Biology subjects

Zhan, H.

Publications and source records attributed to Zhan, H..

33 records · Page 2Linked to original sources

bioRxiv2023

Massive Multiplexing of Spatially Resolved Single Neuron Projections with Axonal BARseq

Neurons in the cortex are heterogeneous, sending diverse axonal projections to multiple brain regions. Unraveling the logic of these projections requires single-neuron resolution. Although a growing number of techniques have enabled high-throughput reconstruction, these techniques are typically limited to dozens or at most hundreds of neurons per brain, requiring that statistical analyses combine data from different specimens. Here we present axonal BARseq, a high-throughput approach based on reading out nucleic acid barcodes using in situ RNA sequencing, which enables analysis of even densely labeled neurons. As a proof of principle, we have mapped the long-range projections of >8,000 mouse primary auditory cortex neurons from a single brain. We identified major cell types based on projection targets and axonal trajectory. The large sample size enabled us to systematically quantify the projections of intratelencephalic (IT) neurons, and revealed that individual IT neurons project to different layers in an area-dependent fashion. Axonal BARseq is a powerful technique for studying the heterogeneity of single neuronal projections at high throughput within individual brains.

neuroscience↗

bioRxiv2023

Mitochondrial Genome Analysis and Phylogeny and Divergence Time Evaluation of the Strix aluco

In this study, a complete mitochondrial genome of the Strix aluco was reported for the first time, with a total length of 18,632 bp. There were 37 genes, including 22 tRNAs, 2 rRNAs, 13 protein-coding genes (PCGs), and 2 non-coding control regions (D-loop). The second-generation sequencing of the complete mitochondrial genome of the S. aluco was conducted using the Illumina platform, and then Tytoninae was used as the out-group, PhyloSuite software was applied to build the ML-tree and BI-tree of the Strigiformes, and finally, the divergence time tree was constructed using Beast2.6.7 software, the age of Miosurnia diurna fossil-bearing sediments (6.0-9.5 Ma) was set as the internal correction point. The common ancestor of the Strix was confirmed to have diverged during the Pleistocene(2.59~0.01Ma). The dramatic uplift of the Qinling Mountains in the Middle Pleistocene and the climate oscillation of the Pleistocene together caused Strix divergence between the northern and southern parts of mainland China. The isolation of glacial-interglacial rotation and glacier refuge was the main reason for the divergence of the common ancestor of the Strix uralensis and the S. aluco during this period. This study provides a reference for the evolution history of the Strix. Summary statementThis study was the first time to assemble the complete mitochondrial genome of Strix aluco, and report the divergence time of Strix. A full discussion was made, and it was inferred that the uplift of the Qinling Mountains and the glacial refuge led to the differentiation of this genus.

evolutionary biology↗

bioRxiv2023

High-throughput sequencing of macaque basolateral amygdala projections reveals dissociable connectional motifs with frontal cortex

The basolateral amygdala (BLA) projects widely across the macaque frontal cortex1-4, and amygdalo-frontal projections are critical for optimal emotional responding5 and decision- making6. Yet, little is known about the single-neuron architecture of these projections: namely, whether single BLA neurons project to multiple parts of the frontal cortex. Here, we use MAPseq7 to determine the projection patterns of over 3000 macaque BLA neurons. We found that one-third of BLA neurons have two or more distinct targets in parts of frontal cortex and of subcortical structures. Further, we reveal non-random structure within these branching patterns such that neurons with four targets are more frequently observed than those with two or three, indicative of widespread networks. Consequently, these multi-target single neurons form distinct networks within medial and ventral frontal cortex consistent with their known functions in regulating mood and decision-making. Additionally, we show that branching patterns of single neurons shape functional networks in the brain as assessed by fMRI-based functional connectivity. These results provide a neuroanatomical basis for the role of the BLA in coordinating brain-wide responses to valent stimuli8 and highlight the importance of high- resolution neuroanatomical data for understanding functional networks in the brain.

neuroscience↗

bioRxiv2022

Nodavirus RNA Replication Crown Architecture Reveals Proto-Crown Precursor and Viral Protein A Conformational Switching

Positive-strand RNA viruses replicate their genomes in virus-induced membrane vesicles, and the resulting RNA replication complexes are a major target for virus control. Nodavirus studies first revealed viral RNA replication proteins forming a 12-fold symmetric "crown" at the vesicle opening to the cytosol, an arrangement recently confirmed to extend to distantly related alphaviruses. Using cryo-electron microscopy (cryo-EM), we show that mature nodavirus crowns comprise two stacked 12-mer rings of multi-domain viral RNA replication protein A. Each ring contains an ~19 nm circle of C-proximal polymerase domains, differentiated by strikingly diverged positions of N-proximal RNA capping/membrane binding domains. The lower ring is a "proto-crown" precursor that assembles prior to RNA template recruitment, RNA synthesis and replication vesicle formation. In this proto-crown, the N-proximal segments interact to form a toroidal central floor, whose 3.1 [A] resolution structure reveals many mechanistic details of the RNA capping/membrane binding domains. In the upper ring, cryo-EM fitting indicates that the N-proximal domains extend radially outside the polymerases, forming separated, membrane-binding "legs." The polymerase and N-proximal domains are connected by a long linker accommodating the conformational switch between the two rings and possibly also polymerase movements associated with RNA synthesis and non-symmetric electron density in the lower center of mature crowns. The results reveal remarkable viral protein multifunctionality, conformational flexibility and evolutionary plasticity and new insights into (+)RNA virus replication and control. SignificancePositive-strand RNA viruses - including coronaviruses, alphaviruses, flaviviruses and many other medically and economically important pathogens - replicate their RNA genomes by virus-encoded machinery that has been poorly characterized. Using an advanced nodavirus model, we identify a major precursor in RNA replication complex assembly and show it to be a 12-mer ring of viral RNA replication protein A, whose single particle cryo-EM structure reveals functional features of its membrane interaction, assembly, polymerase and RNA capping domains. We further show that fully functional RNA replication complexes acquire a second 12-mer ring of protein A in alternate conformation atop the first, and a central density likely to represent another polymerase conformation. These findings provide strong foundations for understanding, controlling and beneficially using such viruses.

microbiology↗

bioRxiv2021

JAK2V617F mutant megakaryocytes contribute to hematopoietic aging in a murine model of myeloproliferative neoplasm

Megakaryocytes (MKs) is an important component of the hematopoietic niche. Abnormal MK hyperplasia is a hallmark feature of myeloproliferative neoplasms (MPNs). The JAK2V617F mutation is present in hematopoietic cells in a majority of patients with MPNs. Using a murine model of MPN in which the human JAK2V617F gene is expressed specifically in the MK lineage, we show that the JAK2V617F-bearing MKs promote hematopoietic stem cell (HSC) aging, manifesting as myeloid-skewed hematopoiesis with an expansion of CD41+ HSCs, a reduced engraftment and self-renewal capacity, and a reduced differentiation capacity. HSCs from 2yr old mice with JAK2V617F-bearing MKs were more proliferative and less quiescent than HSCs from age-matched control mice. Examination of the marrow hematopoietic niche reveals that the JAK2V617F-bearing MKs not only have decreased direct interactions with hematopoietic stem/progenitor cells during aging, but also suppress the vascular niche function during aging. Unbiased RNA expression profiling reveals that HSC aging has a profound effect on MK transcriptomic profiles, while targeted cytokine array shows that the JAK2V617F-bearing MKs can alter the hematopoietic niche through increased levels of pro-inflammatory and anti-angiogenic factors. Therefore, as a hematopoietic niche cell, MKs represent an important connection between the extrinsic and intrinsic mechanisms for HSC aging. Significance StatementThe relative contribution of intrinsic and extrinsic mechanisms to HSC aging remains debated. We find that JAK2V617F mutant MKs can accelerate hematopoietic aging both directly (via decreased MK-HSC interaction) and indirectly (via suppressing vascular niche function). We also show that HSC aging has a profound effect on MK function. Our data suggest that, as a hematopoietic niche cell, MKs represent an important connection between HSC-intrinsic and HSC-extrinsic aging mechanisms.

cancer biology↗

bioRxiv2021

A murine model with JAK2V617F expression in both hematopoietic cells and vascular endothelial cells recapitulates the key features of human myeloproliferative neoplasm

The myeloproliferative neoplasms (MPNs) are characterized by an expansion of the neoplastic hematopoietic stem/progenitor cells (HSPC) and an increased risk of cardiovascular complications. The acquired kinase mutation JAK2V617F is present in hematopoietic cells in a majority of patients with MPNs. Vascular endothelial cells (ECs) carrying the JAK2V617F mutation can also be detected in patients with MPNs. In this study, we show that a murine model with both JAK2V617F-bearing hematopoietic cells and JAK2V617F-bearing vascular ECs recapitulated all the key features of the human MPN disease, which include disease transformation from essential thrombocythemia to myelofibrosis, extramedullary splenic hematopoiesis, and spontaneous cardiovascular complications. During aging and MPN disease progression, there was a loss of both HSPC number and HSPC function in the marrow while the neoplastic hematopoiesis was relatively maintained in the spleen, mimicking the advanced phases of human MPN disease. Different vascular niche of the marrow and spleen could contribute to the different JAK2V617F mutant stem cell functions we have observed in this JAK2V617F-positive murine model. Compared to other MPN murine models reported so far, our studies demonstrate that endothelial dysfunction plays an important role in both the hematologic and cardiovascular abnormalities of MPN. Key PointsO_LIA murine model in which JAK2V617F is expressed in both hematopoietic cells and ECs recapitulated the key features of the human MPN disease C_LIO_LIDifferent vascular niche of the marrow and spleen could contribute to different JAK2V617F HSC functions during MPN disease progression C_LI

cancer biology↗

bioRxiv2021

Cell competition between wild-type and JAK2V617F mutant cells prevents disease relapse after stem cell transplantation in a murine model of myeloproliferative neoplasm

Disease relapse after allogeneic stem cell transplantation is a major cause of treatment-related morbidity and mortality in patients with myeloproliferative neoplasms (MPNs). The cellular and molecular mechanisms for MPN relapse are not well understood. Here, we established a murine model of MPN relapse, in which [~]60% of the MPN recipient mice develop disease relapse after receiving stem cell transplantation with wild-type marrow donor. Using this model, we find that impaired wild-type cell function is associated with MPN disease relapse. We also show that competition between wild-type and JAK2V617F mutant cells can modulate the immune cell composition and PD-L1 expression induced by the JAK2V617F oncogene. These results suggest that cell competition between wild-type donor cells and JAK2V617F mutant recipient cells can prevent MPN disease relapse after stem cell transplantation.

cancer biology↗

bioRxiv2021

Wiring logic of the early rodent olfactory system revealed by high-throughput sequencing of single neuron projections

The structure of neuronal connectivity often provides insights into the relevant stimulus features, such as spatial location, orientation, sound frequency, etc1-6. The olfactory system, however, appears to lack structured connectivity as suggested by reports of broad and distributed connections both from the olfactory bulb to the piriform cortex7-22 and within the cortex23-25. These studies have inspired computational models of circuit function that rely on random connectivity26-33. It remains, nonetheless, unclear whether the olfactory connectivity contains spatial structure. Here, we use high throughput anatomical methods (MAPseq and BARseq)34-38 to analyze the projections of 5,309 bulb and 30,433 piriform cortex output neurons in the mouse at single-cell resolution. We identify previously unrecognized spatial organization in connectivity along the anterior-posterior axis (A-P) of the piriform cortex. We find that both the bulb projections to the cortex and the cortical outputs are not random, but rather form gradients along the A-P axis. Strikingly, these gradients are matched: bulb neurons targeting a given location within the piriform cortex co-innervate extra-piriform regions that receive strong inputs from neurons within that piriform locus. We also identify signatures of local connectivity in the piriform cortex. Our findings suggest an organizing principle of matched direct and indirect olfactory pathways that innervate extra-piriform targets in a coordinated manner, thus supporting models of information processing that rely on structured connectivity within the olfactory system.

neuroscience↗

bioRxiv2020

Cell competition between wild-type and JAK2V617F mutant cells in a murine model of a myeloproliferative neoplasm

The myeloproliferative neoplasms (MPNs) are clonal stem cell disorders characterized by overproduction of mature blood cells and increased risk of transformation to frank leukemia. The acquired kinase mutation JAK2V617F plays a central role in a majority of these disorders. The hematopoietic stem cell (HSC) compartment in MPN is heterogeneous with the presence of both JAK2 wild-type and JAK2V617F mutant cells in most patients with MPN. Utilizing in vitro co-culture assays and in vivo competitive transplantation assays, we found that the presence of wild-type cells altered the behavior of co-existing JAK2V617F mutant cells, and a mutant microenvironment (niche) could overcome the competition between wild-type and mutant cells, leading to mutant clonal expansion and overt MPN. We also demonstrated that competition between wild-type and JAK2V617F mutant cells triggered a significant immune response, and there was a dynamic PD-L1 deregulation in the mutant stem/progenitor cells caused by their interactions with the neighboring wild-type cells and the microenvironment. Therefore, while accumulation of oncogenic mutations is unavoidable during aging, our data suggest that, if we could therapeutically enhance normal cells ability to compete, we might be better able to control neoplastic cell expansion and prevent the development of a full-blown malignancy. Key PointsO_LIThe presence of wild-type cells alters the behavior of co-existing JAK2V617F mutant cells C_LIO_LIA mutant microenvironment overcomes the competition between wild-type and JAK2V617F mutant cells, leading to the development of a MPN C_LI

cancer biology↗

bioRxiv2020

Global reinforcement of DNA methylation through enhancement of RNA-directed DNA methylation ensures sexual reproduction in rice

DNA methylation is an important epigenetic mark that regulates the expression of genes and transposons. RNA-directed DNA methylation (RdDM) is the main molecular pathway responsible for de novo DNA methylation in plants. In Arabidopsis, however, mutations in RdDM genes cause no visible developmental defects, which raising the question of the biological significance of RdDM in plant development. Here, we isolated and cloned Five Elements Mountain 1 (FEM1), which encodes an RNA-dependent RNA polymerase. Mutation in FEM1 substantially decreased genome-wide CHH methylation levels and abolished the accumulation of 24-nt small interfering RNAs. Moreover, male and female reproductive development was disturbed, which led to the sterility of fem1 mutants. In wild-type (WT) plants but not in fem1 mutants, genome-wide CHH DNA methylation levels were greater in panicles, stamens, and pistils than in seedlings. The global increase of methylation in reproductive organs of the WT was attributed to enhancement of RdDM activity including FEM1 activity. More than half of all encoding genes in the rice genome overlapped with hypermethylated regions in the sexual organs of the WT, and many of them appear to be directly regulated by an increase in DNA methylation.Our results demonstrate that a global increase of DNA methylation through enhancement of RdDM activity in reproductive organs ensures sexual reproduction of rice.View Full Text

plant biology↗

bioRxiv2020

PDIP38 is a novel adaptor-like modulator of the mitochondrial AAA+ protease CLPXP

Polymerase {delta} interacting protein of 38 kDa (PDIP38) was originally identified in a yeast two hybrid screen as an interacting protein of DNA polymerase delta, more than a decade ago. Since this time several subcellular locations have been reported and hence its function remains controversial. Our current understanding of PDIP38 function has also been hampered by a lack of detailed biochemical or structural analysis of this protein. Here we show, that human PDIP38 is directed to the mitochondrion, where it resides in the matrix compartment, together with its partner protein CLPX. PDIP38 is a bifunctional protein, composed of two conserved domains separated by an -helical hinge region (or middle domain). The N-terminal (YccV-like) domain of PDIP38 forms an SH3-like {beta}-barrel, which interacts specifically with CLPX, via the adaptor docking loop within the N-terminal Zinc binding domain (ZBD) of CLPX. In contrast, the C-terminal (DUF525) domain forms an Immunoglobin-like {beta}-sandwich fold, which contains a highly conserved hydrophobic groove. Based on the physicochemical properties of this groove, we propose that PDIP38 is required for the recognition (and delivery to CLPXP) of proteins bearing specific hydrophobic degrons, potentially located at the termini of the target protein. Significantly, interaction with PDIP38 stabilizes the steady state levels of CLPX in vivo. Consistent with these data, PDIP38 inhibits the LONM-mediated turnover of CLPX in vitro. Collectively, our findings shed new light on the mechanistic and functional significance of PDIP38, indicating that in contrast to its initial identification as a nuclear protein, PIDP38 is a bona fide mitochondrial adaptor protein for the CLPXP protease.

biochemistry↗

bioRxiv2019

Endothelial JAK2V617F mutation leads to thrombosis, vasculopathy, and cardiomyopathy in a murine model of myeloproliferative neoplasm

RationalThe myeloproliferative neoplasms (MPNs) are clonal hematological malignancies characterized by hematopoietic stem cell expansion and overproduction of mature blood cells. Cardiovascular complications are the leading cause of morbidity and mortality in patients with MPNs. The acquired kinase mutation JAK2V617F plays a central role in these disorders. Mechanisms responsible for cardiovascular dysfunction in MPNs are not fully understood, limiting the effectiveness of current treatment. ObjectiveVascular endothelial cells (ECs) play critical roles in the regulation of hemostasis and thrombosis. ECs carrying the JAK2V617F mutation can be detected in patients with MPNs. The goal of this study was to test the hypothesis that the JAK2V617F mutation alters endothelial function to promote cardiovascular complications in patients with MPNs. Methods and ResultsWe employed murine models of MPN in which the JAK2V617F mutation is expressed in specific cell lineages. When JAK2V617F is expressed in both blood cells and vascular ECs, the mice developed MPN and spontaneous, age-related dilated cardiomyopathy with an increased risk of sudden death as well as a prothrombotic and vasculopathy phenotype on histology evaluation. We showed that JAK2V617F-mutant ECs are required for this cardiovascular disease phenotype and the mutation can alter endothelial cell function. Finally, in a more therapeutically oriented approach, we demonstrated that transplantation with wild-type donor marrow cells can improve cardiac function by reversing the left ventricle remodeling process in this JAK2V617F-positive MPN murine model. ConclusionsThese findings suggest that the JAK2V617F mutation alters vascular endothelial function to promote cardiovascular complications in MPNs. Therefore, targeting the MPN vasculature represents a promising new therapeutic strategy for patients with MPNs.

molecular biology↗

bioRxiv2018

SYNPLA: A synapse-specific method for identifying learning-induced synaptic plasticity loci

Which neural circuits undergo synaptic changes when an animal learns? Although it is widely accepted that changes in synaptic strength underlie many forms of learning and memory, it remains challenging to connect changes in synaptic strength at specific neural pathways to specific behaviors and memories. Here we introduce SYNPLA (SYNaptic Proximity Ligation Assay), a synapse-specific, high-throughput and potentially brain-wide method capable of detecting circuit-specific learning-induced synaptic plasticity.

neuroscience↗

bioRxiv2018

Spatial organization of projection neurons in the mouse auditory cortex identified by in situ barcode sequencing

Understanding neural circuits requires deciphering interactions among myriad cell types defined by spatial organization, connectivity, gene expression, and other properties. Resolving these cell types requires both single neuron resolution and high throughput, a challenging combination with conventional methods. Here we introduce BARseq, a multiplexed method based on RNA barcoding for mapping projections of thousands of spatially resolved neurons in a single brain, and relating those projections to other properties such as gene or Cre expression. Mapping the projections to 11 areas of 3579 neurons in mouse auditory cortex using BARseq confirmed the laminar organization of the three top classes (IT, PT-like and CT) of projection neurons. In depth analysis uncovered a novel projection type restricted almost exclusively to transcriptionally-defined subtypes of IT neurons. By bridging anatomical and transcriptomic approaches at cellular resolution with high throughput, BARseq can potentially uncover the organizing principles underlying the structure and formation of neural circuits.

neuroscience↗

bioRxiv2018

A computational framework for converting high-throughput DNA sequencing data into neural circuit connectivity.

There is growing interest in determining the connectivity of neural circuits-the "connectome"-at single neuron resolution. Most approaches to circuit mapping rely on either microscopy or physiology, but these approaches have very limited throughput. We have recently proposed BOINC (Barcoding of Individual Neuronal Connectivity), a radically different approach to connectivity mapping based on high-throughput DNA sequencing. Here we describe the set of computational algorithms that serve to convert sequencing data into neural connectivity. We apply our computational pipeline to the results of proof-of-principle experiments illustrating an implementation of BOINC based on pseudorabies virus (PRV). PRV is capable of traversing individual synapses and carry DNA barcodes from one cell to another. Using this high-throughput sequencing data, we obtain 456-by-486 connectivity matrix between putative neurons. An inexpensive high-throughput technique for establishing circuit connectivity at single neuron resolution would represent a major advance in neuroscience.

neuroscience↗