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Biology subjects

Proks, M.

Publications and source records attributed to Proks, M..

7 recordsLinked to original sources

Deep Learning Based Models for Preimplantation Mouse and Human Development

The rapid growth of single-cell transcriptomic technology has produced an increasing number of datasets for both embryonic development and in vitro pluripotent stem cell derived models. This avalanche of data about pluripotency and the process of lineage specification has meant it has become increasingly difficult to define specific cell types or states and compare these to in vitro differentiation. Here we utilize a set of deep learning (DL) tools to integrate and classify multiple datasets. This allows for the definition of both mouse and human embryo cell types, lineages and states, thereby maximising the information one can garner from these precious experimental resources. Our approaches are built on recent initiatives for large scale human organ atlases, but here we focus on the difficult to obtain and process material that spans early mouse, and in particular, human development. Using publicly available data for these stages, we test different deep learning approaches and develop both a model to classify cell types in an unbiased fashion and define the set of genes required to identify lineages, cell types and states. We have used our predictions to probe pluripotent stem cell models for both mouse and human development, showcasing the importance of this resource as a dynamic reference for early embryogenesis.

developmental biology↗

nf-core/marsseq: systematic pre-processing pipeline for MARS-seq experiments

MotivationAs a result of advancing single sequencing technology (scRNA-seq), it has become possible to study gene regulatory mechanism(s) and their influence on evolving cell states in time at the level of individual cells. Since 2009, numerous scRNA-seq protocols have been developed, each with its own advantages, disadvantages and library preparation complexities (Ziegenhain et al. 2017). However, the interpretation of data arising from these techniques often shares similar limitations, such as the lack of a standardized pre-processing workflow and consistent data reproducibility. Here we focus on the standardization of the plate based Massively Parallel RNA Single cell Sequencing (MARS-seq, Jaitin et al. 2014) pre-processing pipeline as described in MARS-seq2.0 (Keren-Shaul et al. 2019), which was developed at the Weizmann Institute of Science. ResultsTo overcome the limitations mentioned above, we have taken the original MARS-seq2.0 pipeline and revised it to enable implementation using the nf-core framework (Ewels et al. 2020). By doing so, we have simplified pipeline execution enabling streamlined application, with increased transparency and scalability. Additionally, we have further improved the pipeline by implementing a custom workflow for RNA velocity estimation. Availability and implementationThe pipeline is part of the nf-core bioinformatics community and is freely available at https://github.com/nf-core/marsseq with data analysis at https://github.com/brickmanlab/proks-et-al-2023.

bioinformatics↗

Enhancer status in the primitive endoderm supports unrestricted lineage plasticity in regulative development

Mammalian blastocyst formation involves the specification of trophectoderm followed by the differentiation of the inner cell mass into either epiblast or primitive endoderm. During this time, the embryo maintains a window of plasticity and can redirect its cellular fate when challenged experimentally. In this context, we found that the primitive endoderm alone was sufficient to regenerate a complete blastocyst and continue normal postimplantation development to term. We identify an in vitro population similar to the early primitive endoderm in vivo, that exhibits the same embryonic and extra-embryonic potency, forming three dimensional embryoid structures. Commitment in early primitive endoderm is suppressed by JAK/STAT signalling, collaborating with OCT4 to safeguard enhancer status enabling multi-lineage differentiation. Our observations support the notion that transcription factor persistence underlies plasticity in regulative development and highlights the importance of primitive endoderm in perturbed development.

developmental biology↗

Transcription Factor Co-Expression Mediates Lineage Priming for Embryonic and Extra-Embryonic Differentiation

In early mammalian development, cleavage stage blastomeres and cells of the inner cell mass (ICM) of the blastocyst co-express embryonic and extra-embryonic transcriptional determinants. Using a double protein-based reporter we identify embryonic stem cells (ESC) that co-express the extra-embryonic factor GATA6 alongside the embryonic factor SOX2 in specific conditions. Based on single cell transcriptomics we find these population resemble unsegregated ICM, exhibiting enhanced differentiation potential for endoderm while maintaining epiblast competence and suggesting they represent an ideal model to determine how GATA6 and SOX2 influence each others DNA binding. To relate this binding to future fate, we describe a complete enhancer set in both ESCs and naive extraembryonic endoderm stem cells and ask whether SOX2 and GATA6 recognize these elements in ICM-like ESC sub-population. Both factors support cooperative recognition in these lineages, with GATA6 bound alongside SOX2 on a fraction of pluripotency enhancers and SOX2 alongside GATA6 more extensively on endoderm enhancers. Our findings suggest that cooperative binding between these antagonistic factors both supports self-renewal and prepares progenitor cells for later differentiation.

developmental biology↗

A bipartite function of ESRRB can integrate signaling over time to balance self-renewal and differentiation

Cooperative DNA binding of transcription factors (TFs) integrates external stimuli and context across tissues and time. Naive mouse embryonic stem cells are derived from early development and can sustain the pluripotent identity indefinitely. Here we ask whether TFs associated with pluripotency evolved to directly support this state, or if the state emerges from their combinatorial action. NANOG and ESRRB are key pluripotency factors that co-bind DNA. We find that when both factors are expressed, ESRRB supports pluripotency. However, when NANOG is not present, ESRRB supports a bistable culture of cells with an embryo-like primitive endoderm identity ancillary to pluripotency. The stoichiometry between NANOG and ESRRB quantitatively influences differentiation, and in silico modeling of bipartite TF activity suggests ESRRB safeguards plasticity in differentiation. Thus, the concerted activity of cooperative TFs can transform their effect to sustain intermediate cell identities and allow ex vivo expansion of highly stable stem cell models.

cell biology↗

Expansion of Ventral Foregut Primes the Enhancer Landscape for Organ Specific Differentiation

Cell proliferation is fundamental for almost all stages of development and differentiation that require an increase cell number. Although cell cycle phase has been associated with differentiation, the actual process of proliferation is not seen as having a specific role. Here we exploit human embryonic stem cell derived endodermal progenitors that we find are an in vitro model for the ventral foregut. These cells exhibit expansion dependent increases in differentiation efficiency to pancreatic progenitors that are linked to organ-specific enhancer priming at the level of chromatin accessibility and the decommissioning of lineage inappropriate enhancers. Our findings suggest that cell proliferation in embryonic development is about more than tissue expansion, it is required to ensure equilibration of gene regulatory networks allowing cells to become primed for future differentiation. The use of expansion of lineage specific intermediates may therefore be an important step in high fidelity in vitro differentiation.

developmental biology↗

Transcriptional Heterogeneity and Cell Cycle Regulation as Central Determinants of Primitive Endoderm Priming.

During embryonic development cells acquire identity at the same time as they are proliferating, implying that an intrinsic facet of cell fate choice requires coupling lineage decisions to rates of cell division. How is the cell cycle regulated to promote or suppress heterogeneity and differentiation? We explore this question combining time lapse imaging with single cell RNA-seq in the contexts of self-renewal, priming and differentiation of embryonic stem cells (ESCs) towards the Primitive Endoderm lineage (PrE). Since ESCs are derived from the Inner Cell Mass of the mammalian blastocyst, ESCs in standard culture conditions are transcriptionally heterogeneous containing subfractions that are primed for either of the two ICM lineages, Epiblast and PrE. These subfractions represent dynamic states that can readily interconvert in culture, and the PrE subfraction is functionally primed for endoderm differentiation. Here we find that differential regulation of cell cycle can tip the balance between these primed populations, such that naive ESC culture conditions promote Epiblast-like expansion and PrE differentiation stimulates the selective proliferation of PrE-primed cells. In endoderm differentiation, we find that this change is accompanied by a counter-intuitive increase in G1 length that also appears replicated in vivo. While FGF/ERK signalling is a known key regulator of ESCs and PrE differentiation, we find it is not just responsible for ESCs heterogeneity, but also cell cycle synchronisation, required for the inheritance of similar cell cycles between sisters and cousins. Taken together, our results point to a tight relationship between transcriptional heterogeneity and cell cycle regulation in the context of lineage priming, with primed cell populations providing a pool of flexible cell types that can be expanded in a lineage-specific fashion while allowing plasticity during early determination.

developmental biology↗