, is variably adaptive with both awned and awnless types under widespread cultivation. Although the B1 locus on the long arm of chromosome 5A is a major determinant of awn suppression, no underlying gene or mechanism of action has been identified. Using association mapping, we identified a SNP marker located on the distal end of 5AL (5A28417) predictive of awn status in a panel of 640 U.S. winter wheat breeding lines, indicating that B1 was the determinant of awn suppression in this germplasm. Analysis of historical data available for the panel determined 5A28417 was also significantly associated with grain test weight. Evaluation of spike morphology and kernel traits was undertaken in a 341 RIL population developed from a cross between awned soft winter wheat cultivar LA95135 and awnless cultivar SS-MPV57. Awn suppression in the population co-segregated with 5A28417 and was co-located with QTL for number of spikelets per spike, kernel weight and kernel length. Fine-mapping located B1 to a region containing only two predicted genes, including a C2H2 zinc finger transcription factor 219 bp from 5A28417 that we named AWNS-A1. Deletions encompassing both genes were present in awned mutants of the awnless cultivar Brundage. Polymorphisms in the AWNS-A1 coding region were not observed in diverse wheat germplasm. However, sequencing of wheat lines representing different marker haplotypes in the surrounding region identified a deletion 3 kb downstream of AWNS-A1. A marker for this deletion was highly predictive of awn suppression in a collection of diverse wheat accessions, and indicates that AWNS-A1 is likely the major determinant of awn suppression in global wheat germplasm. AWNS-A1 is more highly expressed in developing spikes of awnless individuals, suggesting a mechanism for awn suppression.

genetics↗

bioRxiv2018

Microbial coexistence through chemical-mediated interactions

Many microbial functions happen within communities of interacting species. Explaining how species with intrinsically disparate fitness can coexist is important for applications such as manipulating host-associated microbiota or engineering industrial communities. Previous coexistence studies have often neglected interaction mechanisms. Here, we formulate and experimentally constrain a model in which chemical mediators of microbial interactions (e.g. metabolites or waste-products) are explicitly incorporated. We construct many instances of coexistence by simulating community assembly through enrichment and ask how species interactions can explain coexistence. We show that growth-facilitating influences between members are favored in assembled communities. Among negative influences, self-restraint, such as production of self-inhibiting waste, contributes to coexistence, whereas inhibition of other species disrupts coexistence. Coexistence is also favored when interactions are mediated by depletable chemicals that get consumed or degraded, rather than by reusable chemicals that are unaffected by recipients. Our model creates null predictions for coexistence driven by chemical-mediated interactions.

ecology↗

bioRxiv2018

Formation of tRNA wobble inosine in humans is perturbed by a millennia-old mutation linked to intellectual disability

The formation of inosine at the wobble position of eukaryotic tRNAs is an essential modification catalyzed by the ADAT2/ADAT3 complex. In humans, a valine to methionine mutation (V144M) in ADAT3 that originated [~]1,600 years ago is the most common cause of autosomal-recessive intellectual disability (ID) in Arabia. Here, we show that ADAT3-V144M exhibits perturbations in subcellular localization and has increased propensity to form aggregates associated with cytoplasmic chaperonins. While ADAT2 co-expression can suppress the aggregation of ADAT3-V144M, the ADAT2/3 complexes assembled with ADAT3-V144M exhibit defects in adenosine deaminase activity. Moreover, extracts from cell lines derived from ID-affected individuals expressing only ADAT3-V144M display a reduction in tRNA deaminase activity. Notably, we find that the same cell lines from ID-affected individuals exhibit decreased wobble inosine in certain tRNAs. These results identify a role for ADAT2-dependent localization and folding of ADAT3 in wobble inosine modification that is crucial for the developing human brain.

molecular biology↗

bioRxiv2018

Anamorphic development and extended parental care in a 520 million-year-old stem-group euarthropod from China

Extended parental care (XPC) is a complex reproductive strategy in which progenitors actively look after their offspring up to - or beyond - the first juvenile stage in order to maximize their fitness. Although the euarthropod fossil record has produced several examples of brood-care, the appearance of XPC within this phylum remains poorly constrained given the scarcity of developmental data for Palaeozoic stem-group representatives that would link juvenile and adult forms in an ontogenetic sequence. Here, we describe the post-embryonic growth of Fuxianhuia protensa from the early Cambrian Chengjiang Lagerstatte, and show parental care in this stem-group euarthropod. We recognize fifteen distinct ontogenetic stages based on the number and shape of the trunk tergites, and their allocation between the morphologically distinct thorax and abdomen. Our data demonstrate anamorphic post-embryonic development in F. protensa, in which tergites were sequentially added from a posterior growth zone. A life assemblage consisting of a sexually mature F. protensa adult alongside four ontogenetically coeval juveniles, constitutes the oldest occurrence of XPC in the panarthropod fossil record. These findings provide the most phylogenetically basal evidence of anamorphosis in the evolutionary history of total-group Euarthropoda, and reveal a complex post-embryonic reproductive ecology for its early representatives.

paleontology↗

bioRxiv2018

Monitoring the 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U) modification in eukaryotic tRNAs via the gamma-toxin endonuclease

The post-transcriptional modification of tRNA at the wobble position is a universal process occurring in all domains of life. In eukaryotes, the wobble uridine of particular tRNAs is transformed to the 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U) modification which is critical for proper mRNA decoding and protein translation. However, current methods to detect mcm5s2U are technically challenging and/or require specialized instrumental expertise. Here, we show that gamma-toxin endonuclease from the yeast Kluyveromyces lactis can be used as a probe for assaying mcm5s2U status in the tRNA of diverse eukaryotic organisms ranging from protozoans to mammalian cells. The assay couples the mcm5s2U-dependent cleavage of tRNA by gamma-toxin with standard molecular biology techniques such as Northern blot analysis or quantitative PCR to monitor mcm5s2U levels in multiple tRNA isoacceptors. The results gained from the gamma-toxin assay reveals the evolutionary conservation of the mcm5s2U modification across eukaryotic species. Moreover, we have employed the gamma-toxin assay to verify uncharacterized eukaryotic Trm9 and Trm112 homologs that catalyze the formation of mcm5s2U. These findings demonstrate the use of gamma-toxin as a detection method to monitor mcm5s2U status in diverse eukaryotic cell types for cellular, genetic and biochemical studies.

molecular biology↗