Biology subjects

Dong, G.

Publications and source records attributed to Dong, G..

At least 55 records · Page 3Linked to original sources

bioRxiv2022

Double NPY motifs at the N-terminus of Sso2 synergistically bind Sec3 to promote membrane fusion

Exocytosis is an active vesicle trafficking process by which eukaryotes secrete materials to the extracellular environment and insert membrane proteins into the plasma membrane. The final step of exocytosis in yeast involves the assembly of two t-SNAREs, Sso1/2 and Sec9, with the v-SNARE, Snc1/2, on secretory vesicles. The rate-limiting step in this process is the formation of a binary complex of the two t-SNAREs. Despite a previous report of acceleration of binary complex assembly by Sec3, it remains unknown how Sso2 is efficiently recruited to the vesicle-docking site marked by Sec3. Here we report a crystal structure of the pleckstrin homology (PH) domain of Sec3 in complex with a nearly full-length version of Sso2 lacking only its C-terminal transmembrane helix. The structure shows a previously uncharacterized binding site for Sec3 at the N-terminus of Sso2, consisting of two highly conserved triple residue motifs (NPY: Asn-Pro-Tyr). We further reveal that the two NPY motifs bind Sec3 synergistically, which together with the previously reported binding interface constitute dual-site interactions between Sso2 and Sec3 to drive the fusion of secretory vesicles at target sites on the plasma membrane. SignificanceSNARE assembly, which involves one v-SNARE with two t-SNARE proteins, drives the fusion of vesicles to target compartments. The rate-limiting step in SNARE assembly is the assembly of the two t-SNARE proteins on the target membrane. Previous studies in yeast showed that Sec3, a component of the exocyst vesicle tethering complex, directly interacts with the t-SNARE protein Sso2 to promote fast assembly of an Sso2-Sec9 binary t-SNARE complex. This paper presents a new crystal structure of the Sec3 PH domain in complex with a nearly full-length version of Sso2, which reveals a previously unknown binding site for Sec3 at the N-terminus of Sso2. Our work demonstrates that the dual-site interactions between Sso2 and Sec3 plays an essential role in promoting the fusion of secretory vesicles at target sites on the plasma membrane.

biochemistry↗

bioRxiv2022

Functional stemness-related genes revealed by single-cell profiling of naïve and stimulated human CD34+ cells from CB and mPB

Hematopoietic stem cells (HSCs) from different sources show varied repopulating capacity, and HSCs lose their stemness after long-time ex vivo culture. However, the underlying mechanisms of the stemness differences because of the cell sources and the culture stimulation are not fully understood. Here, we applied single-cell RNA-seq (scRNA-seq) to analyze the naive and stimulated human CD34+ cells from cord blood (CB) and mobilized peripheral blood (mPB). We collected over 16,000 single-cell data to construct a comprehensive trajectory inference map and characterized the HSCs population on the hierarchy top, which is under quiescent state. Then we compared HSCs in CB to those in mPB and HSCs of naive samples to those of cultured samples, and identified stemness-related genes (SRGs) associated with culture time (CT-SRGs) and cell source (CS-SRGs), respectively. Interestingly, CT-SRGs and CS-SRGs share genes enriched in the signaling pathways such as mRNA catabolic process, Translational initiation, Ribonucleoprotein complex biogenesis and Cotranslational protein targeting to membrane, suggesting dynamic protein translation and processing may be a common requirement for stemness maintenance. Meanwhile, CT-SRGs are enriched in pathways involved in glucocorticoid and corticosteroid response that affect HSCs homing and engraftment. In contrast, CS-SRGs specifically contain genes related purine and ATP metabolic process which is important to initiate hematopoiesis. Finally, we presented an application through a small-scale drug screening using Connectivity Map (CMap) against CT-SRGs and found a small molecule cucurbitacin I, targeting STAT3/JAK2, can efficiently expand HSCs ex vivo while maintaining its stemness. These results indicate SRGs revealed by scRNA-seq can provide helpful insights to understand the stemness differences under diverse circumstances, and CT-SRGs can be a valuable database to identify candidates enhancing functional HSCs expansion during ex vivo culture.

cell biology↗

bioRxiv2022

Single-cell transcriptional profiling reveals cellular and molecular divergence in human maternal-fetal interface

Placenta play essential role in successful pregnancy, as the most important organ connecting and interplaying between mother and fetus. However, the cellular and molecular characteristics of fetal origin and maternal origin cell populations within the fetomaternal interface still is poorly understood. Here, we profiled the transcriptomes of single cells with well-defined maternal-fetal origin that consecutively localized from fetal section (FS), middle section (Mid_S) to maternal section (Mat_S) within the human full-term placenta. Then, we initially identified the cellular and molecular heterogeneity of cytotrophoblast cell (CTB) and stromal cell (STR) with the spatial location and fetal/maternal origin, also highlighted STR cells from fetal origins showed greater proliferation ability in Mat_S compared to cells from FS or Mid_S. Further, by integrating analysis with the first-trimester placental single cell transcriptome data, we revealed that a subpopulation of trophoblast progenitor-like cells (TPLCs) existed in the full-term placenta and mainly distributed in Mid_S, with high expression of pool of putative cell surface makers and unique molecular features. Moreover, through the extravillous cytotrophoblast (EVT) subsets differentiation trajectory and regulation network analysis, we proposed a putative key transcription factor PRDM6 that promoted the differentiation of endovascular extravillous trophoblast cells (enEVT). Finally, based on the integrated analyses of single cell transcriptional profiling of preeclampsia (PE) and match-trimester normal placenta, we highlighted the defective EVT subgroup composition and down-regulation of PRDM6 may lead to an abnormal enEVT differentiation process in PE. Together, our study offers important resources for better understanding of human placenta, stem cell-based therapy as well as PE, and provides new insights on the study of tissue heterogeneity, the clinical prevention and control of PE as well as the maternal-fetal interface.

developmental biology↗

bioRxiv2021

MetaLogo: a generator and aligner for multiple sequence logos

Sequence logos are used to visually display conservations and variations in short sequences. They can indicate the fixed patterns or conserved motifs in a batch of DNA or protein sequences. However, most of the popular sequence logo generators are based on the assumption that all the input sequences are from the same homologous group, which will lead to an overlook of the heterogeneity among the sequences during the sequence logo making process. Heterogeneous groups of sequences may represent clades of different evolutionary origins, or genes families with different functions. Therefore, it is essential to divide the sequences into different phylogenetic or functional groups to reveal their specific sequence motifs and conservation patterns. To solve these problems, we developed MetaLogo, which can automatically cluster the input sequences after multiple sequence alignment and phylogenetic tree construction, and then output sequence logos for multiple groups and aligned them in one figure. User-defined grouping is also supported by MetaLogo to allow users to investigate functional motifs in a more delicate and dynamic perspective. MetaLogo can highlight both the homologous and nonhomologous sites among sequences. MetaLogo can also be used to annotate the evolutionary positions and gene functions of unknown sequences, together with their local sequence characteristics. We provide users a public MetaLogo web server (http://metalogo.omicsnet.org), a standalone Python package (https://github.com/labomics/MetaLogo), and also a built-in web server available for local deployment. Using MetaLogo, users can draw informative, customized and publishable sequence logos without any programming experience to present and investigate new knowledge on specific sequence sets.

bioinformatics↗

bioRxiv2021

Structural and functional studies of the first tripartite protein complex at the Trypanosoma brucei flagellar pocket collar

The flagellar pocket (FP) is the only endo- and exocytic organelle in most trypanosomes and, as such, is essential throughout the life cycle of the parasite. The neck of the FP is maintained enclosed around the flagellum via the flagellar pocket collar (FPC). The FPC is a macromolecular cytoskeletal structure and is essential for the formation of the FP and cytokinesis. FPC biogenesis and structure are poorly understood, mainly due to the lack of information on FPC composition. To date, only two FPC proteins, BILBO1 and FPC4, have been characterized. BILBO1 forms a molecular skeleton upon which other FPC proteins can, theoretically, dock onto. We previously identified FPC4 as the first BILBO1 interacting partner and demonstrated that its C-terminal domain interacts with the BILBO1 N-terminal domain (NTD). Here, we report the characterization of a new FPC component and BILBO1 partner protein, BILBO2 (Tb927.6.3240) by yeast two-hybrid screen, bioinformatics, functional and structural studies. We show that BILBO2 colocalizes with BILBO1 and can modulate the shape of the BILBO1 filament by interacting with the BILBO1 EF-hand domains. Further, we demonstrate that BILBO1 and BILBO2 share a homologous NTD domain and that both domains interact with FPC4. We have determined a 1.9 [A] resolution crystal structure of the BILBO2 NTD in complex with the FPC4 BILBO1-binding domain. Together with mutational analyses, our studies reveal key residues for the function of the BILBO2 NTD and its interaction with FPC4 and evidenced a tripartite interaction between BILBO1, BILBO2, and FPC4. Our work sheds light on the first atomic structure of an FPC protein complex and represents a significant step in deciphering the FPC function in Trypanosoma brucei and other pathogenic kinetoplastids. Author summaryTrypanosomes belong to a group of zoonotic, protist, parasites that are found in Africa, Asia, South America, and Europe and are responsible for severe human and animal diseases. They all have a common structure called the flagellar pocket (FP). In most trypanosomes, all macromolecular exchanges between the trypanosome and the environment occur via the FP. The FP is thus essential for cell viability and evading the host immune response. We have been studying the flagellar pocket collar (FPC), an enigmatic macromolecular structure at the neck of the FP, and demonstrated its essentiality for the biogenesis of the FP. We demonstrated that BILBO1 is an essential protein of the FPC that interacts with other proteins including a microtubule-binding protein FPC4. Here we identify another bona fide FPC protein, BILBO2, so named because of close similarity with BILBO1 in protein organization and functional domains. We demonstrate that BILBO1 and BILBO2 share a common N-terminal domain involved in the interaction with FPC4, and illustrate a tripartite interaction between BILBO1, BILBO2, and FPC4. Our study also provides the first atomic view of two FPC components. These data represent an additional step in deciphering the FPC structure and function in T. brucei.

cell biology↗

bioRxiv2020

Discovery of a Potent and Dual-Selective Bisubstrate Inhibitor for Protein Arginine Methyltransferase 4/5

Protein arginine methyltransferases (PRMTs) have been implicated in the progression of many diseases. Understanding substrate recognition and specificity of individual PRMT would facilitate the discovery of selective inhibitors towards future drug discovery. Herein, we reported the design and synthesis of bisubstrate analogues for PRMTs that incorporate a S-adenosylmethionine (SAM) analogue moiety and a tripeptide through an alkyl substituted guanidino group. Compound AH237 is a potent and selective inhibitor for PRMT4 and PRMT5 with a half-maximal inhibition concentration (IC50) of 2.8 nM and <1.5 nM, respectively. Computational studies provided a plausible explanation for the high potency and selectivity of AH237 for PRMT4/5 over other 40 methyltransferases. This proof-of-principle study outlines an applicable strategy to develop potent and selective bisubstrate inhibitors for PRMTs, providing valuable probes for future structural studies.

biochemistry↗

bioRxiv2020

Restoration of β-globin expression with optimally designed lentiviral vector for β-thalassemia treatment in Chinese patients

{beta}-thalassemia is one of the most prevalent genetic diseases worldwide. The current treatment for {beta}-thalassemia is allogeneic hematopoietic stem cell transplantation (HSCT), which is limited due to lack of matched donors. Gene therapy has been developed as an alternative therapeutic option for transfusion-ependent {beta}-thalassemia (TDT). However, successful gene therapy for {beta}-thalassemia patients in China has not been reported. Here we present the results of preclinical studies of an optimally designed LV named LentiHBBT87Q in hematopoietic stem cells (HSCs) derived from Chinese TDT patients. LentiHBBT87Q was selected from a series of LVs with optimized backbone and de novo cloning strategy. It contains an exogenous T87Q {beta}-globin gene (HBBT87Q) driven by a specific reconstituted locus control region (rLCR) and efficiently express HBB mRNA and HBB protein in erythroblasts derived from cord blood (CB) HSCs. To facilitate clinical transformation, we manufactured clinical grade LentiHBBT87Q (cLentiHBBT87Q) and optimized its transduction procedure. Importantly, transduction of cLentiHBBT87Qrestored expression of HBB monomer and adult hemoglobin (HbA) tetramer to relatively normal level in erythroblasts from bone marrow (BM) HSCs of Chinese TDT patients, that carry the most common mutation types and cover various genotypes, including {beta}0/{beta}0. Furthermore, viral integration sites (VIS) of cLentiHBBT87Q were similar to other LVs safely used in previous clinical trials and the associated risk of tumorigenesis was not observed in cLentiHBBT87Q transduced HSCs through comprehensive analysis. Taken together, we have engineered the cLentiHBBT87Q that can restore {beta}-globin expression in the HSCs-derived erythroblasts of Chinese TDT patients with minimal risk on tumorigenesis, providing a favorable starting point for future clinical application.

cell biology↗

bioRxiv2020

Transcriptome analyses of β-thalassemia -28 (A>G) mutation using isogenic cell models generated by CRISPR/Cas9 and asymmetric single-stranded oligodeoxynucleotides (assODN)

{beta}-thalassemia, caused by mutations in the human hemoglobin (HBB) gene, is one of the most common genetic diseases in the world. HBB -28 (A>G) mutation is one of the five most common mutations in China patients with {beta}-thalassemia. However, few studies have been conducted to understand how this mutation affects the expression of pathogenesis related genes including globin genes due to limited homologous clinical materials. Therefore, we first developed an efficient technique using CRISPR/Cas9 combined with asymmetric single-stranded oligodeoxynucleotides (assODN) to generate a K562 cell model of HBB -28 (A>G) named K562-28 (A>G). Then, we systematically analyzed the differences between K562-28 (A>G) and K562 at the transcriptome level by high-throughput RNA-seq pre- and post-erythrogenic differentiation. We found that HBB -28 (A>G) mutation not only disturbed the transcription of HBB but also decreased the expression of HBG, which may further aggravate the thalassemia phenotype and partially explain the severer clinical outcome of {beta}-thalassemia patients with HBB -28 (A>G) mutation. Moreover, we found K562-28 (A>G) cell line is more sensitive to hypoxia and showed a defective erythrogenic program compared with K562 before differentiation. In agreement, p38MAPK and ERK pathway are hyperactivated in K562-28 (A>G) after differentiation. Importantly, all above mentioned abnormalities in K562-28 (A>G) were reversed after correction of this mutation with CRISPR/Cas and assODN, confirming the specificity of these phenotypes. Overall, this is the first time to analyze the effects of the HBB - 28 (A>G) mutation at whole-transcriptome level based on isogenic cell lines, providing a landscape for further investigation of the mechanism of {beta}-thalassemia with HBB -28 (A>G) mutation.

pathology↗

bioRxiv2020

Cancer-specific associations of driver genes with immunotherapy outcome

Genomic features such as microsatellite instability (MSI) and tumor mutation burden (TMB) are predictive of immune checkpoint inhibitor (ICI) response. However, they do not account for the functional effects of specific driver gene mutations, which may alter the immune microenvironment and influence immunotherapy outcomes. By analyzing a multi-cancer cohort of 1,525 ICI-treated patients, we identified 12 driver genes in 6 cancer types associated with treatment outcomes, including genes involved in oncogenic signaling pathways (NOTCH, WNT, FGFR) and chromatin remodeling. Mutations of PIK3CA, PBRM1, SMARCA4, and KMT2D were associated with worse outcomes across multiple cancer types. In comparison, genes showing cancer-specific associations--such as KEAP1, BRAF, and RNF43--harbored distinct variant types and variants, some of which were individually associated with outcomes. In colorectal cancer, a common RNF43 indel was a putative neoantigen associated with higher immune infiltration and favorable ICI outcomes. Finally, we showed that selected mutations were associated with PD-L1 status and could further stratify patient outcomes beyond MSI or TMB, highlighting their potential as biomarkers for immunotherapy.

genomics↗

bioRxiv2020

NetBoxR: Automated Discovery of Biological Process Modules by Network Analysis in R

SummaryLarge-scale sequencing projects, such as The Cancer Genome Atlas (TCGA) and the International Cancer Genome Consortium (ICGC), have accumulated a variety of high throughput sequencing and molecular profiling data, but it is still challenging to identify potentially causal genetic mutations in cancer as well as in other diseases in an automated fashion. We developed the NetBoxR package written in the R programming language, that makes use of the NetBox algorithm to identify candidate cancer-related processes. The algorithm makes use of a networkbased approach that combines prior knowledge with a network clustering algorithm, obviating the need for and the limitation of functionally curated gene sets. A key aspect of this approach is its ability to combine multiple data types, such as mutations and copy number alterations, leading to more reliable identification of functional modules. We make the tool available in the Bioconductor R ecosystem for applications in cancer research and cell biology. Availability and implementationThe NetBoxR package is free and open-sourced under the GNU GPL-3 license R package available at https://www.bioconductor.org/packages/release/bioc/html/netboxr.html Contactlium2@mskcc.org; aluna@jimmy.harvard.edu; sander.research@gmail.com Supplementary informationNone

bioinformatics↗

bioRxiv2020

Probing the Plasticity in the Active Site of Protein N-terminal Methyltransferase 1 Using Bisubstrate Analogs

The interactions of a series of bisubstrate analogs with protein N-terminal methyltransferase 1 (NTMT1) were examined to probe the molecular properties of the NTMT1 active site through biochemical characterization and structural studies. Our results indicate that a 2-C to 4-C atom linker enables its respective bisubstrate analog to occupy both substrate and cofactor binding sites of NTMT1, but the bisubstrate analog with a 5-C atom linker only interacts with the substrate binding site and functions as a substrate. Furthermore, the 4-C atom linker is the optimal and produces the most potent inhibitor (Ki, app = 130 {+/-} 40 pM) for NTMT1 to date, displaying over 100,000-fold selectivity over other methyltransferases and 3,000-fold even to its homolog NTMT2. This study reveals the molecular basis for the plasticity of the NTMT1 active site. Additionally, our study outlines a general guidance on the development of bisubstrate inhibitors for any methyltransferases.

biochemistry↗

bioRxiv2020

Selective Peptidomimetic Inhibitors of NTMT1/2: Rational design, synthesis, characterization, and crystallographic studies

Protein N-terminal methyltransferases (NTMTs) methylate the -N-terminal amines of proteins starting with the canonical X-P-K/R motif. Genetic studies imply that NTMT1 regulates cell mitosis and DNA damage repair. Herein, we report the rational design and development of the first potent peptidomimetic inhibitors for NTMT1. Biochemical and co-crystallization studies manifest that BM30 (IC50 of 0.89 {+/-} 0.10 {micro}M) is a competitive inhibitor to the peptide substrate and noncompetitive to the cofactor S-adenosylmethionine. BM30 exhibits over 100-fold selectivity to NTMT1/2 among a panel of 41 methyltransferases, indicating the potential to achieve high selectivity when targeting the peptide substrate binding site of NTMT1/2. Its cell-permeable analog DC432 (IC50 of 54 {+/-} 4 nM) decreases the N-terminal methylation level of SET protein in HCT116 cells. This proof-of principle study provides valuable probes for NTMT1/2 and highlights the opportunity to develop more cell-potent inhibitors to elucidate the function of NTMTs in future.

biochemistry↗

bioRxiv2019

A Quantitative Proteome Map of the Human Body

Determining protein levels in each tissue and how they compare with RNA levels is important for understanding human biology and disease as well as regulatory processes that control protein levels. We quantified the relative protein levels from 12,627 genes across 32 normal human tissue types prepared by the GTEx project. Known and new tissue specific or enriched proteins (5,499) were identified and compared to transcriptome data. Many ubiquitous transcripts are found to encode highly tissue specific proteins. Discordance in the sites of RNA expression and protein detection also revealed potential sites of synthesis and action of protein signaling molecules. Overall, these results provide an extraordinary resource, and demonstrate that understanding protein levels can provide insights into metabolism, regulation, secretome, and human diseases.\n\nSummaryQuantitative proteome study of 32 human tissues and integrated analysis with transcriptome data revealed that understanding protein levels could provide in-depth knowledge to post transcriptional or translational regulations, human metabolism, secretome, and diseases.

systems biology↗

bioRxiv2019

Crystal structure of the TbBILBO1 N-terminal domain reveals a ubiquitin fold with a long rigid loop for the binding of its partner

BILBO1 was the first characterized component of the flagellar pocket collar (FPC) in trypanosomes. The N-terminal domain (NTD) of BILBO1 plays an essential role in Trypanosoma brucei FPC biogenesis and is thus vital for the parasites survival. Here we report a 1.6-[A] resolution crystal structure of TbBILBO1-NTD, which revealed a conserved horseshoe-like hydrophobic pocket formed by an unusually long loop. Mutagenesis studies suggested that another FPC protein, FPC4, interacts with TbBILBO1 via mainly contacting the three conserved aromatic residues W71, Y87 and F89 at the center of this pocket. Overall, we have determined the binding site of TbFPC4 on TbBILBO1-NTD, which may provide a basis for rational drug design in the future.

biochemistry↗

bioRxiv2019

Serum-free culture system for spontaneous human mesenchymal stem cell spheroids formation

Human mesenchymal stem cells (hMSCs) are widely used in clinical research because of their multipotential, immunomodulatory, and reparative properties. Previous studies determined that hMSC spheroids from three-dimensional (3D) culture possess higher therapeutic efficacy than conventional hMSCs from monolayer (2D) culture. To date, various 3D culture methods have been developed to form hMSC spheroids, but most of them used culture medium containing fetal bovine serum (FBS), which is not suitable for further clinical use. Here, we demonstrate that dissociated single MSCs seeded in induced pluripotent stems medium (MiPS), adhere loosely to the dish and spontaneously migrate to form spheroids during day 3 to day 6. Through component deletion screening and complementation experiments, the knockout serum replacement (KSR) was identified as necessary and sufficient for hMSC spheroid formation. Transcriptome analysis showed that the overall expression profiles were highly similar between 2D culture with FBS and KSR derived spheroids. Interestingly, genes related to inflammatory response, immune response, and angiogenesis were up-regulated in spheroids at day 6, and qPCR results further validated the increased expression level of related genes, including STC1, CCL7, HGF, IL24, and TGFB3. When spheroids were re-plated in normal FBS medium, cells formed a typical spindle-shaped morphology, and FACS results showed that the recovered cells retained MSC-specific surface markers, such as CD73, CD90, and CD105. In summary, we developed a practical and convenient method to generate hMSC spheroids for clinical research and therapy.

cell biology↗

bioRxiv2018

Improved performance of microbial fuel cells through addition of trehalose lipids

Electron transfer from microorganisms to the electrode is the key process in microbial fuel cells (MFCs). In this study, a trehalose lipid was added to a Rhodococcus pyridinivorans-inoculated MFC to improve the power output by enhancing electron transfer. Upon trehalose lipid addition, the current density and maximum power density were increased by 1.83 times and 5.93 times, respectively. Cyclic voltammetry analysis revealed that the addition of trehalose lipid increased the electron transfer performance, while electrochemical impedance spectroscopy results proved a decrease in internal resistance. Microscopy images showed that the trehalose lipid-treated bacteria interacted more closely with various fagellum-like contacts, while in the pure trehalose lipid (200 mg/L), pores were obviously observed in the cell surface.\n\nImportanceImproving the power output of microbial fuel cells by the addition of bio-surfactants have been proved to be a novel method. However, only rhamnolipid and sophorolipid are certified to be effective. Trehalose lipid is a common material in cosmetic and bio-medicine industry. Our research broaden the application of bio-surfactant in MFC and preliminarily explain the mechanism.\n\nHighlightsO_LITrehalose lipid enhanced MFC power generation\nC_LIO_LITrehalose lipid decrease MFC internal resistance\nC_LIO_LIPores were observed with the addition of trehalose lipid\nC_LIO_LIAddition of bio-surfactant is a promising way to increase MFC performance\nC_LI

biochemistry↗

bioRxiv2018

Deconvolution of single-cell multi-omics layers reveals regulatory heterogeneity

Integrative analysis of multi-omics layers at single cell level is critical for accurate dissection of cell-to-cell variation within certain cell populations. Here we report scCAT-seq, a technique for simultaneously assaying chromatin accessibility and the transcriptome within the same single cell. We show that the combined single cell signatures enable accurate construction of regulatory relationships between cis-regulatory elements and the target genes at single-cell resolution, providing a new dimension of features that helps direct discovery of regulatory patterns specific to distinct cell identities. Moreover, we generated the first single cell integrated maps of chromatin accessibility and transcriptome in human pre-implantation embryos and demonstrated the robustness of scCAT-seq in the precise dissection of master transcription factors in cells of distinct states during embryo development. The ability to obtain these two layers of omics data will help provide more accurate definitions of \"single cell state\" and enable the deconvolution of regulatory heterogeneity from complex cell populations.

genomics↗

bioRxiv2018

The effector of Hippo signaling, Taz, is required for formation of the micropyle and fertilization in zebrafish

The mechanisms that ensure fertilization of eggs by a single sperm are not fully understood. In all teleosts, a channel called the micropyle is the only route of entry for sperm to enter and fertilize the egg. The micropyle forms by penetration of the developing vitelline envelope by a single specialized follicle cell, the micropylar cell, which subsequently degenerates. The mechanisms underlying micropylar cell specification and micropyle formation are poorly understood. Here, we show that an effector of the Hippo signaling pathway, the Transcriptional co-activator with a PDZ-binding domain (Taz), plays crucial roles in micropyle formation and fertilization in zebrafish. Genome editing mutants affecting taz can grow to adults, however, eggs from homozygous taz females are not fertilized even though oocytes in mutant females are histologically normal with intact animal-vegetal polarity, complete meiosis and proper ovulation. However, taz mutant eggs have no micropyle. We show that Taz protein is specifically enriched from mid-oogenesis onwards in two follicle cells located at the animal pole of the oocyte, and co-localizes with the actin and tubulin cytoskeleton. Taz protein and micropylar cell are not detected in taz mutant ovaries. Our work identifies a novel role for the Hippo/Taz pathway in micropylar cell specification in zebrafish, and uncovers the molecular basis of micropyle formation in teleosts.

developmental biology↗