The role of functional calcium handling during the early stages of mouse heart development

During development the heart is the first organ to form and function in the embryo proper. Along with being fundamental for contraction, Ca2+ is also a key signalling molecule known to regulate cardiac genes, however, it is unclear how Ca2+-handling feeds-back onto the initiation of beating and whether this directly impacts on early embryonic heart development per se. The aim of this study was to investigate how initial contractions of the early mouse heart are established and what the downstream consequences of function are on cardiac differentiation and heart development. Using ex vivo Ca2+ imaging we found evidence of randomly distributed Ca2+ transients in the forming cardiac crescent, prior to contraction, suggesting early Ca2+ handling is essential for cardiomyocyte differentiation and subsequent heart development. To study the downstream effects of early Ca2+ we used a murine embryonic stem cell (mESC) model of cardiomyocyte differentiation which recapitulated heart development in vivo. Assessment of Ca2+ handling proteins indicated that the Na+-Ca2+ exchanger (NCX) was one of the earliest sarcolemmal transporters to be expressed, prior to the L-type Ca2+ channel (LTCC). Pharmacological inhibition of NCX revealed an essential early role in establishing and maintaining the first Ca2+ transients through to initiation of contraction; a role superseded by the LTCC as differentiation progressed. Upon NCX blockade cardiomyocyte differentiation was inhibited, coincident with the down-regulation of signature cardiac genes and calmodulin kinase signalling, which culminated in the failure of beating cardiomyocytes to form. This study points to a novel mechanism by which form and function are intricately linked such that, from the outset, Ca2+ acts to initiate contraction as well as regulate cardiac differentiation and the formation of the heart, adding an important layer onto our current understanding of mammalian cardiovascular development

Calcium handling precedes cardiac differentiation to initiate the first heartbeat

The mammalian heartbeat is thought to begin just prior to the linear heart tube stage of development. How the initial contractions are established and the downstream consequences of the earliest contractile function on cardiac differentiation and morphogenesis have not been described. Using high-resolution live imaging of mouse embryos, we observed randomly distributed spontaneous asynchronous Ca2+-oscillations (SACOs) in the forming cardiac crescent (stage E7.75) prior to overt beating. Nascent contraction initiated at around E8.0 and was associated with sarcomeric assembly and rapid Ca2+ transients, underpinned by sequential expression of the Na+-Ca2+ exchanger (NCX1) and L-type Ca2+ channel (LTCC). Pharmacological inhibition of NCX1 and LTCC revealed rapid development of Ca2+ handling in the early heart and an essential early role for NCX1 in establishing SACOs through to the initiation of beating. NCX1 blockade impacted on CaMKII signalling to down-regulate cardiac gene expression, leading to impaired differentiation and failed crescent maturation

Single-cell RNAseq profiling of early heart developmental physiology

The heart is the first organ to form and function during mammalian development. Recently we established that in the mouse embryo, spontaneous asynchronous calcium oscillations (SACOs) occour in the cardiac crescent prior to the onset of cardiac contractions. Blocking Sodium Calcium Exchanger (NCX1) function inhibits these oscillations and cardiac differentiation

Calcium handling precedes cardiac differentiation to initiate the first heart beat

The mammalian heartbeat is thought to begin just prior to the linear heart tube stage of development. How the initial contractions are established and the downstream consequences of the earliest contractile function on cardiac differentiation and morphogenesis have not been described. Using high-resolution live imaging of mouse embryos, we observed randomly distributed spontaneous asynchronous Ca2+-oscillations (SACOs) in the forming cardiac crescent (stage E7.75) prior to overt beating. Nascent contraction initiated at around E8.0 and was associated with sarcomeric assembly and rapid Ca2+ transients, underpinned by sequential expression of the Na+-Ca2+ exchanger (NCX1) and L-type Ca2+ channel (LTCC). Pharmacological inhibition of NCX1 and LTCC revealed rapid development of Ca2+ handling in the early heart and an essential early role for NCX1 in establishing SACOs through to the initiation of beating. NCX1 blockade impacted on CaMKII signalling to down-regulate cardiac gene expression, leading to impaired differentiation and failed crescent maturation

Integration of spatial and single-cell transcriptomic data elucidates mouse organogenesis.

Funder: EMBL International PhD ProgrammeFunder: Support from the Francis Crick Institute, which receives core funding from CRUK, the UK Medical Research Council, and the Wellcome Trust (all under FC001051).Funder: Core support from the MRC and the Wellcome Trust to the Wellcome-MRC Cambridge Stem Cell Institute.Funder: Paul G Allen Frontiers Foundation Discovery Center for Cell Lineage Tracing (grant UWSC10142).Funder: Core funding from EMBLMolecular profiling of single cells has advanced our knowledge of the molecular basis of development. However, current approaches mostly rely on dissociating cells from tissues, thereby losing the crucial spatial context of regulatory processes. Here, we apply an image-based single-cell transcriptomics method, sequential fluorescence in situ hybridization (seqFISH), to detect mRNAs for 387 target genes in tissue sections of mouse embryos at the 8-12 somite stage. By integrating spatial context and multiplexed transcriptional measurements with two single-cell transcriptome atlases, we characterize cell types across the embryo and demonstrate that spatially resolved expression of genes not profiled by seqFISH can be imputed. We use this high-resolution spatial map to characterize fundamental steps in the patterning of the midbrain-hindbrain boundary (MHB) and the developing gut tube. We uncover axes of cell differentiation that are not apparent from single-cell RNA-sequencing (scRNA-seq) data, such as early dorsal-ventral separation of esophageal and tracheal progenitor populations in the gut tube. Our method provides an approach for studying cell fate decisions in complex tissues and development

Integration of spatial and single-cell transcriptomic data elucidates mouse organogenesis.

Funder: EMBL International PhD ProgrammeFunder: Support from the Francis Crick Institute, which receives core funding from CRUK, the UK Medical Research Council, and the Wellcome Trust (all under FC001051).Funder: Core support from the MRC and the Wellcome Trust to the Wellcome-MRC Cambridge Stem Cell Institute.Funder: Paul G Allen Frontiers Foundation Discovery Center for Cell Lineage Tracing (grant UWSC10142).Funder: Core funding from EMBLMolecular profiling of single cells has advanced our knowledge of the molecular basis of development. However, current approaches mostly rely on dissociating cells from tissues, thereby losing the crucial spatial context of regulatory processes. Here, we apply an image-based single-cell transcriptomics method, sequential fluorescence in situ hybridization (seqFISH), to detect mRNAs for 387 target genes in tissue sections of mouse embryos at the 8-12 somite stage. By integrating spatial context and multiplexed transcriptional measurements with two single-cell transcriptome atlases, we characterize cell types across the embryo and demonstrate that spatially resolved expression of genes not profiled by seqFISH can be imputed. We use this high-resolution spatial map to characterize fundamental steps in the patterning of the midbrain-hindbrain boundary (MHB) and the developing gut tube. We uncover axes of cell differentiation that are not apparent from single-cell RNA-sequencing (scRNA-seq) data, such as early dorsal-ventral separation of esophageal and tracheal progenitor populations in the gut tube. Our method provides an approach for studying cell fate decisions in complex tissues and development