Biology subjects

Christie, L.

Publications and source records attributed to Christie, L..

9 recordsLinked to original sources

bioRxiv2025

A blastocyst-derived in vitro model of the human chorion

The placenta supports the foetus by mediating nutrient and gas exchange, hormone production, and immune protection. Yet, human placental biology remains poorly understood due to limited in vitro models. The foetal part of the placenta, the chorion, consists of a trophoblast-derived layer and a mesenchymal zone containing extraembryonic mesoderm cells. Here, we show that human blastocysts can give rise to both trophoblast organoids and extraembryonic mesoderm cells under the same culture conditions. Trophoblast organoids originate from both outer trophoblast cells and inner cells of the blastocyst, while extraembryonic mesoderm cells derive exclusively from inner cells. These organoids recapitulate the cellular composition, morphology, and function of the in vivo trophoblast. Moreover, given the high rate of aneuploidy at the blastocyst stage, aneuploid trophoblast organoids can be readily generated. These models reflect the trophoblasts unique ability to tolerate aneuploidy and offer a valuable platform to study human placental development and pathophysiology.

developmental biology↗

bioRxiv2025

NETSseq Reveals Inflammatory and Aging Mechanisms in Distinct Cell Types Driving Cerebellar Decline in Ataxia Telangiectasia

The cellular and molecular changes driving the neurological abnormalities associated with ataxia - telangiectasia (A-T) are not well understood. Here, we applied our proprietary Nuclear Enriched Transcript Sort sequencing (NETSseq) platform to investigate changes in cell type composition and gene expression in human cerebellar post-mortem tissue from A-T and control donors. Compared to single-nuclei technologies, NETSseq provided a more robust detection of genes with low abundance, a higher cell type specific expression pattern, and significantly lower levels of cross-contamination. We found dysregulation in neurotransmitter signaling in granule neurons, potentially underlying the impaired motor coordination in A-T. Astrocytes and microglia have evidence of accelerated aging, with astrocytes being characterized by neurotoxic signatures, while microglia showed activation of DNA damage response pathways. These findings highlight the importance of NETSseq as a resource for investigating mechanisms and biological processes associated with disease, providing high-sensitivity, cell-specific insights to advance targeted therapies for neurodegenerative diseases.

neuroscience↗

bioRxiv2024

Live imaging human embryos reveals mitotic errors and lineage specification prior to implantation

Meiotic and mitotic chromosome segregation errors in human development have been studied mostly prior to and at the time of fertilisation. Despite chromosomal errors being a leading cause of miscarriage and infertility, chromosome missegregation has not been extensively studied at later stages of human development. Here we optimised labelling, light-sheet live imaging and semi-quantitative analysis of human embryos and reveal chromosome segregation errors just prior to implantation. We found that human embryos exhibited a number of chromosome missegregation events including multipolar spindle formation, lagging chromosomes, misalignment and chromosome slippage. We found that the majority of lagging chromosomes were passively inherited by one of the daughter cells, instead of reincorporating into the nuclei, suggesting a distinct pattern of micronuclei inheritance. By semi-automated segmentation, we tracked the position of labelled cells in human embryos and observed that while most labelled cells remained segregated to the outside, and therefore restricted to a placental-progenitor fate, there was evidence of a rare cell migration to cells positioned on the inside, which suggests that there may be plasticity. Altogether, we found that mitotic chromosome segregation errors arise just prior to implantation, which has implications for our understanding of biological events that contribute to aneuploidy mosaicism.

developmental biology↗

bioRxiv2024

Suppression of ERK signalling promotes pluripotent epiblast in the human blastocyst

Studies in the mouse demonstrate the importance of fibroblast growth factor (FGF) and extra-cellular receptor tyrosine kinase (ERK) in specification of embryo-fated epiblast and yolk-sac-fated hypoblast cells from uncommitted inner cell mass (ICM) cells prior to implantation. Molecular mechanisms regulating specification of early lineages in human development are comparatively unclear. Here we show that exogenous FGF stimulation leads to expanded hypoblast molecular marker expression, at the expense of the epiblast. Conversely, we show that specifically inhibiting ERK activity leads to expansion of epiblast cells functionally capable of giving rise to naive human pluripotent stem cells. Single-cell transcriptomic analysis indicates that these epiblast cells downregulate FGF signalling and upregulate molecular markers associated with naive pluripotency. Our functional study demonstrates for the first time the molecular mechanisms governing ICM specification in human development, whereby segregation of the epiblast and hypoblast lineages occurs during maturation of the mammalian embryo in an ERK signal-dependent manner.

developmental biology↗

bioRxiv2023

Convergent Insulin and TGF-β signalling drives cancer cachexia by promoting aberrant fatbody ECM accumulation in a Drosophila tumour model

Cancer cachexia is a wasting disease suffered by advanced stage cancer patients and ultimately causes [~]30% of cancer mortalities. Clinical observations have shown that extracellular matrix (ECM) remodelling which leads to fibrosis in the adipose tissue is a key feature of cancer cachexia. However, the molecular regulators of adipose ECM remodelling are not known and how this leads to muscle wasting is unclear. In this study, using a Drosophila cachexia model, we found that in the adipose tissue of both wildtype and tumour bearing animals, insulin and TGF-{beta} signalling converge via a BMP antagonist short gastrulation (sog) to regulate ECM remodelling. In tumour bearing animals, the aberrant ECM accumulation in the fatbody, contributes towards muscle detachment by preventing ECM secretion and subsequently depleting muscles of fatbody-secreted ECM proteins. Strikingly, activation of insulin signalling, inhibition of TGF-{beta} signalling, or modulation of ECM secretion via SPARC or Rab10 in the fatbody, was able to rescue tissue wasting in the presence of tumour. Together, our study highlights the importance of adipose ECM remodelling in the context of cancer cachexia.

developmental biology↗

bioRxiv2023

MICA: A multi-omics method to predict gene regulatory networks in early human embryos

Recent advances in single-cell-omics have been transformative to characterise cell types in challenging to study biological contexts. It is technically difficult to infer transcription factor-gene regulatory interactions from these datasets, especially in contexts with limited single-cell sample number such as the early human embryo. Here we systematically assessed the application of four different linear or non-linear gene regulatory network (GRN) prediction strategies to single-cell simulated and human embryo transcriptome datasets. We also compared how gene expression normalisation methods impact on regulatory network predictions, finding that TPM (transcripts per million reads) outperformed alternative methods. We identified more reproducible GRN inferences using a non-linear method based on mutual information (MI) applied to single-cell transcriptome datasets refined with chromatin accessibility (CA) (called MICA), compared to alternative network prediction methods tested. MICA captures complex non-monotonic dependencies and feedback loops. Using MICA, we generated the first GRN inferences in early human development. MICA predicted co-localisation of the AP-1 transcription factor subunit proto-oncogene JUND and the TFAP2C transcription factor AP-2{psi} in human preimplantation embryos. These interactions were supported by previous findings in other developmental and stem cell contexts. Overall, our comparative analysis of gene regulatory network prediction methods defines a pipeline that can be applied to single-cell multi-omics datasets in especially challenging contexts to infer interactions between transcription factor expression and target gene regulation.

developmental biology↗

bioRxiv2022

A conserved role of Hippo signaling in initiation of the first lineage specification event across mammals

Our understanding of the molecular events driving cell specification in early mammalian development relies mainly on mouse studies, and it remains unclear whether these mechanisms are conserved across mammals, including humans. We have recently shown that the establishment of cell polarity via aPKC is a conserved event in the initiation of the trophectoderm (TE) placental program in mouse, cow, and human embryos. However, the molecular mechanisms transducing cell polarity into cell fate in cow and human embryos is unknown. Here, we have examined the evolutionary conservation of the molecular cascade downstream of aPKC in four different mammalian species: mouse, rat, cow, and human. Surprisingly, by morphokinetic and immunofluorescence analyses, we observe that rat embryos more closely recapitulate human and cow developmental dynamics, in comparison to the mouse. Nevertheless, in all four species, inhibition of the Hippo pathway by targeting LATS kinases is sufficient to drive ectopic TE initiation and downregulation of SOX2, a marker of the inner cell mass. Our comparative embryology approach uncovered intriguing differences as well as similarities in a fundamental developmental process among mammals, reinforcing the importance of cross-species investigations.

developmental biology↗

bioRxiv2021

Direct reprogramming of human embryonic to trophoblast stem cells

During the first week of development, human embryos form a blastocyst comprised of an inner cell mass and trophectoderm (TE) cells, the latter of which are progenitors of placental trophoblast. Here we investigated the expression of transcripts in the human TE from early to late blastocyst stages. We identified enrichment of transcription factors GATA2, GATA3, TFAP2C and KLF5 and characterised their protein expression dynamics across TE development. By inducible overexpression and mRNA transfection we determined that these factors, together with MYC, are sufficient to establish induced trophoblast stem cells (iTSCs) from primed human embryonic stem cells. These iTSCs self-renew and recapitulate morphological characteristics, gene expression profiles, and directed differentiation potential similar to existing human TSCs. Systematic omission of each, or combinations of factors, revealed the critical importance of GATA2 and GATA3 for iTSC transdifferentiation. Altogether, these findings provide insights into the transcription factor network that may be operational in the human TE and broaden the methods for establishing cellular models of early human placental progenitor cells, which may be useful in the future to model placental-associated diseases. Summary statementTranscriptional analysis of human blastocysts reveals transcription factors sufficient to derive induced trophoblast stem cells from primed human embryonic stem cells.

developmental biology↗

bioRxiv2020

Potentiating TMEM16A channel function has no effect on airway goblet cells or bronchial and pulmonary vascular smooth muscle function

The calcium-activated chloride channel TMEM16A enables chloride secretion across several transporting epithelia, including in the airway where it represents a therapeutic target for the treatment of cystic fibrosis. Additional roles for TMEM16A have also been proposed, including enhancing goblet cell exocytosis, increasing goblet cell numbers and stimulating smooth muscle contraction. The aim of the present study was to test whether the pharmacological regulation of TMEM16A channel function, both potentiation and inhibition, could affect any of these proposed biological roles. In vitro, a recently described potent and selective TMEM16A potentiator (ETX001) failed to stimulate mucin release from primary human bronchial epithelial (HBE) cells over a 24h exposure period using both biochemical and imaging endpoints. In addition, treatment of HBE cells with ETX001 or a potent and selective TMEM16A inhibitor (Ani9) for 4 days did not influence mucin release or goblet cell formation. In vivo, a TMEM16A potentiator was without effect on goblet cell emptying in an IL-13 driven goblet cell metaplasia model. Using freshly isolated human bronchi and pulmonary arteries, neither ETX001 or Ani9 had any effect on the contractile or relaxant responses of the tissues. In vivo, ETX001 also failed to influence either lung or cardiovascular function when delivered directly into the airways of telemetered rats. Together, these studies do not support a role for TMEM16A in the regulation of goblet cell numbers or mucin release, or on the regulation of airway or pulmonary artery smooth muscle contraction.

physiology↗