Biology subjects

Lancrin, C.

Publications and source records attributed to Lancrin, C..

9 recordsLinked to original sources

bioRxiv2024

Genetic gradual reduction of OGT activity unveils the essential role of O-GlcNAc in the mouse embryo

The reversible glycosylation of nuclear and cytoplasmic proteins (O-GlcNAcylation) is catalyzed by a single enzyme, namely O-GlcNAc transferase (OGT). The mammalian Ogt gene is X-linked and it is essential for embryonic development and for the viability of proliferating cells. We perturbed OGTs function in vivo by creating a murine allelic series of four single amino acid substitutions reducing OGTs catalytic activity to a range of degrees. The severity of the embryonic lethality was proportional to the degree of impairment of OGTs catalysis, demonstrating that the O-GlcNAc modification itself is required for early development. We identified milder hypomorphic Ogt alleles that perturb O-GlcNAc homeostasis while being compatible with embryogenesis. The analysis of the transcriptomes of the mutant embryos at different stages suggested a sexually-dimorphic developmental delay caused by the decrease in O-GlcNAc. Furthermore, a mild reduction of OGTs enzymatic activity was sufficient to loosen the silencing of endogenous retroviruses in vivo.

developmental biology↗

bioRxiv2024

Targeted CRISPR-Cas9 screening identifies transcription factor network controlling murine haemato-endothelial fate commitment

Haematopoiesis is a tightly coordinated process that forms and maintains all blood cells. During development blood generation begins in the yolk sac with the differentiation of haemato-endothelial mesoderm giving rise to haematopoietic progenitors. Which molecular regulators are crucial for haemato-endothelial mesoderm formation remains unclear and has not been studied in an unbiased way. Here we employ a mouse embryonic stem cell model that recapitulates embryonic blood development and perform targeted CRISPR-Cas9 knock out screens focusing on transcription factors and chromatin regulators. Focusing on the transition of primitive towards haematoendothelial mesoderm we identified the known master regulator Etv2 and novel transcription factors including Smad1, Ldb1, Six4 and Zbtb7b acting as crucial drivers or repressors of mesodermal commitment. Our transcriptome analysis highlights that each factor has a precise impact on the gene expression signature of the developing mesoderm resulting in the formation of mesodermal subsets with a defined lineage differentiation bias. Our study reveals novel molecular pathways governing mesodermal development crucial to allow endothelial and haematopoietic lineage specification.

developmental biology↗

bioRxiv2021

Identifying a novel role for the master regulator Tal1 in the Endothelial to Hematopoietic Transition

Recent progress in the generation of bona-fide Hematopoietic Stem and Progenitor Cells (HSPCs) in vitro and ex vivo has been built on the knowledge of developmental hematopoiesis, underscoring the importance of understanding in detail this developmental process. Here, we sought to elucidate the function of the hematopoietic regulators Tal1, Lmo2 and Lyl1 in the Endothelial to Hematopoietic Transition (EHT), the process through which HSPCs are generated from endothelial precursors during embryogenesis. We used a mouse embryonic-stem cell (mESC)-based differentiation system to model hematopoietic development, and combined gain-of-function experiments in sorted vascular smooth muscle cells (VSM) with multi-omics to obtain mechanistic insights into the mode of action of Tal1, Lmo2 and Lyl1. We found that these factors promote the silencing of the VSM transcriptional program and the activation of the hematopoietic one. Through this approach and the use of a Tet-on system to control the expression of Tal1 during hematopoietic specification from mESCs, we discovered that its expression in endothelial cells is crucial for the EHT to occur.

developmental biology↗

bioRxiv2021

Single-cell transcriptome analysis of embryonic and adult endothelial cells allows to rank the hemogenic potential of post-natal endothelium

Hematopoietic stem cells are crucial for the continuous production of blood cells during life. The transplantation of these cells is one of the most common treatments to cure patient suffering of blood diseases. However, the lack of suitable donors is a major limitation. One option to get hematopoietic stem cells matching perfectly a patient is cellular reprogramming. Hematopoietic stem cells emerge from endothelial cells in blood vessels during embryogenesis through the endothelial to hematopoietic transition. Here, we used single-cell transcriptomics analysis to compare embryonic and post-natal endothelial cells to investigate the potential of adult vasculature to be reprogrammed in hematopoietic stem cells. Although transcriptional similarities have been found between embryonic and adult endothelial cells, we found some key differences in term of transcription factors expression. There is a deficit of expression of Runx1, Tal1, Lyl1 and Cbfb in adult endothelial cells compared to their embryonic counterparts. Using a combination of gene expression profiling and gene regulatory network analysis, we found that endothelial cells from the pancreas, brain, kidney and liver appear to be the most suitable targets for cellular reprogramming into hematopoietic stem cells. Overall, our work provides an important resource for the rational design of a reprogramming strategy for the generation of hematopoietic stem cells.

developmental biology↗

bioRxiv2020

Operation of a TCA cycle subnetwork in the mammalian nucleus

Nucleic acid and histone modifications critically depend on central metabolism for substrates and co-factors. Although a few enzymes related to the formation of these required metabolites have been reported in the nucleus, the corresponding metabolic pathways are considered to function elsewhere in the cell. Here we show that a substantial part of the mitochondrial tricarboxylic acid (TCA) cycle, the biosynthetic hub of epigenetic modification factors, is operational also in the nucleus. Using 13C-tracer analysis, we identified activity of glutamine-to-fumarate, citrate-to-succinate, and glutamine-to-aspartate routes in the nuclei of HeLa cells. Proximity labeling mass-spectrometry revealed a spatial vicinity of the involved enzymes with core nuclear proteins, supporting their nuclear location. We further show nuclear localization of aconitase 2 and 2-oxoglutarate dehydrogenase in mouse embryonic stem cells. Together, our results demonstrate operation of an extended metabolic pathway in the nucleus warranting a revision of the canonical view on metabolic compartmentalization and gene expression regulation.

systems biology↗

bioRxiv2019

Single-cell atlas of major haematopoietic tissues sheds light on blood cell formation from embryonic endothelium

The Yolk Sac (YS) and Aorta-Gonad-Mesonephros (AGM) are two major haematopoietic regions during embryonic development. Interestingly, AGM is the only one generating haematopoietic stem cells (HSCs). To identify the difference between AGM and YS, we compared them using single-cell RNA sequencing between 9.5 and 11.5 days of mouse embryonic development and identified cell populations using CONCLUS, a new computational tool. The AGM was the only one containing neurons and a specific mesenchymal population, while the YS major component was an epithelial population expressing liver marker genes. In addition, the YS contained a major endothelial population expressing Stab2, a hyaluronan receptor, also highly expressed by liver endothelium. We demonstrated that the YS haematopoietic potential was restricted to Stab2-negative cells and that ectopic expression of Stab2 could reduce blood cell formation from endothelium. Our results indicate that the AGM is a tissue more favourable to HSCs development than the YS because of its microenvironment and the nature of its endothelial cells.

developmental biology↗

bioRxiv2018

Iron deficiency affects early stages of embryonic hematopoiesis but not the endothelial to hematopoietic transition

Iron is an essential micronutrient for hematopoiesis and previous research suggested that iron deficiency in the pregnant female could cause anemia in the offspring. Since the development of all embryonic and adult blood cells begins in the embryo, we aimed to resolve the role of iron in embryonic hematopoiesis. For this purpose, we used an experimental system of mouse embryonic stem cells differentiation into embryonic hematopoietic progenitors. We modulated the iron status in cultures by adding either an iron chelator DFO for iron deficiency, or ferric ammonium citrate for iron excess, and followed the emergence of developing hematopoietic progenitors by flow cytometry. We found interestingly that iron deficiency by DFO did not block the endothelial to hematopoietic transition, the first step of hematopoiesis. However, it had a differential effect on the proliferation, survival and clonogenic capacity of hematopoietic progenitors. Surprisingly, iron deficiency affected erythro-myeloid Kitpos CD41+ progenitors significantly more than the primitive erythroid Kitneg CD41+. The Kitpos progenitors paradoxically died more, proliferated less and had more reduction in colony formation than Kitneg after 24 hours of DFO treatment. Kitpos progenitors expressed less transferrin-receptor on the cell surface and had less labile iron compared to Kitneg, which could reduce their capacity to compete for scarce iron and survive iron deficiency. We suggest that iron deficiency could disturb hematopoiesis already at an early embryonic stage by compromising survival, proliferation and differentiation of definitive hematopoietic progenitors.

developmental biology↗

bioRxiv2018

Single-cell transcriptomics identifies CD44 as a new marker and regulator of haematopoietic stem cells development

The endothelial to haematopoietic transition (EHT) is the process whereby haemogenic endothelium differentiates into haematopoietic stem and progenitor cells (HSPCs). The intermediary steps of this process are unclear, in particular the identity of endothelial cells that give rise to HSPCs is unknown. Using single-cell transcriptome analysis and antibody screening we identified CD44 as a new marker of EHT enabling us to isolate robustly the different stages of EHT in the aorta gonad mesonephros (AGM) region. This allowed us to provide a very detailed phenotypical and transcriptional profile for haemogenic endothelial cells, characterising them with high expression of genes related to Notch signalling, TGFbeta/BMP antagonists (Smad6, Smad7 and Bmper) and a downregulation of genes related to glycolysis and the TCA cycle. Moreover, we demonstrated that by inhibiting the interaction between CD44 and its ligand hyaluronan we could block EHT, identifying a new regulator of HSPC development.

developmental biology↗

bioRxiv2018

Discovery of a new path for red blood cell generation in the mouse embryo

Erythropoiesis occurs through several waves during embryonic development. Although the source of the primitive wave is well characterized, the origin of erythrocytes later in embryogenesis is less clear due to overlaps between the different erythroid waves. Using the miR144/451-GFP mouse model to track cells expressing the erythroid microRNAs miR144/451, we identified cells co-expressing VE-Cadherin and GFP in the yolk sac between E9.5 and E12. This suggested the existence of hemogenic endothelial cells committed to erythropoiesis (Ery-HEC). We showed that these cells were capable of generating erythrocytes ex vivo and we demonstrated that the formation of Ery-HEC was independent of the Runx1 gene expression. Using transcriptome analysis, we demonstrated that these cells coexpressed endothelial and erythroid genes such as Hbb-bh1 and Gata1 but we were surprised to detect the primitive erythroid genes Aqp3 and Aqp8 suggesting the formation of primitive erythrocytes at a much later time point than initially thought. Finally, we showed that enforced expression of Gata1 in endothelial cells was enough to initiate the erythroid transcriptional program.

developmental biology↗