Publications by authors named "Patrick P L Tam"

133 Publications

Defining cell identity beyond the premise of differential gene expression.

Cell Regen 2021 May 1;10(1):20. Epub 2021 May 1.

School of Mathematics and Statistics, The University of Sydney, Sydney, NSW, 2006, Australia.

Identifying genes that define cell identity is a requisite step for characterising cell types and cell states and predicting cell fate choices. By far, the most widely used approach for this task is based on differential expression (DE) of genes, whereby the shift of mean expression are used as the primary statistics for identifying gene transcripts that are specific to cell types and states. While DE-based methods are useful for pinpointing genes that discriminate cell types, their reliance on measuring difference in mean expression may not reflect the biological attributes of cell identity genes. Here, we highlight the quest for non-DE methods and provide an overview of these methods and their applications to identify genes that define cell identity and functionality.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s13619-021-00083-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8087741PMC
May 2021

Opportunities and challenges with stem cell-based embryo models.

Stem Cell Reports 2021 May 4;16(5):1031-1038. Epub 2021 Mar 4.

Children's Medical Research Institute, University of Sydney, and School of Medical Sciences, Faculty of Medicine and Health, University of Sydney, Sydney, Australia. Electronic address:

Stem cell-based embryo models open an unprecedented avenue for modeling embryogenesis, cell lineage differentiation, tissue morphogenesis, and organogenesis in mammalian development. Experimentation on these embryo models can lead to a better understanding of the mechanisms of development and offers opportunities for functional genomic studies of disease-causing mechanisms, identification of therapeutic targets, and preclinical modeling of advanced therapeutics for precision medicine. An immediate challenge is to create embryo models of high fidelity to embryogenesis and organogenesis in vivo, to ensure that the knowledge gleaned is biologically meaningful and clinically relevant.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.stemcr.2021.02.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8185371PMC
May 2021

Conserved Epigenetic Regulatory Logic Infers Genes Governing Cell Identity.

Cell Syst 2020 12 4;11(6):625-639.e13. Epub 2020 Dec 4.

Institute for Molecular Bioscience, The University of Queensland, Brisbane, Australia. Electronic address:

Determining genes that orchestrate cell differentiation in development and disease remains a fundamental goal of cell biology. This study establishes a genome-wide metric based on the gene-repressive trimethylation of histone H3 at lysine 27 (H3K27me3) across hundreds of diverse cell types to identify genetic regulators of cell differentiation. We introduce a computational method, TRIAGE, which uses discordance between gene-repressive tendency and expression to identify genetic drivers of cell identity. We apply TRIAGE to millions of genome-wide single-cell transcriptomes, diverse omics platforms, and eukaryotic cells and tissue types. Using a wide range of data, we validate the performance of TRIAGE in identifying cell-type-specific regulatory factors across diverse species including human, mouse, boar, bird, fish, and tunicate. Using CRISPR gene editing, we use TRIAGE to experimentally validate RNF220 as a regulator of Ciona cardiopharyngeal development and SIX3 as required for differentiation of endoderm in human pluripotent stem cells. A record of this paper's transparent peer review process is included in the Supplemental Information.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.cels.2020.11.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7781436PMC
December 2020

Author Correction: Molecular architecture of lineage allocation and tissue organization in early mouse embryo.

Nature 2020 Oct;586(7827):E7

State Key Laboratory of Cell Biology, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Shanghai, China.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41586-020-2755-1DOI Listing
October 2020

Prenet: Predictive network from ATAC-SEQ data.

J Bioinform Comput Biol 2020 02;18(1):2040003

Embryology Unit, Children's Medical Research Institute, The University of Sydney, Westmead, NSW 2145, Australia.

Assays for transposase-accessible chromatin sequencing (ATAC-seq) provides an innovative approach to study chromatin status in multiple cell types. Moreover, it is also possible to efficiently extract differentially accessible chromatin (DACs) regions by using state-of-the-art algorithms (e.g. DESeq2) to predict gene activity in specific samples. Furthermore, it has recently been shown that small dips in sequencing peaks can be attributed to the binding of transcription factors. These dips, also known as footprints, can be used to identify trans-regulating interactions leading to gene expression. Current protocols used to identify footprints (e.g. pyDNAse and HINT-ATAC) have shown limitations resulting in the discovery of many false positive footprints. We generated a novel approach to identify genuine footprints within any given ATAC-seq dataset. Herein, we developed a new pipeline embedding DACs together with footprints resulting in the generation of a dictive gene regulatory work (PreNet) simply from ATAC-seq data. We further demonstrated that PreNet can be used to unveil meaningful molecular regulatory pathways in a given cell type.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1142/S021972002040003XDOI Listing
February 2020

TWIST1 Homodimers and Heterodimers Orchestrate Lineage-Specific Differentiation.

Mol Cell Biol 2020 05 14;40(11). Epub 2020 May 14.

Embryology Unit, Children's Medical Research Institute, The University of Sydney, Sydney, Australia.

The extensive array of basic helix-loop-helix (bHLH) transcription factors and their combinations as dimers underpin the diversity of molecular function required for cell type specification during embryogenesis. The bHLH factor TWIST1 plays pleiotropic roles during development. However, which combinations of TWIST1 dimers are involved and what impact each dimer imposes on the gene regulation network controlled by TWIST1 remain elusive. In this work, proteomic profiling of human TWIST1-expressing cell lines and transcriptome analysis of mouse cranial mesenchyme have revealed that TWIST1 homodimers and heterodimers with TCF3, TCF4, and TCF12 E-proteins are the predominant dimer combinations. Disease-causing mutations in TWIST1 can impact dimer formation or shift the balance of different types of TWIST1 dimers in the cell, which may underpin the defective differentiation of the craniofacial mesenchyme. Functional analyses of the loss and gain of TWIST1-E-protein dimer activity have revealed previously unappreciated roles in guiding lineage differentiation of embryonic stem cells: TWIST1-E-protein heterodimers activate the differentiation of mesoderm and neural crest cells, which is accompanied by the epithelial-to-mesenchymal transition. At the same time, TWIST1 homodimers maintain the stem cells in a progenitor state and block entry to the endoderm lineage.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/MCB.00663-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7225560PMC
May 2020

Cellular diversity and lineage trajectory: insights from mouse single cell transcriptomes.

Development 2020 01 24;147(2). Epub 2020 Jan 24.

School of Biomedical Sciences, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, China.

Single cell RNA-sequencing (scRNA-seq) technology has matured to the point that it is possible to generate large single cell atlases of developing mouse embryos. These atlases allow the dissection of developmental cell lineages and molecular changes during embryogenesis. When coupled with single cell technologies for profiling the chromatin landscape, epigenome, proteome and metabolome, and spatial tissue organisation, these scRNA-seq approaches can now collect a large volume of multi-omic data about mouse embryogenesis. In addition, advances in computational techniques have enabled the inference of developmental lineages of differentiating cells, even without explicitly introduced genetic markers. This Spotlight discusses recent advent of single cell experimental and computational methods, and key insights from applying these methods to the study of mouse embryonic development. We highlight challenges in analysing and interpreting these data to complement and expand our knowledge from traditional developmental biology studies in relation to cell identity, diversity and lineage differentiation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1242/dev.179788DOI Listing
January 2020

Author Correction: Molecular architecture of lineage allocation and tissue organization in early mouse embryo.

Nature 2020 01;577(7791):E6

State Key Laboratory of Cell Biology, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Shanghai, China.

An Amendment to this paper has been published and can be accessed via a link at the top of the paper.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41586-019-1887-7DOI Listing
January 2020

Transcriptional network dynamics during the progression of pluripotency revealed by integrative statistical learning.

Nucleic Acids Res 2020 02;48(4):1828-1842

Charles Perkins Centre, School of Mathematics and Statistics, University of Sydney, Sydney, NSW 2006, Australia.

The developmental potential of cells, termed pluripotency, is highly dynamic and progresses through a continuum of naive, formative and primed states. Pluripotency progression of mouse embryonic stem cells (ESCs) from naive to formative and primed state is governed by transcription factors (TFs) and their target genes. Genomic techniques have uncovered a multitude of TF binding sites in ESCs, yet a major challenge lies in identifying target genes from functional binding sites and reconstructing dynamic transcriptional networks underlying pluripotency progression. Here, we integrated time-resolved 'trans-omic' datasets together with TF binding profiles and chromatin conformation data to identify target genes of a panel of TFs. Our analyses revealed that naive TF target genes are more likely to be TFs themselves than those of formative TFs, suggesting denser hierarchies among naive TFs. We also discovered that formative TF target genes are marked by permissive epigenomic signatures in the naive state, indicating that they are poised for expression prior to the initiation of pluripotency transition to the formative state. Finally, our reconstructed transcriptional networks pinpointed the precise timing from naive to formative pluripotency progression and enabled the spatiotemporal mapping of differentiating ESCs to their in vivo counterparts in developing embryos.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/nar/gkz1179DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7038952PMC
February 2020

Modeling the early development of a primate embryo.

Authors:
Patrick P L Tam

Science 2019 11;366(6467):798-799

Embryology Unit, Children's Medical Research Institute, University of Sydney and School of Medical Sciences, Faculty of Medicine and Health, University of Sydney, Westmead, NSW 2145, Australia.

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1126/science.aaz6976DOI Listing
November 2019

Ularcirc: visualization and enhanced analysis of circular RNAs via back and canonical forward splicing.

Nucleic Acids Res 2019 11;47(20):e123

Victor Chang Cardiac Research Institute.

Circular RNAs (circRNA) are a unique class of transcripts that can only be identified from sequence alignments spanning discordant junctions, commonly referred to as backsplice junctions (BSJ). Canonical splicing is also linked with circRNA biogenesis either from the parental transcript or internal to the circRNA, and is not fully utilized in circRNA software. Here we present Ularcirc, a software tool that integrates the visualization of both BSJ and forward splicing junctions and provides downstream analysis of selected circRNA candidates. Ularcirc utilizes the output of CIRI, circExplorer, or raw chimeric output of the STAR aligner and assembles BSJ count table to allow multi-sample analysis. We used Ularcirc to identify and characterize circRNA from public and in-house generated data sets and demonstrate how it can be used to (i) discover novel splicing patterns of parental transcripts, (ii) detect internal splicing patterns of circRNA, and (iii) reveal the complexity of BSJ formation. Furthermore, we identify circRNA that have potential open reading frames longer than their linear sequence. Finally, we detected and validated the presence of a novel class of circRNA generated from ApoA4 transcripts whose BSJ derive from multiple non-canonical splicing sites within coding exons. Ularcirc is accessed via https://github.com/VCCRI/Ularcirc.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/nar/gkz718DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6846653PMC
November 2019

Molecular architecture of lineage allocation and tissue organization in early mouse embryo.

Nature 2019 08 7;572(7770):528-532. Epub 2019 Aug 7.

State Key Laboratory of Cell Biology, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Shanghai, China.

During post-implantation development of the mouse embryo, descendants of the inner cell mass in the early epiblast transit from the naive to primed pluripotent state. Concurrently, germ layers are formed and cell lineages are specified, leading to the establishment of the blueprint for embryogenesis. Fate-mapping and lineage-analysis studies have revealed that cells in different regions of the germ layers acquire location-specific cell fates during gastrulation. The regionalization of cell fates preceding the formation of the basic body plan-the mechanisms of which are instrumental for understanding embryonic programming and stem-cell-based translational study-is conserved in vertebrate embryos. However, a genome-wide molecular annotation of lineage segregation and tissue architecture of the post-implantation embryo has yet to be undertaken. Here we report a spatially resolved transcriptome of cell populations at defined positions in the germ layers during development from pre- to late-gastrulation stages. This spatiotemporal transcriptome provides high-resolution digitized in situ gene-expression profiles, reveals the molecular genealogy of tissue lineages and defines the continuum of pluripotency states in time and space. The transcriptome further identifies the networks of molecular determinants that drive lineage specification and tissue patterning, supports a role of Hippo-Yap signalling in germ-layer development and reveals the contribution of visceral endoderm to the endoderm in the early mouse embryo.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41586-019-1469-8DOI Listing
August 2019

Establishment of porcine and human expanded potential stem cells.

Nat Cell Biol 2019 06 3;21(6):687-699. Epub 2019 Jun 3.

Key Laboratory of Regenerative Biology of Chinese Academy of Sciences, Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, China.

We recently derived mouse expanded potential stem cells (EPSCs) from individual blastomeres by inhibiting the critical molecular pathways that predispose their differentiation. EPSCs had enriched molecular signatures of blastomeres and possessed developmental potency for all embryonic and extra-embryonic cell lineages. Here, we report the derivation of porcine EPSCs, which express key pluripotency genes, are genetically stable, permit genome editing, differentiate to derivatives of the three germ layers in chimeras and produce primordial germ cell-like cells in vitro. Under similar conditions, human embryonic stem cells and induced pluripotent stem cells can be converted, or somatic cells directly reprogrammed, to EPSCs that display the molecular and functional attributes reminiscent of porcine EPSCs. Importantly, trophoblast stem-cell-like cells can be generated from both human and porcine EPSCs. Our pathway-inhibition paradigm thus opens an avenue for generating mammalian pluripotent stem cells, and EPSCs present a unique cellular platform for translational research in biotechnology and regenerative medicine.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41556-019-0333-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7035105PMC
June 2019

Mechanistic insights from the LHX1-driven molecular network in building the embryonic head.

Dev Growth Differ 2019 Jun 21;61(5):327-336. Epub 2019 May 21.

Embryology Unit, Children's Medical Research Institute, The University of Sydney, Westmead, New South Wales, Australia.

Development of an embryo is driven by a series of molecular instructions that control the differentiation of tissue precursor cells and shape the tissues into major body parts. LIM homeobox 1 (LHX1) is a transcription factor that plays a major role in the development of the embryonic head of the mouse. Loss of LHX1 function disrupts the morphogenetic movement of head tissue precursors and impacts on the function of molecular factors in modulating the activity of the WNT signaling pathway. LHX1 acts with a transcription factor complex to regulate the transcription of target genes in multiple phases of development and in a range of embryonic tissues of the mouse and Xenopus. Determining the interacting factors and transcriptional targets of LHX1 will be key to unraveling the ensemble of factors involved in head development and building a head gene regulatory network.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/dgd.12609DOI Listing
June 2019

Dynamics of Wnt activity on the acquisition of ectoderm potency in epiblast stem cells.

Development 2019 04 5;146(7). Epub 2019 Apr 5.

Embryology Unit, Children's Medical Research Institute, Westmead, NSW 2145, Australia.

During embryogenesis, the stringent regulation of Wnt activity is crucial for the morphogenesis of the head and brain. The loss of function of the Wnt inhibitor Dkk1 results in elevated Wnt activity, loss of ectoderm lineage attributes from the anterior epiblast, and the posteriorisation of anterior germ layer tissue towards the mesendoderm. The modulation of Wnt signalling may therefore be crucial for the allocation of epiblast cells to ectoderm progenitors during gastrulation. To test this hypothesis, we examined the lineage characteristics of epiblast stem cells (EpiSCs) that were derived and maintained under different signalling conditions. We showed that suppression of Wnt activity enhanced the ectoderm propensity of the EpiSCs. Neuroectoderm differentiation of these EpiSCs was further empowered by the robust re-activation of Wnt activity. Therefore, during gastrulation, the tuning of the signalling activities that mediate mesendoderm differentiation is instrumental for the acquisition of ectoderm potency in the epiblast.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1242/dev.172858DOI Listing
April 2019

Single-Cell RNA-Seq Reveals Cellular Heterogeneity of Pluripotency Transition and X Chromosome Dynamics during Early Mouse Development.

Cell Rep 2019 03;26(10):2593-2607.e3

Department of Physiology and Pharmacology, Karolinska Institutet, 17177 Solna, Sweden; Center for Molecular Medicine, Karolinska University Hospital, 17176 Solna, Sweden; School of Life Sciences and Technology, Tongji University, 200092 Shanghai, China. Electronic address:

Following implantation, the epiblast (EPI) cells transit from the naive to primed pluripotency, accompanied by dynamic changes in X chromosome activity in females. To investigate the molecular attributes of this process, we performed single-cell RNA-seq analysis of 1,724 cells of E5.25, E5.5, E6.25, and E6.5 mouse embryos. We identified three cellular states in the EPI cells that capture the transition along the pluripotency continuum and the acquisition of primitive streak propensity. The transition of three EPI states was driven by inductive signaling activity emanating from the visceral endoderm (VE). In the EPI of female embryos, X chromosome reactivation (XCR) was initiated prior to the completion of imprinted X chromosome inactivation (XCI), and the ensuing random XCI was highly asynchronous. Moreover, imprinted paternal XCI proceeded faster in the VE than the extraembryonic ectoderm. Our study has provided a detailed molecular roadmap of the emergent lineage commitment before gastrulation and characterized X chromosome dynamics during early mouse development.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.celrep.2019.02.031DOI Listing
March 2019

Gene Editing of Mouse Embryonic and Epiblast Stem Cells.

Methods Mol Biol 2019 ;1940:77-95

The University of Sydney, Children's Medical Research Institute, Westmead, NSW, Australia.

Efficient and reliable methods for gene editing are critical for the generation of loss-of-gene function stem cells and genetically modified mice. Here, we outline the application of CRISPR-Cas9 technology for gene editing in mouse embryonic stem cells (mESCs) to generate knockout ESC chimeras for the fast-tracked analysis of gene function. Furthermore, we describe the application of gene editing directly to mouse epiblast stem cells (mEpiSCs) for modelling germ layer differentiation in vitro.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/978-1-4939-9086-3_6DOI Listing
July 2019

Mouse gastrulation: Attributes of transcription factor regulatory network for epiblast patterning.

Dev Growth Differ 2018 Oct;60(8):463-472

State Key Laboratory of Cell Biology, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Shanghai, China.

Gastrulation is a key milestone in early mouse development when multipotent epiblast cells are allocated to progenitors of diverse tissue lineages that constitute the ensemble of building blocks of the body plan. The analysis of gene function revealed that the activity of transcription factors is likely to be the fundamental driving force underpinning the lineage specification and tissue patterning in the primary germ layers. The developmental-spatial transcriptome of the gastrulating embryo revealed the concerted and interactive activity of the gene regulatory network anchored by development-related transcription factors. The findings of the network structure offer novel insights into the regionalization of tissue fates and enable tracking of the progression of epiblast patterning, leading to the construction of molecularly annotated fate maps of epiblast during gastrulation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/dgd.12568DOI Listing
October 2018

Single-Cell Transcriptomic Analysis of Cardiac Differentiation from Human PSCs Reveals HOPX-Dependent Cardiomyocyte Maturation.

Cell Stem Cell 2018 10;23(4):586-598.e8

Institute for Molecular Bioscience, The University of Queensland, Brisbane, QLD 4072, Australia; Centre for Cardiac and Vascular Biology, The University of Queensland, Brisbane, QLD 4072, Australia; School of Biomedical Sciences, The University of Queensland, Brisbane, QLD 4072, Australia. Electronic address:

Cardiac differentiation of human pluripotent stem cells (hPSCs) requires orchestration of dynamic gene regulatory networks during stepwise fate transitions but often generates immature cell types that do not fully recapitulate properties of their adult counterparts, suggesting incomplete activation of key transcriptional networks. We performed extensive single-cell transcriptomic analyses to map fate choices and gene expression programs during cardiac differentiation of hPSCs and identified strategies to improve in vitro cardiomyocyte differentiation. Utilizing genetic gain- and loss-of-function approaches, we found that hypertrophic signaling is not effectively activated during monolayer-based cardiac differentiation, thereby preventing expression of HOPX and its activation of downstream genes that govern late stages of cardiomyocyte maturation. This study therefore provides a key transcriptional roadmap of in vitro cardiac differentiation at single-cell resolution, revealing fundamental mechanisms underlying heart development and differentiation of hPSC-derived cardiomyocytes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.stem.2018.09.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6220122PMC
October 2018

A gene regulatory network anchored by LIM homeobox 1 for embryonic head development.

Genesis 2018 09 14;56(9):e23246. Epub 2018 Sep 14.

Embryology Unit, Children's Medical Research Institute, The University of Sydney, Sydney, New South Wales, Australia.

Development of the embryonic head is driven by the activity of gene regulatory networks of transcription factors. LHX1 is a homeobox transcription factor that plays an essential role in the formation of the embryonic head. The loss of LHX1 function results in anterior truncation of the embryo caused by the disruption of morphogenetic movement of tissue precursors and the dysregulation of WNT signaling activity. Profiling the gene expression pattern in the Lhx1 mutant embryo revealed that tissues in anterior germ layers acquire posterior tissue characteristics, suggesting LHX1 activity is required for the allocation and patterning of head precursor tissues. Here, we used LHX1 as an entry point to delineate its transcriptional targets and interactors and construct a LHX1-anchored gene regulatory network. Using a gain-of-function approach, we identified genes that immediately respond to Lhx1 activation. Meta-analysis of the datasets of LHX1-responsive genes and genes expressed in the anterior tissues of mouse embryos at head-fold stage, in conjunction with published Xenopus embryonic LHX1 (Xlim1) ChIP-seq data, has pinpointed the putative transcriptional targets of LHX1 and an array of genetic determinants functioning together in the formation of the mouse embryonic head.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/dvg.23246DOI Listing
September 2018

Suppressing Nodal Signaling Activity Predisposes Ectodermal Differentiation of Epiblast Stem Cells.

Stem Cell Reports 2018 07 28;11(1):43-57. Epub 2018 Jun 28.

State Key Laboratory of Cell Biology, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, University of Chinese Academy of Sciences, 320 Yue Yang Road, Shanghai 200031, China; School of Life Science and Technology, ShanghaiTech University, 100 Haike Road, Shanghai 201210, China. Electronic address:

The molecular mechanism underpinning the specification of the ectoderm, a transient germ-layer tissue, during mouse gastrulation was examined here in a stem cell-based model. We captured a self-renewing cell population with enhanced ectoderm potency from mouse epiblast stem cells (EpiSCs) by suppressing Nodal signaling activity. The transcriptome of the Nodal-inhibited EpiSCs resembles that of the anterior epiblast of embryonic day (E)7.0 and E7.5 mouse embryo, which is accompanied by chromatin modifications that reflect the priming of ectoderm lineage-related genes for expression. Nodal-inhibited EpiSCs show enhanced ectoderm differentiation in vitro and contribute to the neuroectoderm and the surface ectoderm in postimplantation chimeras but lose the propensity for mesendoderm differentiation in vitro and in chimeras. Our findings show that specification of the ectoderm progenitors is enhanced by the repression of Nodal signaling activity, and the ectoderm-like stem cells provide an experimental model to investigate the molecular characters of the epiblast-derived ectoderm.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.stemcr.2018.05.019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6067151PMC
July 2018

Exploring early human embryo development.

Science 2018 06;360(6393):1075-1076

Embryology Unit, Children's Medical Research Institute and School of Medical Sciences, Faculty of Medicine and Health, University of Sydney, Westmead, NSW 2145, Australia.

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1126/science.aas9302DOI Listing
June 2018

Homozygous Dkk1 Knockout Mice Exhibit High Bone Mass Phenotype Due to Increased Bone Formation.

Calcif Tissue Int 2018 01 6;102(1):105-116. Epub 2017 Nov 6.

Orthopaedic Research & Biotechnology Unit, The Children's Hospital at Westmead, Research Building, Locked Bag 4001, Westmead, NSW, 2145, Australia.

Wnt antagonist Dkk1 is a negative regulator of bone formation and Dkk1 heterozygous mice display a high bone mass phenotype. Complete loss of Dkk1 function disrupts embryonic head development. Homozygous Dkk1 mice that were heterozygous for Wnt3 loss of function mutation (termed Dkk1 KO) are viable and allowed studying the effects of homozygous inactivation of Dkk1 on bone formation. Dkk1 KO mice showed a high bone mass phenotype exceeding that of heterozygous mice as well as a high incidence of polydactyly and kinky tails. Whole body bone density was increased in the Dkk1 KO mice as shown by longitudinal dual-energy X-ray absorptiometry. MicroCT analysis of the distal femur revealed up to 3-fold increases in trabecular bone volume and up to 2-fold increases in the vertebrae, compared to wild type controls. Cortical bone was increased in both the tibiae and vertebrae, which correlated with increased strength in tibial 4-point bending and vertebral compression tests. Dynamic histomorphometry identified increased bone formation as the mechanism underlying the high bone mass phenotype in Dkk1 KO mice, with no changes in bone resorption. Mice featuring only Wnt3 heterozygosity showed no evident bone phenotype. Our findings highlight a critical role for Dkk1 in the regulation of bone formation and a gene dose-dependent response to loss of DKK1 function. Targeting Dkk1 to enhance bone formation offers therapeutic potential for osteoporosis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s00223-017-0338-4DOI Listing
January 2018

Establishment of mouse expanded potential stem cells.

Nature 2017 10 11;550(7676):393-397. Epub 2017 Oct 11.

Division of Stem Cell Therapy, Institute of Medical Science, University of Tokyo, Tokyo 108-8639, Japan.

Mouse embryonic stem cells derived from the epiblast contribute to the somatic lineages and the germline but are excluded from the extra-embryonic tissues that are derived from the trophectoderm and the primitive endoderm upon reintroduction to the blastocyst. Here we report that cultures of expanded potential stem cells can be established from individual eight-cell blastomeres, and by direct conversion of mouse embryonic stem cells and induced pluripotent stem cells. Remarkably, a single expanded potential stem cell can contribute both to the embryo proper and to the trophectoderm lineages in a chimaera assay. Bona fide trophoblast stem cell lines and extra-embryonic endoderm stem cells can be directly derived from expanded potential stem cells in vitro. Molecular analyses of the epigenome and single-cell transcriptome reveal enrichment for blastomere-specific signature and a dynamic DNA methylome in expanded potential stem cells. The generation of mouse expanded potential stem cells highlights the feasibility of establishing expanded potential stem cells for other mammalian species.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/nature24052DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5890884PMC
October 2017

Identification of liver-specific enhancer-promoter activity in the 3' untranslated region of the wild-type AAV2 genome.

Nat Genet 2017 Aug 19;49(8):1267-1273. Epub 2017 Jun 19.

Gene Therapy Research Unit, Children's Medical Research Institute and Sydney Children's Hospitals Network, University of Sydney, Sydney, New South Wales, Australia.

Vectors based on adeno-associated virus type 2 (AAV2) are powerful tools for gene transfer and genome editing applications. The level of interest in this system has recently surged in response to reports of therapeutic efficacy in human clinical trials, most notably for those in patients with hemophilia B (ref. 3). Understandably, a recent report drawing an association between AAV2 integration events and human hepatocellular carcinoma (HCC) has generated controversy about the causal or incidental nature of this association and the implications for AAV vector safety. Here we describe and functionally characterize a previously unknown liver-specific enhancer-promoter element in the wild-type AAV2 genome that is found between the stop codon of the cap gene, which encodes proteins that form the capsid, and the right-hand inverted terminal repeat. This 124-nt sequence is within the 163-nt common insertion region of the AAV genome, which has been implicated in the dysregulation of known HCC driver genes and thus offers added insight into the possible link between AAV integration events and the multifactorial pathogenesis of HCC.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/ng.3893DOI Listing
August 2017

Pluripotency of embryo-derived stem cells from rodents, lagomorphs, and primates: Slippery slope, terrace and cliff.

Stem Cell Res 2017 03 17;19:104-112. Epub 2017 Jan 17.

Embryology Unit, Children's Medical Research Institute, Westmead, NSW 2145, Australia; School of Medical Sciences, Sydney Medical School, University of Sydney, NSW 2006, Australia.

The diverse cell states and in vitro conditions for the derivation and maintenance of the mammalian embryo-derived pluripotent stem cells raise the questions of whether there are multiple states of pluripotency of the stem cells of each species, and if there are innate species-specific variations in the pluripotency state. We will address these questions by taking a snapshot of our knowledge of the properties of the pluripotent stem cells, focusing on the maintenance of pluripotency and inter-conversion of the different types of pluripotent stem cells from rodents, lagomorphs and primates. We conceptualize pluripotent stem cells acquiring a series of cellular states represented as terraces on a slope of descending gradient of pluripotency. We propose that reprogramming pluripotent stem cells from a primed to a naive state is akin to moving upstream over a steep cliff to a higher terrace.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.scr.2017.01.008DOI Listing
March 2017

New Insights into Early Human Development: Lessons for Stem Cell Derivation and Differentiation.

Cell Stem Cell 2017 01;20(1):18-28

Embryology Unit, Children's Medical Research Institute and School of Medical Sciences, Sydney Medical School, University of Sydney, Westmead, NSW 2145, Australia. Electronic address:

Pathways underlying mouse embryonic development have always informed efforts to derive, maintain, and drive differentiation of human pluripotent stem cells. However, direct application of mouse embryology to the human system has not always been successful because of fundamental developmental differences between species. The naive pluripotent state of mouse embryonic stem cells (ESCs), in particular, has been difficult to capture in human ESCs, and appears to be transitory in the human embryo itself. Further studies of human and non-human primate embryo development are needed to untangle the complexities of pluripotency networks across mammalian species.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.stem.2016.12.004DOI Listing
January 2017

Interactome of the inhibitory isoform of the nuclear transporter Importin 13.

Biochim Biophys Acta Mol Cell Res 2017 Mar 16;1864(3):546-561. Epub 2016 Dec 16.

Department of Biochemistry & Molecular Biology, Monash University, Clayton, Victoria, Australia. Electronic address:

Importin 13 (Imp13) is a bidirectional nuclear transporter of proteins involved in a range of important cellular processes, with an N-terminally truncated inhibitory isoform (tImp13) specifically expressed in testis. To gain insight into tImp13 function, we performed a yeast-2-hybrid screen from a human testis cDNA library, identifying for the first time a suite of interactors with roles in diverse cellular process. We validated the interaction of tImp13 with Eukaryotic translation initiation factor 4γ2 (EIF4G2) and High mobility group containing protein 20A (HMG20A), benchmarking that with glucocorticoid receptor (GR), a known Imp13 interactor expressed in testis. Coimmunoprecipitation assays indicated association of both tImp13 and Imp13 with EIF4G2, HMG20A and GR. Quantitative confocal microscopic analysis revealed the ability of tImp13 to inhibit the nuclear localisation of EIF4G2, HMG20A and GR, as well as that of Imp13 to act as a nuclear exporter for both EIF4G2 and HMG20A, and as a nuclear importer for GR. The physiological relevance of these results was highlighted by the cytoplasmic localisation of EIF4G2, HMG20A and GR in pachytene spermatocytes/round spermatids in the murine testis where tImp13 is present at high levels, in contrast to the nuclear localisation of HMG20A and GR in spermatogonia, where tImp13 is largely absent. Interestingly, Imp13, EIF4G2, HMG20A and GR were found together in the acrosome vesicle of murine epididymal spermatozoa. Collectively, our findings show, for the first time, that tImp13 may have a functional role in the mature spermatozoa, in addition to that in the meiotic germ cells of the testis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bbamcr.2016.12.017DOI Listing
March 2017
-->