Biology subjects

Wu, Y.

Publications and source records attributed to Wu, Y..

At least 757 records · Page 42Linked to original sources

bioRxiv2022

Synergistic effect of mutations in the Parkinson's disease-linked proteins synaptojanin 1 and auxilin

Parkinsons disease (PD) is a neurodegenerative disorder characterized by defective dopaminergic (DAergic) input to the striatum. Mutations in two genes encoding synaptically enriched clathrin-uncoating factors, synaptojanin 1 (SJ1) and auxilin, have been implicated in atypical Parkinsonism. SJ1 knock-in (SJ1-KIRQ) mice carrying a disease-linked mutation display neurological manifestations reminiscent of Parkinsonism. Here we report that auxilin knockout (Aux-KO) mice display dystrophic changes of a subset of nigrostriatal DAergic terminals similar to those of SJ1-KIRQ mice. Furthermore, Aux-KO/SJ1-KIRQ double mutant mice have shorter lifespan and more severe synaptic defects than single mutant mice. These include increase in dystrophic striatal nerve terminals positive for DAergic markers and for the PD risk protein SV2C, as well as adaptive changes in striatal interneurons. The synergistic effect of the two mutations demonstrates a special lability of DAergic neurons to defects in clathrin uncoating, with implications for PD pathogenesis in at least some forms of this condition.

neuroscience↗

bioRxiv2022

Pan-cancer proteomic map of 949 human cell lines reveals principles of cancer vulnerabilities

The proteome provides unique insights into biology and disease beyond the genome and transcriptome. Lack of large proteomic datasets has restricted identification of new cancer biomarkers. Here, proteomes of 949 cancer cell lines across 28 tissue types were analyzed by mass spectrometry. Deploying a clinically-relevant workflow to quantify 8,498 proteins, these data capture evidence of cell type and post-transcriptional modifications. Integrating multi-omics, drug response and CRISPR-Cas9 gene essentiality screens with a deep learning-based pipeline revealed thousands of protein-specific biomarkers of cancer vulnerabilities. Proteomic data had greater power to predict drug response than the equivalent portion of the transcriptome. Further, random downsampling to only 1,500 proteins had limited impact on predictive power, consistent with protein networks being highly connected and co-regulated. This pan-cancer proteomic map (ProCan-DepMapSanger), available at https://cellmodelpassports.sanger.ac.uk, is a comprehensive resource revealing principles of protein regulation with important implications for future clinical studies.

systems biology↗

bioRxiv2022

Omicron-specific mRNA vaccine elicits potent immune responses in mice, hamsters, and nonhuman primates

SARS-CoV-2 has infected more than 400 million people around the globe and caused millions of deaths. Since its identification in November 2021, Omicron, a highly transmissible variant, has become the dominant variant in most countries. Omicrons highly mutated spike protein, the main target of vaccine development, significantly compromises the immune protection from current vaccination. We develop an mRNA vaccine (SOmicron-6P) based on an Omicron-specific sequence. In mice, SOmicron-6P shows superior neutralizing antibodies inducing abilities to a clinically approved inactivated virus vaccine, a clinically approved protein subunit vaccine, and an mRNA vaccine (SWT-2P) with the same sequence of BNT162b2 RNA. Significantly, SOmicron-6P induces a 14.4[~]27.7-fold and a 28.3[~]50.3-fold increase of neutralizing activity against the pseudovirus of Omicron and authentic Omicron compared to SWT-2P, respectively. In addition, two doses SOmicron-6P significantly protects Syrian hamsters against challenge with SARS-CoV-2 Omicron variant and elicits high titers of nAbs in a dose-dependent manner in macaques. Our results suggest that SOmicron-6P offers advantages over current vaccines, and it will be helpful for those with weak immunity.

microbiology↗

bioRxiv2022

The brassinosteroid biosynthesis gene TaD11-2A controls grain size and its elite haplotype improves wheat grain yields

Brassinosteroids (BRs) control many important agronomic traits, therefore the manipulation of BR components could improve crop productivity and performance. However, the potential effects of BR-related genes on yield-related traits and stress tolerance in wheat (Triticum aestivum L.) remain poorly understood. Here, we identified TaD11 genes in wheat (rice D11 orthologs) that encoded enzymes involved in BR biosynthesis. TaD11 genes were highly expressed in roots (Zadoks scale: Z11) and grains (Z75), while expression was significantly suppressed by exogenous BR (24-epiBL). Ectopic expression of TaD11-2A rescued the abnormal panicle structure and plant height (PH) of the cpb1 mutant, and also increased endogenous BR levels, resulting in improved grain yields and grain quality in rice. Natural variations in TaD11-2A were associated with significant differences in yield-related traits, including PH, grain width (GW), 1000-grain weight (TGW), and grain yield per plant (GYPP), and its favorable haplotype, TaD11-2A-HapI was subjected to positive selection during wheat breeding. Additionally, TaD11-2A influenced root length and salt tolerance in rice and wheat at seedling stages. These results indicated the important role of BR TaD11 biosynthetic genes in controlling grain size and root length, and also highlighted their potential in the molecular biological analysis of wheat. HighlightThe brassinosteroid biosynthesis gene TaD11-2A regulates grain size and root length and its haplotype favorably improves grain yields and salt tolerance in wheat.

plant biology↗

bioRxiv2022

Dexamethasone Inhibits Cytokine-Induced, DUOX2-Related VEGF-A Expression and DNA damage in Human Pancreatic Cancer Cells and Growth of Pancreatic Cancer Xenografts

Previously, we demonstrated that pro-inflammatory cytokines enhance dual oxidase 2 (DUOX2)-dependent production of reactive oxygen species by human pancreatic ductal carcinoma (PDAC) cells, and that DUOX2 expression is significantly increased in patients with early stages of PDAC. In genetically-engineered mouse models of PDAC, dexamethasone (Dex) decreases formation of pancreatic intraepithelial neoplasia (PanIn) foci as well as PDAC invasiveness. Herein, we report that Dex, in a concentration- and time-dependent fashion, inhibited pro-inflammatory cytokine (IFN-{gamma}/LPS/IL-17A/IL-4)-mediated enhancement of DUOX2 expression in BxPC-3, CFPAC-1, and AsPC-1 human PDAC cell lines, as well as DUOX2-induced DNA damage. The inhibitory effects of Dex were abolished by pre-treatment with the Dex antagonist RU-486. Examination of the human DUOX2 promoter in silico revealed a putative negative glucocorticoid receptor (GR) binding element (IRnGRE). Western analysis, using nuclear extracts from Dex-treated PDAC cells, demonstrated that Dex activated the glucocorticoid receptor in PDAC cell nuclei in the presence of certain co-repressors, such as NCoR-1/2 and histone deacetylases (HDAC1, 2, and 3). Dex produced no anti-proliferative effects on PDAC cells in vitro. However, Dex significantly decreased the growth of BxPC-3 xenografts while decreasing inflammatory and immune cell infiltration of the microenvironment, as well as the mRNA expression of DUOX2 and VEGF-A, in BxPC-3 tumors. In contrast, Dex had no effect on the growth of xenografts developed from MIA-PaCa cells that are unresponsive to pro-inflammatory cytokines in culture. In summary, these studies suggest that suppression of inflammation-related DUOX2 expression by Dex could diminish the oxidative milieu supporting PDAC growth and development.

pharmacology and toxicology↗

bioRxiv2022

An evolutionarily conserved mechanism for control the translation of long proteins

Plants have evolved sophisticated mechanisms to regulate gene expression to activate immune responses against pathogen infections. However, how the translation system contributes to plant immunity is largely unknown. The evolutionarily conserved thiolation modification of tRNA ensures efficient decoding during translation. Here we show that tRNA thiolation is required for plant immunity in Arabidopsis. We identify a cgb Arabidopsis mutant, which is hyper-susceptible to the pathogen Pseudomonas syringae. CGB encodes ROL5, a homolog of yeast NCS6 required for tRNA thiolation. ROL5 physically interacts with CTU2, a homolog of yeast NCS2. Mutations in either ROL5 or CTU2 result in loss of tRNA thiolation. Further analyses reveal that both transcriptional reprogramming and translational reprogramming during immune responses are compromised in cgb. Notably, the translation of the salicylic acid receptor NPR1 is reduced in cgb, resulting in reduced salicylic acid signaling. Our study not only reveals a new regulatory mechanism for plant immunity but also uncovers a new biological function of tRNA thiolation.

biochemistry↗

bioRxiv2022

Nitric oxide signaling controls collective contractions in a colonial choanoflagellate

Although signaling by the gaseous molecule nitric oxide (NO) regulates key physiological processes in animals, including contractility1-3, immunity4,5, development6-9 and locomotion10,11, the early evolution of animal NO signaling remains unclear. To reconstruct the role of NO in the animal stem lineage, we set out to study NO signaling in choanoflagellates, the closest living relatives of animals12. In animals, NO produced by the nitric oxide synthase (NOS) canonically signals through cGMP by activating soluble guanylate cyclases (sGCs)13,14. We surveyed the distribution of the NO signaling pathway components across the diversity of choanoflagellates and found three species that express NOS, sGCs, and downstream genes previously shown to be involved in the NO/cGMP pathway. One of these, Choanoeca flexa, forms multicellular sheets that undergo collective contractions controlled by cGMP15. We found that treatment with NO induces sustained contractions in C. flexa by signaling through an sGC/cGMP pathway. Biochemical assays show that NO directly binds C. flexa sGC1 and stimulates its cyclase activity. The NO/cGMP pathway acts independently from other inducers of C. flexa contraction, including mechanical stimuli and heat, but sGC activity is required for contractions induced by light-to-dark transitions. The output of NO signaling in C. flexa - contractions resulting in a switch from feeding to swimming - resembles the effect of NO in sponges1-3 and cnidarians11,16,17, where it interrupts feeding and activates contractility. These data provide insights into the biology of the first animals and the evolution of NO signaling.

evolutionary biology↗

bioRxiv2022

Cerebral organoids for modeling of HSV-1-induced-multiscale neuropathology associated with Alzheimers disease and phenotypic rescue

Herpes simplex virus type I (HSV-1) infection is a potential risk factor involved in the pathogenesis of Alzheimers disease (AD) and contributes to the AD viral hypothesis. However, further understanding of the neuropathological effects of the HSV-1 infection is hampered by the limitations of existing infection models due to the distinct differences between human brains and other mammalians brains. Here we generated cerebral organoid models derived from pluripotent stem cells to investigate the HSV-induced pathogenesis associated with AD and the role of antiviral drugs in the phenotypic rescue. Our results identified that the HSV-1-infected cerebral organoids recapitulated multiscale neuropathology pertaining to the onset and progression of AD across the genetic, cellular, and tissue levels, including the multicellular A{beta} deposition, dysregulated endogenous AD mediators, reactive gliosis, neuroinflammation, and neural loss. Furthermore, we identified that two antiviral drugs, namely Ribavirin (RBV) and Valacyclovir (VCV), inhibited HSV-1 replication and rescued the neuropathological phenotypes associated with AD in the HSV-1-infected cerebral organoids, implying their therapeutic potential to slow down the progression of AD. Our study provides a high-fidelity human-relevant in-vitro HSV-1 infection model to reconstitute the multiscale neuropathological features associated with AD and discover therapeutic drug candidates relevant to the AD viral hypothesis. IMPORTANCEAD is a progressive neurodegenerative disorder characterized by neuronal death, brain atrophy, and cognitive impairment. HSV-1 is a neurotropic double-stranded DNA virus resulting in long-term neurological sequelae in adults. There is accumulating evidence indicating that AD may be associated with HSV-1 infections. In this study, we generated human-physiologically related cerebral organoids derived from human embryonic stem cells (hESCs) to investigate the influence of HSV-1 infection on the neuropathology associated with AD. We found that the HSV-1-infected cerebral organoids could model typical neuropathological features associated with AD. Additionally, we discovered that both RBV and VCV significantly rescued the HSV-1-induced pathological phenotypes associated with AD. These findings may open new avenues of consideration related to HSV-1 infection and AD. Furthermore, the contribution of these antiviral drugs to AD linked to HSV-1 infection will provide new clues to potential new therapies.

neuroscience↗

bioRxiv2022

Genome-wide analyses of PAM-relaxed Cas9 genome editors reveal substantial off-target effects by ABE8e in rice

PAM-relaxed Cas9 nucleases, cytosine base editors and adenine base editors are promising tools for precise genome editing in plants. However, their genome-wide off-target effects are largely undetermined. Here, we conduct whole-genome sequencing (WGS) analyses of transgenic plants edited by xCas9, Cas9-NGv1, Cas9-NG, SpRY, nCas9-NG-PmCDA1, nSpRY-PmCDA1 and nSpRY-ABE8e in rice. Our results reveal different guide RNA (gRNA)-dependent off-target effects with different editors. De novo generated new gRNAs by SpRY editors lead to additional but not substantial off-target mutations. Strikingly, ABE8e results in ~500 genome-wide A-to-G off-target mutations at TA motif sites per transgenic plant. The preference of the TA motif by ABE8e is also observed at the target sites. Finally, we investigate the timeline and mechanism of somaclonal variation due to tissue culture, which chiefly contributes to the background mutations. This study provides a comprehensive understanding on the scales and mechanisms of off-target and background mutations during PAM-relaxed genome editing in plants.

genetics↗

bioRxiv2022

Interference with lipoprotein maturation sensitizes methicillin-resistant Staphylococcus aureus to human group IIA secreted phospholipase A2 and daptomycin.

Methicillin-resistant Staphylococcus aureus (MRSA) has been classified as a high priority pathogen by the World Health Organization underlining the high demand for new therapeutics to treat infections. Human group IIA secreted phospholipase A2 (hGIIA) is among the most potent bactericidal proteins against Gram-positive bacteria, including S. aureus. To determine hGIIA-resistance mechanisms of MRSA we screened the Nebraska Transposon Mutant Library using a sublethal concentration of recombinant hGIIA. We identified and confirmed the role of lspA, encoding the lipoprotein signal peptidase LspA, as a new hGIIA resistance gene in both in vitro assays and an infection model in hGIIA-transgenic mice. Increased susceptibility of the lspA mutant was associated with faster and increased cell wall penetration of hGIIA. Moreover, lspA deletion also increased susceptibility to daptomycin, a last-resort antibiotic to treat MRSA infections. Exposure of MRSA wild-type to the LspA-specific inhibitors globomycin and myxovirescin A1 induced a lspA mutant phenotype with regard to hGIIA and daptomycin killing. Analysis of >26,000 S. aureus genomes showed that LspA is highly sequence-conserved, suggesting that LspA inhibition could be applied universally. The role of LspA in hGIIA resistance was not restricted to MRSA since Streptococcus mutans and Enterococcus faecalis were also more hGIIA-susceptible after lspA deletion or LspA inhibition, respectively. Overall, our data suggest that pharmacological blocking of LspA may disarm Gram-positive pathogens, including MRSA, to enhance clearance by innate host defense molecules and clinically-applied antibiotics.

microbiology↗

bioRxiv2022

mSigHdp: hierarchical Dirichlet process mixture modeling for mutational signature discovery

Mutational signatures are characteristic patterns of mutations caused by endogenous or exogenous mutational processes. These signatures can be discovered by analyzing mutations in large sets of samples - usually somatic mutations in tumor samples. Most programs for discovering mutational signatures are based on non-negative matrix factorization (NMF). Alternatively, signatures can be discovered using hierarchical Dirichlet process (HDP) mixture models, an approach that has been explored less. These models assign mutations to clusters and view each cluster as being generated from the signature of a particular mutational process. Here we describe mSigHdp, an improved approach to using HDP mixture models to discover mutational signatures. We benchmarked mSigHdp and state-of-the-art NMF-based approaches on 4 realistic synthetic data sets. These data sets encompassed 18 cancer types. In total they contained 3.5x107 single-base-substitution mutations representing 32 signatures and 6.1x106 small-insertion-and-deletion mutations representing 13 signatures. For 3 of the 4 data sets, mSigHdp had the best positive predictive value for discovering mutational signatures, and for all 4 data sets, it had the best true positive rate. Its CPU usage was similar to that of the NMF-based approaches. Thus, mSigHdp is an important and practical addition to the set of tools available for discovering mutational signatures. Data and code availabilitymSigHdp is available at public repositories https://github.com/steverozen/mSigHdp and https://github.com/steverozen/hdpx. The synthetic data, code for generating the synthetic data, code for running the mutational-signature discovery programs, the main outputs of the programs, and code for analyzing their results and for generating the data figures in this paper are available at https://github.com/Rozen-Lab/mSigHdp sup files. A singularity container with mSigHdp can be downloaded from cloud.sylabs.io with the shell command "singularity pull library://rozen-lab/msighdp/msighdp:2.1.2". A toy-example Rscript for using this container is at https://github.com/steverozen/mSigHdp/blob/master/data-raw/container_scripts/test_mSigHdp.R. Supplementary materialOne excel file of supplementary tables and one PDF file of supplementary figures have been submitted along with this manuscript.

bioinformatics↗

bioRxiv2022

Highly efficient, all-organic bioluminescence-photosensitizer conjugate eradicates early-stagetumors and prevents metastasis in mice

Photodynamic therapy (PDT) is an established treatment modality using light-activatable drugs. Despite its unique cytotoxic mechanism, the shallow penetration of light has been a serious drawback limiting the applications of PDT. Here, we report bioluminescence-activated PDT (BL-PDT) using efficient bioluminescence resonance energy transfer (BRET) conjugates of clinically approved photosensitizers, Ce6, and luciferase proteins. A high photon-to-Ce6 conversion efficiency (80%), along with intracellular delivery by membrane-fusion liposomes, enabled effective cancer cell killing in vitro. In a syngeneic mouse model of aggressive 4T1 triple-negative breast cancer, as well as a xenograft model of MDA-MB-231 tumors, BL-PDT resulted in complete tumor remission and prevention of metastasis, as well as neo-adjuvant effects. Our result shows the promise of molecularly activable, clinically viable, depth-unlimited phototherapy.

cancer biology↗

bioRxiv2022

Impact of various vaccine boosters on neutralization against Omicron following prime vaccinations with inactivated or adenovirus-vectored vaccine

Since the first report on November 24, 2021, the Omicron SARS-CoV-2 variant is now overwhelmingly spreading across the world. Two SARS-CoV-2 inactivated vaccines (IAVs), one recombinant protein subunit vaccine (PRV), and one adenovirus-vectored vaccine (AdV) have been widely administrated in many countries including China to pursue herd immunity. Here we investigated cross-neutralizing activities in 341 human serum specimens elicited by full-course vaccinations with IAV, PRV and AdV, and by various vaccine boosters following prime IAV and AdV vaccinations. We found that all types of vaccines induced significantly lower neutralizing antibody titers against the Omicron variant than against the prototype strain. For prime vaccinations with IAV and AdV, heterologous boosters with AdV and PRV, respectively, elevated serum Omicron-neutralizing activities to the highest degrees. In a mouse model, we further demonstrated that among a series of variant-derived RBD-encoding mRNA vaccine boosters, it is only the Omicron booster that significantly enhanced Omicron neutralizing antibody titers compared with the prototype booster following a prime immunization with a prototype S-encoding mRNA vaccine candidate. In summary, our systematical investigations of various vaccine boosters inform potential booster administrations in the future to combat the Omicron variant.

immunology↗

bioRxiv2022

cir-DNA sequencing revealed the landscape of extrachromosomal circular DNA in articular cartilage and the potential roles in osteoarthritis

ObjectiveExtrachromosomal circular DNA (eccDNA) has been shown to be involved in several physiological and pathological processes including immunity, inflammation, aging and tumor. However, the expression of eccDNA in cartilage has been not reported until now. In this study, we aimed to investigate the landscape of eccDNA in articular cartilage and analyze the potential roles in Osteoarthritis (OA). MethodsThe samples of articular cartilage were obtained from total knee arthroplasty (TKA) donors with OA. The mtDNAs (mitochondrial DNA) and the linear DNAs from chondrocytes of articular cartilage were removed. Then the eccDNAs were enriched for cir-DNA sequencing. After quality control evaluation, we systematically revealed the identified eccDNA data including size distribution, the size range and sequence pattern. Moreover, we explored and disscussed the potential roles of eccDNA in OA via motif analysis and GO/KEGG pathways analysis. ResultsThe chondrocytes from OA cartilage contained an abundance of eccDNAs, which was termed as OC-eccDNAs (OA Cartilage-derived eccDNA). The characteristics of OC-eccDNAs were tissue specific, including the distribution, the size range and sequence pattern. Moreover, the functional analysis indicated that eccDNA may be involved in the homeostasis maintenance of chondrocytes and participated in the process of OA. ConclutionsOur data firstly showed the landscape of eccDNA in articular cartilage, and preliminarily indicated the potential roles of eccDNA in OA.

molecular biology↗

bioRxiv2022

Lineage-mosaic and mutation-patched spike proteins for broad-spectrum COVID-19 vaccine

The widespread SARS-CoV-2 in humans results in the continuous emergence of new variants. Recently emerged Omicron variant with multiple spike mutations sharply increases the risk of breakthrough infection or reinfection, highlighting the urgent need for new vaccines with broad-spectrum antigenic coverage. Using inter-lineage chimera and mutation patch strategies, we engineered a recombinant monomeric spike variant (STFK1628x), which showed high immunogenicity and mutually complementary antigenicity to its prototypic form (STFK). In hamsters, a bivalent vaccine comprised of STFK and STFK1628x elicited high titers of broad-spectrum antibodies to neutralize all 14 circulating SARS-CoV-2 variants, including Omicron; and fully protected vaccinees from intranasal SARS-CoV-2 challenges of either the ancestral strain or immune-evasive Beta variant. Strikingly, the vaccination of hamsters with the bivalent vaccine completely blocked the within-cage virus transmission to unvaccinated sentinels, for either the ancestral SARS-CoV-2 or Beta variant. Thus, our study provides new insights and antigen candidates for developing next-generation COVID-19 vaccines.

microbiology↗

bioRxiv2022

Harringtonine has the effects of double blocking SARS-CoV-2 membrane fusion

Fusion with host cell membrane is the main mechanism of infection of SARS-CoV-2. Here, we propose a new strategy to double block SARS-CoV-2 membrane fusion by using Harringtonine (HT), a small-molecule antagonist. By using cell membrane chromatography (CMC), we found that HT specifically targeted the SARS-CoV-2 S protein and host cell TMPRSS2, and then confirmed that HT can inhibit pseudotyped virus membrane fusion. Furthermore, HT successfully blocked SARS-CoV-2 infection, especially in the delta and Omicron mutant. Since HT is a small-molecule antagonist, it is minimally affected by the continuous variation of SARS-CoV-2. Our findings show that HT is a potential small-molecule antagonist with a new mechanism of action against SARS-CoV-2 infection, and thus HT mainly targets the S protein, and thus, greatly reduces the damage of the S proteins autotoxicity to the organ system, has promising advantages in the clinical treatment of COVID-19.

pharmacology and toxicology↗

bioRxiv2022

Post-embryonic maturation of the C. elegans motor circuit

During development, animals can maintain behavioral output even as the underlying circuits structurally remodel. After hatching, C. elegans undergoes substantial motor neuron expansion and synapse re-wiring while the animal continuously moves with an undulatory pattern. To understand how the circuit transitions from its juvenile to mature configuration without disrupting functional output, we reconstructed the C. elegans motor circuit by electron microscopy across larval development. We observed: 1) Embryonic motor neurons transiently interact with the developing post-embryonic motor neurons prior to remodeling of their juvenile wiring; 2) Post-embryonic neurons initiate synapse development with their future partners as their neurites navigate through the juvenile nerve cords; 3) Embryonic and post-embryonic neurons sequentially build structural machinery needed for the adult circuit before the embryonic neurons relinquish their roles to post-embryonic neurons; 4) This transition is repeated region by region along the body in an anterior to posterior sequence, following the birth order of post-embryonic neurons. Through this orchestrated, programmed and gradual rewiring, the motor circuit transforms from asymmetric to symmetric wiring. These maturation strategies support the continuous maintenance of motor patterns as the juvenile circuit develops into the adult configuration. HighlightsO_LIPost-embryonic motor circuit maturation was reconstructed by synapse-resolution serial EM. C_LIO_LIMotor patterns are maintained as the circuit matures from asymmetric to symmetric configuration. C_LIO_LIProgrammed rewiring gradually and sequentially transforms the circuit structure. C_LIO_LIPreparatory and communicative wiring-rewiring allows maturation without functional disruption. C_LI

neuroscience↗

bioRxiv2022

Chondroitin sulfates enhances the barrier function of basement membrane assembled by heparan sulfates

Glycosaminoglycans (GAGs) are ubiquitously expressed polysaccharides attached to proteoglycans, but their functions in the retina are poorly understood. Here we generated conditional knockouts of biosynthetic enzymes for heparan sulfate (HS) and chondroitin sulfate (CS) in retinal progenitor cells. We showed that ablation of HS polymerase Ext1 did not affect initial progression of retinal angiogenesis, but it disrupted the pruning of blood vessels and establishment of arterioles and venules. In the absence of retinal HS, blood vessels were also vulnerable to high oxygen tension in early postnatal stages, which can be rescued by exogenous VEGF, consistent with the role of retinal HS in the fine-tuning of VEGF signaling. Furthermore, we observed that the retinal inner limiting membrane (ILM) was disrupted by deletion of Ext1 in a timing specific manner, suggesting that retinal HS is required for the assembly but not the maintenance of the ILM. Lastly, we showed that further deletion of C4st1, a CS sulfation enzyme, did not affect the assembly of the ILM, but aggravated the ILM permeability when combined with Ext1 deletion. These results demonstrated an important role of CS and HS in establishing the barrier function of basement membrane.

developmental biology↗