Abstract 44: Interaction Of Osr1 And Tbx5 Is Involved In The Mouse Limb And Heart Development

Mutations of TBX5 cause Holt-Oram syndrome (HOS) in human, a disease characterized by upper limb and heart defects. Mouse embryos of Osr1 knockout caused similar heart defects, while the upper limb defects have never been reported. By genetically marking Osr1 expressing cells in mice, using Osr1:CreERT2 , we showed that Osr1 expression cells contribute to the atrial septum progenitors between E8.0 and E11.0, and to the forelimb after E9.0. The expression of Osr1 in the forelimb showed a gradient decreasing pattern from the digit 5 to digit 1. Conditional- Tbx5 haploinsuffiency, using Osr1:CreERT2 , compound with Osr1 haploinsuffiency induced more incidence of atrial septal defects (ASDs) and double outlet right ventricle (DORV). Forty percent of these embryos also had digit defects: the digits are either missing, fused or lack normal identity, which were not observed in mouse embryos of either Osr1 or Tbx5 haploinsuffiency. Detailed study of the cardiac progenitors of the compound haploinsufficinecy for Tbx5 and Osr1 showed decreased proliferation in the posterior second heart field, which was associated with lower number of cells transiting from G2 to M phase and less gene expression of Cdk6 and CyclinD2 . In summary, our study demonstrated that interaction of Osr1 and Tbx5 is involved in the mouse limb and heart development and provides a potential mechanism for HOS.

Download Full-text

Hey2 restricts cardiac progenitor addition to the developing heart

10.1101/199638 ◽

2017 ◽

Author(s):

Natalie Gibb ◽

Savo Lazic ◽

Ashish R. Deshwar ◽

Xuefei Yuan ◽

Michael D. Wilson ◽

...

Keyword(s):

Heart Development ◽

Heart Defects ◽

Myocardial Cell ◽

Cell Number ◽

Tube Formation ◽

Heart Tube ◽

Cardiac Progenitors ◽

Developing Heart ◽

Heart Field ◽

A Cell

ABSTRACTA key event in vertebrate heart development is the timely addition of second heart field (SHF) progenitor cells to the poles of the heart tube. This accretion process must occur to the proper extent to prevent a spectrum of congenital heart defects (CHDs). However, the factors that regulate this critical process are poorly understood. Here we demonstrate that Hey2, a bHLH transcriptional repressor, restricts SHF progenitor accretion to the zebrafish heart. hey2 expression demarcated a distinct domain within the cardiac progenitor population. In the absence of Hey2 function an increase in myocardial cell number and SHF progenitors was observed. We found that Hey2 limited proliferation of SHF-derived cardiomyocytes in a cell-autonomous manner, prior to heart tube formation, and further restricted the developmental window over which SHF progenitors were deployed to the heart. Taken together, our data suggests a role for Hey2 in controlling the proliferative capacity and cardiac contribution of late-differentiating cardiac progenitors.

Download Full-text

Tbx1 regulates extracellular matrix-cell interactions in the second heart field

Human Molecular Genetics ◽

10.1093/hmg/ddz058 ◽

2019 ◽

Vol 28 (14) ◽

pp. 2295-2308 ◽

Cited By ~ 10

Author(s):

Daniela Alfano ◽

Alessandra Altomonte ◽

Claudio Cortes ◽

Marchesa Bilio ◽

Robert G Kelly ◽

...

Keyword(s):

Extracellular Matrix ◽

Cultured Cells ◽

Time Window ◽

Outflow Tract ◽

Mouse Embryos ◽

Cell Interactions ◽

Deletion Syndrome ◽

Cardiac Progenitors ◽

Second Heart Field ◽

Heart Field

Abstract Tbx1, the major candidate gene for DiGeorge or 22q11.2 deletion syndrome, is required for efficient incorporation of cardiac progenitors of the second heart field (SHF) into the heart. However, the mechanisms by which TBX1 regulates this process are still unclear. Here, we have used two independent models, mouse embryos and cultured cells, to define the role of TBX1 in establishing morphological and dynamic characteristics of SHF in the mouse. We found that loss of TBX1 impairs extracellular matrix (ECM)-integrin-focal adhesion (FA) signaling in both models. Mosaic analysis in embryos suggested that this function is non-cell autonomous, and, in cultured cells, loss of TBX1 impairs cell migration and FAs. Additionally, we found that ECM-mediated integrin signaling is disrupted upon loss of TBX1. Finally, we show that interfering with the ECM-integrin-FA axis between E8.5 and E9.5 in mouse embryos, corresponding to the time window within which TBX1 is required in the SHF, causes outflow tract dysmorphogenesis. Our results demonstrate that TBX1 is required to maintain the integrity of ECM-cell interactions in the SHF and that this interaction is critical for cardiac outflow tract development. More broadly, our data identifies a novel TBX1 downstream pathway as an important player in SHF tissue architecture and cardiac morphogenesis.

Download Full-text

Ventricular, atrial and outflow tract heart progenitors arise from spatially and molecularly distinct regions of the primitive streak

10.1101/2020.07.12.198994 ◽

2020 ◽

Author(s):

Kenzo Ivanovitch ◽

Pablo Soro-Barrio ◽

Probir Chakravarty ◽

Rebecca A Jones ◽

S. Neda Mousavy Gharavy ◽

...

Keyword(s):

Single Cell ◽

Heart Development ◽

Primitive Streak ◽

Outflow Tract ◽

Lineage Tracing ◽

Molecular Signature ◽

Genetic Lineage ◽

Cardiac Progenitors ◽

Heart Field ◽

Genetic Lineage Tracing

AbstractThe heart develops from two sources of mesoderm progenitors, the first and second heart field (FHF and SHF). Using a single cell transcriptomic assay in combination with genetic lineage tracing, we find the FHF and SHF are subdivided into distinct pools of progenitors in gastrulating mouse embryos at earlier stages than previously thought. Each subpopulation has a distinct origin in the primitive streak. The first progenitors to leave the primitive streak contribute to the left ventricle, shortly after right ventricle progenitor emigrate, followed by the outflow tract and atrial progenitors. Although cells allocated to the outflow tract and atrium leave the primitive streak at a similar stage, they arise from different regions. Outflow tract originate from distal locations in the primitive streak while atrial progenitors are positioned more proximally. Moreover, single cell RNA sequencing demonstrates that the primitive streak cells contributing to the ventricles have a distinct molecular signature from those forming the outflow tract and atrium. We conclude that cardiac progenitors are pre-patterned within the primitive streak and this prefigures their allocation to distinct anatomical structures of the heart. Together, our data provide a new molecular and spatial map of mammalian cardiac progenitors that will support future studies of heart development, function and disease.

Download Full-text

Ventricular, atrial, and outflow tract heart progenitors arise from spatially and molecularly distinct regions of the primitive streak

PLoS Biology ◽

10.1371/journal.pbio.3001200 ◽

2021 ◽

Vol 19 (5) ◽

pp. e3001200

Author(s):

Kenzo Ivanovitch ◽

Pablo Soro-Barrio ◽

Probir Chakravarty ◽

Rebecca A. Jones ◽

Donald M. Bell ◽

...

Keyword(s):

Single Cell ◽

Heart Development ◽

Primitive Streak ◽

Outflow Tract ◽

Lineage Tracing ◽

Molecular Signature ◽

Genetic Lineage ◽

Cardiac Progenitors ◽

Heart Field ◽

Genetic Lineage Tracing

The heart develops from 2 sources of mesoderm progenitors, the first and second heart field (FHF and SHF). Using a single-cell transcriptomic assay combined with genetic lineage tracing and live imaging, we find the FHF and SHF are subdivided into distinct pools of progenitors in gastrulating mouse embryos at earlier stages than previously thought. Each subpopulation has a distinct origin in the primitive streak. The first progenitors to leave the primitive streak contribute to the left ventricle, shortly after right ventricle progenitor emigrate, followed by the outflow tract and atrial progenitors. Moreover, a subset of atrial progenitors are gradually incorporated in posterior locations of the FHF. Although cells allocated to the outflow tract and atrium leave the primitive streak at a similar stage, they arise from different regions. Outflow tract cells originate from distal locations in the primitive streak while atrial progenitors are positioned more proximally. Moreover, single-cell RNA sequencing demonstrates that the primitive streak cells contributing to the ventricles have a distinct molecular signature from those forming the outflow tract and atrium. We conclude that cardiac progenitors are prepatterned within the primitive streak and this prefigures their allocation to distinct anatomical structures of the heart. Together, our data provide a new molecular and spatial map of mammalian cardiac progenitors that will support future studies of heart development, function, and disease.

Download Full-text

From Stripes to a Beating Heart: Early Cardiac Development in Zebrafish

Journal of Cardiovascular Development and Disease ◽

10.3390/jcdd8020017 ◽

2021 ◽

Vol 8 (2) ◽

pp. 17

Author(s):

Cassie L. Kemmler ◽

Fréderike W. Riemslagh ◽

Hannah R. Moran ◽

Christian Mosimann

Keyword(s):

Congenital Heart Defects ◽

Heart Development ◽

Congenital Heart ◽

Cardiac Development ◽

Heart Defects ◽

Lineage Tracing ◽

Genetic Lineage ◽

Zebrafish Model ◽

Lateral Plate ◽

Cardiac Progenitors

The heart is the first functional organ to form during vertebrate development. Congenital heart defects are the most common type of human birth defect, many originating as anomalies in early heart development. The zebrafish model provides an accessible vertebrate system to study early heart morphogenesis and to gain new insights into the mechanisms of congenital disease. Although composed of only two chambers compared with the four-chambered mammalian heart, the zebrafish heart integrates the core processes and cellular lineages central to cardiac development across vertebrates. The rapid, translucent development of zebrafish is amenable to in vivo imaging and genetic lineage tracing techniques, providing versatile tools to study heart field migration and myocardial progenitor addition and differentiation. Combining transgenic reporters with rapid genome engineering via CRISPR-Cas9 allows for functional testing of candidate genes associated with congenital heart defects and the discovery of molecular causes leading to observed phenotypes. Here, we summarize key insights gained through zebrafish studies into the early patterning of uncommitted lateral plate mesoderm into cardiac progenitors and their regulation. We review the central genetic mechanisms, available tools, and approaches for modeling congenital heart anomalies in the zebrafish as a representative vertebrate model.

Download Full-text

Abstract 16013: Direct Nkx2-5 Transcriptional Repression of Isl1 Controls Cardiomyocyte Subtype Identity

Circulation ◽

10.1161/circ.130.suppl_2.16013 ◽

2014 ◽

Vol 130 (suppl_2) ◽

Author(s):

Alexander Goedel ◽

Tatjana Dorn ◽

Jason T Lam ◽

Franziska Herrmann ◽

Jessica Haas ◽

...

Keyword(s):

Heart Development ◽

Transcriptional Repression ◽

Embryonic Stem ◽

Cardiac Differentiation ◽

Heart Tube ◽

Cardiac Progenitors ◽

Gene Enhancer ◽

Heart Field ◽

Homeobox Protein ◽

Islet 1

During heart development the second heart field (SHF) provides progenitor cells for most cardiomyocytes and expresses the LIM-homeodomain transcription factor Islet-1 (Isl1) and the homeobox protein Nkx2-5. Here, we show that a direct repression of Isl1 transcription by Nkx2-5 is necessary for proper specification and maturation of ventricular and atrial chamber-specific myocardial lineages. Overexpression of Nkx2-5 in mouse embryonic stem cells (ESCs) delayed specification of cardiac progenitors and inhibited expression of Isl1 and its downstream targets in the Isl1+ precursor population. These effects were partially rescued by Isl1 overexpression. Embryos deficient for Nkx2-5 in the Isl1+ lineage failed to downregulate Isl1 protein in cardiomyocytes of the heart tube (Figure 1A). We demonstrated that Nkx2-5 directly binds to an Isl1 gene enhancer and represses the transcriptional activity of Isl1. Furthermore, we showed that overexpression of Isl1 does not prevent cardiac differentiation of ESCs and in Xenopus laevis embryos. Instead, Isl1 overexpression in ESCs leads to enhanced specification of cardiac progenitors, earlier cardiac differentiation, and increased number of cardiomyocytes (Figure 1B). Functional and molecular analysis of Isl1-overexpressing cardiomyocytes revealed higher beating frequencies in both ESC-derived contracting areas and Xenopus Isl1-gain-of-function hearts (Figure 1C), which was associated with upregulation of nodal-specific genes and downregulation of transcripts of working myocardium. Our findings provide an Isl1/Nkx2-5-mediated mechanism that coordinately regulates the specification of cardiac progenitors towards the different myocardial lineages and ensures proper acquisition of myocyte subtype-identity (Figure 1D).

Download Full-text

Embryonic organoids recapitulate early heart organogenesis

10.1101/802181 ◽

2019 ◽

Cited By ~ 1

Author(s):

Giuliana Rossi ◽

Andrea Boni ◽

Romain Guiet ◽

Mehmet Girgin ◽

Robert G. Kelly ◽

...

Keyword(s):

Heart Development ◽

Cardiac Tissue ◽

Embryonic Stem ◽

Coordinated Development ◽

Cardiac Progenitors ◽

Tissue Interactions ◽

Heart Field ◽

Morphogenetic Potential ◽

Key Aspects

AbstractOrganoids are powerful models for studying tissue development, physiology, and disease. However, current culture systems disrupt the inductive tissue-tissue interactions needed for the complex morphogenetic processes of native organogenesis. Here we show that mouse embryonic stem cells (mESCs) can be coaxed to robustly undergo the fundamental steps of early heart organogenesis with an in vivo-like spatiotemporal fidelity. These axially patterned embryonic organoids support the generation of cardiovascular progenitors, as well as first and second heart field compartments. The cardiac progenitors self-organize into an anterior domain reminiscent of a cardiac crescent before forming a beating cardiac tissue near a putative primitive gut-like tube, from which it is separated by an endocardial-like layer. These findings unveil the surprising morphogenetic potential of mESCs to execute key aspects of organogenesis through the coordinated development of multiple tissues. This platform could be an excellent tool for studying heart development in unprecedented detail and throughput.

Download Full-text

Tbx1 regulates extracellular matrix-cell interactions in the second heart field

10.1101/267906 ◽

2018 ◽

Author(s):

Daniela Alfano ◽

Alessandra Altomonte ◽

Claudio Cortes ◽

Marchesa Bilio ◽

Robert G. Kelly ◽

...

Keyword(s):

Cultured Cells ◽

Time Window ◽

Focal Adhesions ◽

Outflow Tract ◽

Mouse Embryos ◽

Cell Interactions ◽

Deletion Syndrome ◽

Cardiac Progenitors ◽

Second Heart Field ◽

Heart Field

SUMMARYTbx1, the major candidate gene for DiGeorge or 22q11.2 deletion syndrome, is required for efficient incorporation of cardiac progenitors (CPs) of the second heart field (SHF) into the heart. However, the mechanisms by which TBX1 regulates this process are still unclear. Here, we have used two independent models, mouse embryos and cultured cells, to define the role of TBX1 in establishing morphological and dynamic characteristics of SHF in the mouse. We found that loss of TBX1 impairs extra cellular matrix (ECM)-integrin-focal adhesion (FA) signaling in both models. Mosaic analysis in embryos showed that this function is non-cell autonomous and, in cultured cells, loss of TBX1 impairs cell migration and focal adhesions. Additionally, we found that ECM-mediated outside-in integrin signaling is disrupted upon loss of TBX1. Finally, we show that interfering with the ECM-integrin-FA axis between E8.5 and E9.5 in mouse embryos, corresponding to the time window within which TBX1 is required in the SHF, causes outflow tract dysmorphogenesis. Our results demonstrate that TBX1 is required to maintain the integrity of ECM-cell interactions in the SHF, and that this interaction is critical for cardiac outflow tract development. More broadly, our data identifies a novel TBX1 downstream pathway as an important player in SHF tissue architecture and cardiac morphogenesis.

Download Full-text

A Role for the Cytoskeleton in Heart Looping

The Scientific World JOURNAL ◽

10.1100/tsw.2007.87 ◽

2007 ◽

Vol 7 ◽

pp. 280-298 ◽

Cited By ~ 23

Author(s):

Kersti K. Linask ◽

Michael VanAuker

Keyword(s):

Heart Development ◽

Cell Biology ◽

Heart Defects ◽

Heart Tube ◽

Second Heart Field ◽

The Past ◽

Complex Process ◽

Nonmuscle Myosin Ii ◽

Heart Looping ◽

Heart Field

Over the past 10 years, key genes involved in specification of left-right laterality pathways in the embryo have been defined. The read-out for misexpression of laterality genes is usually the direction of heart looping. The question of how dextral looping direction occurred mechanistically and how the heart tube bends remains unknown. It is becoming clear from our experiments and those of others that left-right differences in cell proliferation in the second heart field (anterior heart field) drives the dextral direction. Evidence is accumulating that the cytoskeleton is at the center of laterality, and the bending and rotational forces associated with heart looping. If laterality pathways are modulated upstream, the cytoskeleton, including nonmuscle myosin II (NMHC-II), is altered downstream within the cardiomyocytes, leading to looping abnormalities. The cytoskeleton is associated with important mechanosensing and signaling pathways in cell biology and development. The initiation of blood flow during the looping period and the inherent stresses associated with increasing volumes of blood flowing into the heart may help to potentiate the process. In recent years, the steps involved in this central and complex process of heart development that is the basis of numerous congenital heart defects are being unraveled.

Download Full-text

Cardiac progenitors auto-regulate second heart field cell fate via Wnt secretion

10.1101/2021.01.31.428968 ◽

2021 ◽

Author(s):

Matthew Miyamoto ◽

Suraj Kannan ◽

Hideki Uosaki ◽

Tejasvi Kakani ◽

Sean Murphy ◽

...

Keyword(s):

Single Cell ◽

Cell Fate ◽

Congenital Heart ◽

Heart Defects ◽

Cardiac Marker ◽

Cardiac Progenitors ◽

Second Heart Field ◽

Heart Field ◽

Heart Formation

Proper heart formation requires coordinated development of two anatomically distinct groups of cells - the first and second heart fields (FHF and SHF). Given that congenital heart defects are often restricted to derivatives of the FHF or SHF, it is crucial to understand the mechanisms controlling their development. Wnt signaling has previously been implicated in SHF proliferation; however, the source of Wnts remains unknown. Through comparative gene analysis, we found upregulation of Wnts and Wnt receptor/target genes in the FHF and SHF, respectively, raising the possibility that early cardiac progenitors may secrete Wnts to influence SHF cell fate. To probe this further, we deleted Wntless (Wls), a gene required for Wnt ligand secretion, in various populations of precardiac cells. Deletion of Wls in Mesp1+ cells resulted in formation of a single chamber heart with left ventricle identity, implying compromised SHF development. This phenotype was recapitulated by deleting Wls in cells expressing Islet1, a pan-cardiac marker. Similarly, Wls deletion in cells expressing Nkx2.5, a later-expressed pan-cardiac marker, resulted in hypoplastic right ventricle, a structure derived from the SHF. However, no developmental defects were observed when deleting Wls in SHF progenitors. To gain mechanistic insights, we isolated Mesp1-lineage cells from developing embryos and performed single-cell RNA-sequencing. Our comprehensive single cell transcriptome analysis revealed that Wls deletion dysregulates developmental trajectories of both anterior and posterior SHF cells, marked by impaired proliferation and premature differentiation. Together, these results demonstrate a critical role of local precardiac mesodermal Wnts in SHF fate decision, providing fundamental insights into understanding heart field development and chamber formation.Significance StatementThere is significant interest in understanding the mechanisms underlying heart formation to develop treatments and cures for patients suffering from congenital heart disease. In particular, we were interested in the intricacies of first (FHF) and second heart field (SHF) development, as many congenital heart defects present with heart field-specific etiologies. Here, we uncovered a novel relationship between specified cardiac progenitor cells and second heart field progenitors. Through genetic manipulation of Wnt secretion in developing mouse embryos, we identified a population of cardiac progenitor cells that acts as a local source of Wnts which are necessary for proper SHF development. Our single cell transcriptomic analysis of developing anterior mesoderm showed cardiac progenitor-secreted Wnts function through regulation of differentiation and proliferation among SHF progenitors. Thus, this study provides insight into the source and timing of Wnts required for SHF development, and points to the crucial role of co-developing cell populations in heart development.

Download Full-text