Biology subjects

Puceat, M.

Publications and source records attributed to Puceat, M..

7 recordsLinked to original sources

bioRxiv2022

A single cell transcriptomic and clonal analysis depicts valvulogenesis

Congenital heart malformations often include mitral valve defects which remain largely unexplained. During embryogenesis, a restricted population of endocardial cells within the atrioventricular canal (AVC) undergoes endothelial to mesenchymal transition (EndMT) to give rise to mitral valvular cells. However, the identity, fate decisions of these progenitors as well as the distribution of their derivatives in valve leaflets remain unknown. Here, we use scRNA-seq of genetically labeled mouse AVC endocardial cells and of micro-dissected embryonic and postnatal mitral valves to characterize the developmental road. We uncovered the genetic, cell signaling and metabolic processes underlying specification of the progenitors and how they contribute to subtypes of endothelial and interstitial embryonic and postnatal valvular cells. Using clonal genetic tracing with multicolor reporter, we describe specific modes of growth of endocardial cell-derived clones which build up in a proper manner functional valve leaflets. Our data reveal how both genetic and metabolic specification mechanisms specifically drive the fate of a subset of endocardial cells toward valve progenitors and their distinct clonal contribution to the formation of the valve.

developmental biology↗

bioRxiv2022

Inhibition of TGFB pathway prevents short body size and cardiac defects in Nipbl-deficient mice, a mouse model of Cornelia de Lange syndrome

Cornelia de Lange Syndrome (CdLS) largely caused by mutation of the cohesin loader NIPBL is a rare developmental disorder affecting the formation of many organs. Besides a short body size and neurological defects, more than half of CdLS children feature various cardiac malformations. To mimic the disease and test a therapeutic strategy, we generated a C57/Bl6 Nipbl+/- mouse model of the disease. These mice featured a severe delay in both embryonic and postnatal growth. The Nipbl-deficient embryonic and neonatal hearts developed ventricular hypertrophy, aortic and valve defects associated with a persistent truncus arteriosus and a ventricular septal defect. Muscles derived from the second heart field were deficient in the Nipbl haplo-insufficient mouse embryos. The adult hearts then featured a severe aortic senescence phenotype and a stenosis resulting in an increase in aortic flux velocity and persistent left ventricular hypertrophy. Using proteomics and RNA-sequencing in embryos, we identified a dysregulated TGF{beta} pathway in the outflow tract of embryonic hearts as well as the presence of senescent cells as early as in E13.5 Nipbl+/- embryonic hearts, limb primordium cartilage as well as in different post-natal tissues including muscle and brain cortex. Treatment of pregnant Nipbl+/- mice with a TGF{beta}R (ALK5) inhibitor from E9.5 to E13.5 prevented cell -senescence and rescued the cardiac phenotype as well as the body size of mice at birth. Altogether our data revealed that an exacerbated TGF{beta} pathway associated with cell senescence is at the origin of many defects in a CdL mouse model. This druggable pathway opens the path toward a potential preventive and/or therapeutic strategy for post-natal CdLS patients.

developmental biology↗

bioRxiv2020

Maternal vitamin D deficiency impairs heart formation in mouse offspring through a change in 3D-chromatin structure

The origins of congenital heart diseases, the most common congenital diseases are still largely unknown. Environmental factors are now emerging as major causes of these diseases. Vitamin D deficiency has become a public health burden, notably for childbearing age, pregnant and breastfeeding women. Since maternal 25-hydroxyvitamin D (25(OH)D) determined fetal and neonatal 25(OH)D status, foetuses exposed to insufficient levels of vitamin D, may feature developmental defects. Herein, we investigated the effects of maternal vitamin D deficiency on cardiovascular defects in early and later life of offsprings in two generations as well as the molecular mechanisms underlying vitamin D effect. Eight weeks before and during pregnancy, C57BL/6JRj female mice received a sufficient or vitamin D deficient diet ((1.0 IU/g in control vs 0.0 IU/g in Vitamin D Deficient (VDD) group). E16.5 Embryos of maternal VDD diet featured hypertrophic heart revealed by a thicker left ventricular (LV) wall and septum. RNAseq analysis of LV revealed 1555 transcripts differentially expressed in the VDD group and among them cardiac transcription factors and constitutive cardiac genes (tbx5, gata4, myl2). Anti-Vitamin D receptor (VDR) Chip-seq from chromatin of E16.5 LV uncovered different targeting of tbx5 and tbx3 loci by VDR in the VDD vs control embryos. Anti-CTCF ChIP-loop experiments focusing on the Tbx3 and Tbx5 loci uncovered a change in the Topology Associated Domains associated with these loci. Echocardiography of 2-months-old VDD offspring revealed a significantly thicker left ventricle and increased fractional shortening while 6-months-old mice featured cardiac decompensation and in turn failing LV. Maternal vitamin D deficiency severely affects heart formation following a change in chromatin conformation on cardiac gene loci and impacts function of adult hearts in two generations. These defects are likely to be at the origin of cardiovascular diseases in the adulthood.

developmental biology↗

bioRxiv2020

SCHNAPPs - Single Cell sHiNy APPlication(s)

MotivationSingle-cell RNA-sequencing (scRNAseq) experiments are becoming a standard tool for bench-scientists to explore the cellular diversity present in all tissues. On one hand, the data produced by scRNASeq is technically complex, with analytical workflows that are still very much an active field of bioinformatics research, and on the other hand, a wealth of biological background knowledge is often needed to guide the investigation. Therefore, there is an increasing need to develop applications geared towards bench-scientists to help them abstract the technical challenges of the analysis, so that they can focus on the Science at play. It is also expected that such applications should support closer collaboration between bioinformaticians and bench-scientists by providing reproducible science tools. ResultsWe present SCHNAPPs, a computer program designed to enable bench-scientists to autonomously explore and interpret single cell RNA-seq expression data and associated annotations. The Shiny-based application allows selecting genes and cells of interest, performing quality control, normalization, clustering, and differential expression analyses, applying standard workflows from Seurat (Stuart et al., 2019) or Scran (Lun et al., 2016) packages, and most of the common visualizations. An R-markdown report can be generated that tracks the modifications, and selected visualizations facilitating communication and reproducibility between bench-scientist and bioinformatician. The modular design of the tool allows to easily integrate new visualizations and analyses by bioinformaticians. We still recommend that a data analysis specialist oversees the analysis and interpretation. AvailabilityThe SCHNAPPs application, docker file, and documentation are available on GitHub: https://c3bi-pasteur-fr.github.io/UTechSCB-SCHNAPPs; Example contribution are available at the following GitHub site: https://github.com/baj12/SCHNAPPsContributions.

bioinformatics↗

bioRxiv2020

Hox-dependent coordination of cardiac progenitor cell patterning and differentiation

Perturbation of addition of second heart field (SHF) cardiac progenitor cells to the poles of the heart tube results in congenital heart defects (CHD). The transcriptional programs and upstream regulatory events operating in different subpopulations of the SHF remain unclear. Here, we profile the transcriptome and chromatin accessibility of anterior and posterior SHF sub-populations at genome-wide levels and demonstrate that Hoxb1 negatively regulates differentiation in the posterior SHF. Spatial mis-expression of Hoxb1 in the anterior SHF results in hypoplastic right ventricle. Activation of Hoxb1 in embryonic stem cells arrests cardiac differentiation, whereas Hoxb1-deficient embryos display premature cardiac differentiation. Moreover, ectopic differentiation in the posterior SHF of embryos lacking both Hoxb1 and its paralog Hoxa1 results in atrioventricular septal defects. Our results show that Hoxb1 plays a key role in patterning cardiac progenitor cells that contribute to both cardiac poles and provide new insights into the pathogenesis of CHD.

developmental biology↗

bioRxiv2018

Direct reprogramming of human epithelial cells into organoids by miR-106a-3p

The 3D cultures provide more insight into cell-to-cell and cell-to-matrix interactions, better mimicking the environment where stem cells reside compared to traditional 2D cultures. Although the precise molecular pathways involved in the regulation of stem and progenitor cell fate remain unknown, it is widely accepted that transcription factors play a crucial role as intrinsic regulators in these fate decisions. In this study, we carried out a microRNA screen to track the behaviour of adult stem/progenitor cells derived from human mammary epithelial cells grown in 3D cultures. We identified miR-106a-3p, which enriches the adult stem cell-like lineage and promotes the expansion of 3D cultures. Transcriptomic analysis showed that this miRNA regulates transcription factors such as REST, CBFB, NF-YA, and GATA3, thereby enhancing the maintenance of adult stem/progenitor cells in human epithelial cells. These data reveal a clear transcriptional program that governs the maintenance of adult stem/progenitor cells and controls their fate.

cell biology↗

bioRxiv2017

Geometrical confinement guides Brachyury self-patterning in embryonic stem cells.

During embryogenesis, signaling molecules initiate cell diversification, sometimes via stochastic processes, other times via the formation of long range gradients of activity which pattern entire fields of cells. Such mechanisms are not insensitive to noise (Lander, 2011), yet embryogenesis is a remarkably robust process suggesting that multiple layers of regulations secure patterning during development. In the present study, we present a proof of concept according to which an asymmetric pattern of gene expression obtained from a spatially disorganised population of cells can be guided by the geometry of the environment in a reproducible and robust manner. We used ESC as a model system whithin which multiple developmental cell states coexist (MacArthur and Lemischka, 2013; Smith, 2017; Torres-Padilla and Chambers, 2014). We first present evidence that a reciprocal regulation of genes involved in the establishment of antero-posterior polarity during peri-implantation stages of mouse development is spontaneously occuring within ESC. We then show that a population of cells with primitive streak characteristics localise in regions of high curvature and low cell density. Finally, we show that this patterning did not depend on self-organised gradients of morphogen activity but instead could be attributed to positional rearrangements. Our findings unveil a novel role for tissue geometry in guiding the self-patterning of primitive streak cells and provide a framework to further refine our understanding of symmetry breaking events occuring in ESC aggregates. Finally, this work demonstrates that the self-patterning of a specific population of ESC, Brachyury positive cells in this case, can be directed by providing engineered external geometrical cues.

developmental biology↗