Patrick Tam

Profiling spatial gene activity in marmoset embryos

Publisher: Springer Science and Business Media LLC

Date: 20-07-2022

DOI: 10.1038/S41422-022-00703-0

Technical challenges of studying early human development

Publisher: The Company of Biologists

Date: 06-2023

DOI: 10.1242/DEV.201797

Abstract: Recent years have seen exciting progress across human embryo research, including new methods for culturing embryos, transcriptional profiling of embryogenesis and gastrulation, mapping lineage trajectories, and experimenting on stem cell-based embryo models. These advances are beginning to define the dynamical principles of development across stages, tissues and organs, enabling a better understanding of human development before birth in health and disease, and potentially leading to improved treatments for infertility and developmental disorders. However, there are still significant roadblocks en route to this goal. Here, we highlight technical challenges to studying early human development and propose ways and means to overcome some of these constraints.

TWIST1 and chromatin regulatory proteins interact to guide neural crest cell differentiation

Publisher: eLife Sciences Publications, Ltd

Date: 08-02-2021

DOI: 10.7554/ELIFE.62873

Abstract: Protein interaction is critical molecular regulatory activity underlining cellular functions and precise cell fate choices. Using TWIST1 BioID-proximity-labeling and network propagation analyses, we discovered and characterized a TWIST-chromatin regulatory module (TWIST1-CRM) in the neural crest cells (NCC). Combinatorial perturbation of core members of TWIST1-CRM: TWIST1, CHD7, CHD8, and WHSC1 in cell models and mouse embryos revealed that loss of the function of the regulatory module resulted in abnormal differentiation of NCCs and compromised craniofacial tissue patterning. Following NCC delamination, low level of TWIST1-CRM activity is instrumental to stabilize the early NCC signatures and migratory potential by repressing the neural stem cell programs. High level of TWIST1 module activity at later phases commits the cells to the ectomesenchyme. Our study further revealed the functional interdependency of TWIST1 and potential neurocristopathy factors in NCC development.

Author response: TWIST1 and chromatin regulatory proteins interact to guide neural crest cell differentiation

Publisher: eLife Sciences Publications, Ltd

Date: 25-01-2021

DOI: 10.7554/ELIFE.62873.SA2

Uncovering cell identity through differential stability with Cepo

Publisher: Springer Science and Business Media LLC

Date: 20-12-2021

DOI: 10.1038/S43588-021-00172-2

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

Publisher: Wiley

Date: 10-2018

DOI: 10.1111/DGD.12568

Abstract: Gastrulation is a key milestone in early mouse development when multipotent epiblast cells are allocated to progenitors of erse 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.

Integration of Computational Analysis and Spatial Transcriptomics in Single-cell Studies

Publisher: Elsevier BV

Date: 02-2023

DOI: 10.1016/J.GPB.2022.06.006

Meta-Analysis Identifies Novel Common Genes Differently Altered in Cross-Species Models of Rett Syndrome

Publisher: MDPI AG

Date: 04-07-2022

DOI: 10.20944/PREPRINTS202207.0030.V1

Abstract: Rett syndrome (RTT) is a rare disease and one of the most abundant causes for intellectual disa-bilities in females. Single mutations in the gene coding for methyl-CpG-binding protein 2 (MECP2), are responsible for the disease. MeCP2 regulates gene expression as a transcriptional regulator as well as through epigenetic imprinting and chromatin condensation. Consequently, numerous biological pathways on multiple levels are influenced however, the exact molecular pathways from genotype to phenotype are currently not fully elucidated. Treatment of RTT is purely symptomatic where no curative options for RTT have yet to reach the clinic. The paucity of this is mainly due to an incomplete understanding of the underlying pathophysiology of the dis-order with no clinically useful common disease drivers, biomarkers or therapeutic targets being identified. With the premise of identifying universal and robust disease drivers and therapeutic targets, here we interrogated a range of RTT transcriptomic studies spanning different species, models and MECP2 mutations. A meta-analysis using RNA sequencing data from brains of RTT mouse models human post-mortem brain tissue and patient-derived induced pluripotent stem cells (iPSC) neurons was performed using Weighted Gene Correlation Network Analysis (WGC-NA). This study identified a module of genes common to all datasets with the following ten hub genes driving the expression ATRX, ADCY7, ADCY9, SOD1, CACNA1A, PLCG1, CCT5, RPS9, BDNF and MECP2. Here we discuss the potential benefits of these genes as therapeutic targets.

Replication stress tolerance and management differs between naïve and primed pluripotent cells

Publisher: Cold Spring Harbor Laboratory

Date: 13-05-2022

DOI: 10.1101/2022.05.12.491744

Abstract: Replication stress is an endemic threat to genome stability. For reasons unknown, replication stress response factors become essential during peri-implantation development. This coincides with a stem cell potency switch from the naïve to the primed state. Using genetically matched, chimera-derived mouse naïve embryonic (mESC) and primed epiblast stem cells (mEpiSC) we found that replication stress management differs between potency states. Primed mEpiSCs rely on Atr activity to prevent replication catastrophe, minimize genomic damage, avoid apoptosis, and re-enter the cell cycle. Conversely, under replications stress, mESCs readily activate Atm regardless of Atr activity, undergo replication catastrophe, and induce apoptosis. Primed pluripotent cells therefore engage Atr to counteract replication difficulties and maintain viability, whereas cells in the naïve state are more readily cleared under the same conditions. We anticipate these ergent strategies enable pluripotent cells of different potency states to meet associated proliferative or developmental demands during early development.

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

Publisher: The Company of Biologists

Date: 15-01-2020

DOI: 10.1242/DEV.179788

Abstract: 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, ersity and lineage differentiation.

Establishment of porcine and human expanded potential stem cells

Publisher: Springer Science and Business Media LLC

Date: 06-2019

DOI: 10.1038/S41556-019-0333-2

A cis-regulatory-directed pipeline for the identification of genes involved in cardiac development and disease.

Publisher: Springer Science and Business Media LLC

Date: 12-2021

DOI: 10.1186/S13059-021-02539-0

Abstract: Congenital heart diseases are the major cause of death in newborns, but the genetic etiology of this developmental disorder is not fully known. The conventional approach to identify the disease-causing genes focuses on screening genes that display heart-specific expression during development. However, this approach would have discounted genes that are expressed widely in other tissues but may play critical roles in heart development. We report an efficient pipeline of genome-wide gene discovery based on the identification of a cardiac-specific cis -regulatory element signature that points to candidate genes involved in heart development and congenital heart disease. With this pipeline, we retrieve 76% of the known cardiac developmental genes and predict 35 novel genes that previously had no known connectivity to heart development. Functional validation of these novel cardiac genes by RNAi-mediated knockdown of the conserved orthologs in Drosophila cardiac tissue reveals that disrupting the activity of 71% of these genes leads to adult mortality. Among these genes, RpL14 , RpS24 , and Rpn8 are associated with heart phenotypes. Our pipeline has enabled the discovery of novel genes with roles in heart development. This workflow, which relies on screening for non-coding cis -regulatory signatures, is amenable for identifying developmental and disease genes for an organ without constraining to genes that are expressed exclusively in the organ of interest.

Time space and single-cell resolved tissue lineage trajectories and laterality of body plan at gastrulation

Publisher: Springer Science and Business Media LLC

Date: 14-09-2023

DOI: 10.1038/S41467-023-41482-5

Mammalian gastrulation: signalling activity and transcriptional regulation of cell lineage differentiation and germ layer formation

Publisher: Portland Press Ltd.

Date: 18-11-2022

DOI: 10.1042/BST20220256

Abstract: The interplay of signalling input and downstream transcriptional activity is the key molecular attribute driving the differentiation of germ layer tissue and the specification of cell lineages within each germ layer during gastrulation. This review delves into the current understanding of signalling and transcriptional control of lineage development in the germ layers of mouse embryo and non-human primate embryos during gastrulation and highlights the inter-species conservation and ergence of the cellular and molecular mechanisms of germ layer development in the human embryo.

Meta-Analysis Identifies BDNF and Novel Common Genes Differently Altered in Cross-Species Models of Rett Syndrome

Publisher: MDPI AG

Date: 22-09-2022

DOI: 10.3390/IJMS231911125

Abstract: Rett syndrome (RTT) is a rare disorder and one of the most abundant causes of intellectual disabilities in females. Single mutations in the gene coding for methyl-CpG-binding protein 2 (MeCP2) are responsible for the disorder. MeCP2 regulates gene expression as a transcriptional regulator as well as through epigenetic imprinting and chromatin condensation. Consequently, numerous biological pathways on multiple levels are influenced. However, the exact molecular pathways from genotype to phenotype are currently not fully elucidated. Treatment of RTT is purely symptomatic as no curative options for RTT have yet to reach the clinic. The paucity of this is mainly due to an incomplete understanding of the underlying pathophysiology of the disorder with no clinically useful common disease drivers, biomarkers, or therapeutic targets being identified. With the premise of identifying universal and robust disease drivers and therapeutic targets, here, we interrogated a range of RTT transcriptomic studies spanning different species, models, and MECP2 mutations. A meta-analysis using RNA sequencing data from brains of RTT mouse models, human post-mortem brain tissue, and patient-derived induced pluripotent stem cell (iPSC) neurons was performed using weighted gene correlation network analysis (WGCNA). This study identified a module of genes common to all datasets with the following ten hub genes driving the expression: ATRX, ADCY7, ADCY9, SOD1, CACNA1A, PLCG1, CCT5, RPS9, BDNF, and MECP2. Here, we discuss the potential benefits of these genes as therapeutic targets.

LoxCode in vivo barcoding resolves epiblast clonal fate to fetal organs

Publisher: Cold Spring Harbor Laboratory

Date: 03-01-2023

DOI: 10.1101/2023.01.02.522501

Abstract: Contribution of the mammalian epiblast to fetal organs during embryogenesis has been investigated using reporters of marker genes, or through single cell or spatial RNA sequencing to infer differentiation trajectories. However, much remains to be learned about the clonal fate of mammalian epiblast cells in vivo . Here we develop a high ersity, high throughput, Cre recombinase-driven DNA LoxCode barcoding technology for in vivo clonal lineage tracing. Using this LoxCode mouse model, cells in E5.5 pre-gastrulation embryos were barcoded in utero and assessed in bulk via PCR or via single-cell RNA sequencing for their contribution to a comprehensive range of tissues and cell types in the E12.5 organogenesis-stage embryo. While a few typically large clones contributed to a erse range of cell types of multiple germ layer derivatives, many clones displayed reproducible patterns of lineage restriction. Most prominent were clonal fate biases towards either blood, ectoderm lineages, mesenchymal tissues or limbs, likely reflecting branch points during development. In the context of a stochastic agent-based model of tissue development, clonal fate biases could be explained by early differentiation events occurring shortly after barcoding, and clonal similarities between tissues arose as a consequence of shared differentiation paths. At the single-cell level, clones exhibited heterogeneity in terms of tissue contributions, gene expression profiles, and in some instances left-right asymmetries and/or anterior-posterior segregation. Our study demonstrates the power and versatility of LoxCode barcoding in investigating native clonal fate and provides a deep clonal interrogation of the contribution of the mammalian epiblast to fetal organs.

Material-mediated histogenesis using mechano-chemically microstructured cell niches

Publisher: Cold Spring Harbor Laboratory

Date: 11-02-2021

DOI: 10.1101/2021.02.10.430691

Abstract: Stem-cell derived tissue models are commonly cultured under globally-delivered stimuli that trigger histogenesis via self-organizing activity. However, the culture of such tissue models is prone to stochastic behavior, limiting the reproducibility of cellular composition and resulting in non-physiological architectures. To overcome these shortcomings, we developed a method for printing cell niche microenvironments with microstructured cues that mediate local histogenic processes, including mechanosensing and differentiation of selected cell types. Microstructured cues include independently tunable mechano-chemical properties, with conjugated peptides, proteins, and morphogens across a range of Young’s moduli. By rationally designing niches, we mediate the structure of tissues derived from stem-cell-progenitor sources, including a bone-fat assembly from stromal mesenchyme, and embryonic tissues derived from hiPSC. We show that microstructured cues can recapitulate mechano-chemical signals resembling early embryonic histogenesis. This outcome includes a role for niche mechanics in human embryonic organization, where soft niche mechanics bias markers of mesendodermal differentiation and epithelial-to-mesenchymal-transition (EMT), as well as a demonstration of a material-mediated morphogen signaling centers able to induce foci of mesenchymal and EMT differentiation. Thus, microstructured materials can mediate local histogenic processes to enhance the structure and composition of tissue models.

An integrated cell barcoding and computational analysis pipeline for scalable analysis of differentiation at single-cell resolution

Publisher: Cold Spring Harbor Laboratory

Date: 14-10-2022

DOI: 10.1101/2022.10.12.511862

Abstract: This study develops a versatile cell multiplexing and data analysis platform to gain knowledge gain into mechanisms of cell differentiation. We engineer a cell barcoding system in human cells enabling multiplexed single-cell RNA sequencing for high throughput perturbation of customisable and erse experimental conditions. This is coupled with a new computational analysis pipeline that overcomes the limitations of conventional algorithms by using an unsupervised, genome-wide, orthogonal biological reference point to reveal the cell ersity and regulatory networks in the input scRNA-seq data set. We implement this pipeline by engineering transcribed barcodes into induced pluripotent stem cells and multiplex 62 independent experimental conditions comprising eight differentiation time points and nine developmental signalling perturbations in duplicates. We identify and deconstruct the temporal, signalling, and gene regulatory imperatives of iPSC differentiation into cell types of ectoderm, mesoderm, and endoderm lineages. This study provides a cellular and computational pipeline to study cell differentiation applicable to studies in developmental biology, drug discovery, and disease modelling.

Children's Medical Research Institute

Location: Australia

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University Of Sydney

Location: Australia

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Discovery Projects - Grant ID: DP0985052

Start Date: 02-2009

End Date: 02-2012

Amount: $421,000.00

Funder: Australian Research Council

Discovery Projects - Grant ID: DP1094008

Start Date: 06-2010

End Date: 06-2013

Amount: $542,000.00

Funder: Australian Research Council

Discovery Projects - Grant ID: DP130100779

Start Date: 03-2013

End Date: 03-2016

Amount: $540,000.00

Funder: Australian Research Council

Discovery Projects - Grant ID: DP190102793

Start Date: 2019

End Date: 12-2021

Amount: $580,000.00

Funder: Australian Research Council

Discovery Projects - Grant ID: DP120100099

Start Date: 04-2012

End Date: 12-2014

Amount: $300,000.00

Funder: Australian Research Council

Discovery Projects - Grant ID: DP160100933

Start Date: 2016

End Date: 12-2018

Amount: $486,700.00

Funder: Australian Research Council

Special Research Initiatives - Grant ID: SR0354622

Start Date: 11-2003

End Date: 03-2005

Amount: $20,000.00

Funder: Australian Research Council

Research Networks - Grant ID: RN0457079

Start Date: 09-2004

End Date: 08-2009

Amount: $1,500,000.00

Funder: Australian Research Council

Discovery Projects - Grant ID: DP160103651

Start Date: 2016

End Date: 12-2018

Amount: $594,400.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE100100150

Start Date: 2010

End Date: 12-2012

Amount: $500,000.00

Funder: Australian Research Council