Abstract 052: Epicardial-derived Adrenomedullin Drives Cardiac Hyperplasia During Embryogenesis

2013 ◽  
Vol 113 (suppl_1) ◽  
Author(s):  
Sarah E Wetzel-Strong ◽  
Manyu Li ◽  
Toshio Nishikimi ◽  
Kathleen M Caron
Keyword(s):  
Embryonic Development ◽  
Heart Development ◽  
Cardiac Development ◽  
Lymphatic Vessels ◽  
Heart Weight ◽  
Cardiovascular Development ◽  
Proliferating Cells ◽  
Over Expression ◽  
Knockout Animals

The multi-functional peptide adrenomedullin ( Adm = gene, AM = protein) plays important roles in embryonic development and disease. Previous studies demonstrated that Adm knockout mice die at embryonic day 13.5 with small, disorganized hearts and hypoplastic lymphatic vessels, highlighting the importance of this peptide in normal cardiovascular development. Since Adm knockout animals are embryonic lethal, our goal was to generate and characterize a novel model of Adm over-expression to study the role of Adm during development and disease processes. Through gene targeting techniques, we generated a novel mouse model of Adm over-expression, abbreviated as Adm hi/hi . When we assessed gene expression of Adm from 10 different tissues, we found Adm hi/hi mice express 3- to 15-fold more Adm than wildtype littermates. Additionally, peptide levels of AM in lung and kidney, as well as circulating plasma levels of AM were elevated 3-fold over wildtype mice, indicating a functional increase in AM. Our initial analysis revealed that adult Adm hi/hi mice have larger heart weight to body weight ratios than wildtype littermates (4.93±0.23 vs. 5.96±0.29, n = 11-12). We found that compared to wildtype, Adm hi/hi embryos have more proliferating cells during heart development (14.46±1.11 vs. 31.97±2.84, n=4), indicating that hyperplasia drives Adm hi/hi heart enlargement. By crossing the Adm hi/hi line to different tissue-specific Cre lines, we were able to excise the stabilizing bovine growth hormone 3’UTR, thereby returning Adm expression levels back to wildtype in cells with active Cre recombinase. Using this approach, we identified the epicardium as a major source of AM during cardiac development. In conclusion, we found that AM derived primarily from the epicardium drives cardiac hyperplasia during embryonic development resulting in persistent, enlarged hearts of adult Adm hi/hi mice. Since our Adm hi/hi mice recapitulate the 3-fold plasma elevation of AM observed during human disease, this mouse line will be a useful tool for studying the role of elevated AM during disease.

Abstract 257: Clonal Analysis Of Multipotent Cardiovascular Progenitors That Generate Cardiomyocytes During Development

2014 ◽  
Vol 115 (suppl_1) ◽  
Author(s):  
Konstantina Ioanna Sereti ◽  
Paniz Kamran Rashani ◽  
Peng Zhao ◽  
Reza Ardehali
Keyword(s):  
Single Cell ◽  
Heart Development ◽  
Cardiac Development ◽  
Clonal Analysis ◽  
Cardiac Cells ◽  
Single Cell Level ◽  
Reporter System ◽  
Cell Level ◽  
Cardiac Progenitors

It has been proposed that cardiac development in lower vertebrates is driven by the proliferation of cardiomyocytes. Similarly, cycling myocytes have been suggested to direct cardiac regeneration in neonatal mice after injury. Although, the role of cardiomyocyte proliferation in cardiac tissue generation during development has been well documented, the extent of this contribution as well as the role of other cell types, such as progenitor cells, still remains controversial. Here we used a novel stochastic four-color Cre-dependent reporter system (Rainbow) that allows labeling at a single cell level and retrospective analysis of the progeny. Cardiac progenitors expressing Mesp1 or Nkx2.5 were shown to be a source of cardiomyocytes during embryonic development while the onset of αMHC expression marked the developmental stage where the capacity of cardiac cells to proliferate diminishes significantly. Through direct clonal analysis we provide strong evidence supporting that cardiac progenitors, as opposed to mature cardiomyocytes, are the main source of cardiomyocytes during cardiac development. Moreover, we have identified quadri-, tri-, bi, and uni-potent progenitors that at a single cell level can generate cardiomyocytes, fibroblasts, endothelial and smooth muscle cells. Although existing cardiomyocytes undergo limited proliferation, our data indicates that it is mainly the progenitors that contribute to heart development. Furthermore, we show that the limited proliferation capacity of cardiomyocytes observed during normal development was enhanced following neonatal cardiac injury allowing almost complete regeneration of the scared tissue. However, this ability was largely absent in adult injured hearts. Detailed characterization of dividing cardiomyocytes and proliferating progenitors would greatly benefit the development of novel therapeutic options for cardiovascular diseases.

The zebrafish as a model for cardiac development and regeneration

2018 ◽  
Author(s):  
Bill Chaudhry ◽  
José Luis de la Pompa ◽  
Nadia Mercader
Keyword(s):  
Heart Development ◽  
Cardiac Development ◽  
Model Organism ◽  
Laboratory Model ◽  
Developmental Studies ◽  
Sequencing Technologies ◽  
Technological Advances ◽  
The Common ◽  
Zebrafish Heart

The zebrafish has become an established laboratory model for developmental studies and is increasingly used to model aspects of human development and disease. However, reviewers and grant funding bodies continue to speculate on the utility of this Himalayan minnow. In this chapter we explain the similarities and differences between the heart from this distantly related vertebrate and the mammalian heart, in order to reveal the common fundamental processes and to prevent misleading extrapolations. We provide an overview of zebrafish including their husbandry, development, peculiarities of their genome, and technological advances, which make them a highly tractable laboratory model for heart development and disease. We discuss the controversies around morphants and mutants, and relate the development and structures of the zebrafish heart to mammalian counterparts. Finally, we give an overview of regeneration in the zebrafish heart and speculate on the role of the model organism in next-generation sequencing technologies.

Transient Over-Expression of HOX-Regulating Genes CDX2 and MLL during Human Embryoid Body Differentiation Greatly Augments the Generation of Multipotent Hematopoietic Progenitors.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1243-1243
Author(s):  
Michal A. Levine ◽  
Christopher Roxbury ◽  
Elias T. Zambidis
Keyword(s):  
Stem Cell ◽  
Embryonic Development ◽  
Transient Expression ◽  
Embryoid Body ◽  
Hematopoietic Progenitors ◽  
Hematopoietic Development ◽  
Rna Molecules ◽  
Over Expression ◽  
Definitive Hematopoiesis

Abstract Homeobox (HOX) genes play critical roles in normal anterior-posterior patterning of embryonic development, and in hematopoietic stem cell (HSC) development. Conversely, dysregulated expressions of HOX-regulating factors such as CDX2 (Caudal) and MLL (Mixed Lineage Leukemia) are directly linked to development of acute leukemia. Although CDX family (e.g. cdx4) and mll factors play important roles in murine HSC development, their role in normal human embryonic blood development is obscure. The role of CDX genes (e.g., CDX1, CDX2, CDX4), expressed exclusively during embryonic development, is difficult to evaluate in human hematopoietic development, since fetal tissue is difficult to obtain. Our group has developed a human embryonic stem cell (hESC) differentiation system that recapitulates the 2nd-6th gestational weeks of human yolk sac (YS) development, and initiates from an embryonic hemangioblastic progenitor of primitive and definitive hematopoiesis. The role of HOX-regulating genes (and also HOX-regulating microRNAs (miRNAs), e.g., mir196) that regulate the earliest stages of human hematopoietic development can therefore be studied directly in vitro using our hESC model. We tested the effects of pulsatile, transient over-expression of HOX-regulating factors and miRNAs on the generation of primitive and definitive hematopoeitic progenitors during human embryoid body (hEB) differentiation. Since expression of HOX-regulating genes and miRNAs follow temporal, transient expression patterns during normal embryonic, and also hEB development, we developed a methodology that allows similar transient expression of DNA and RNA molecules at multiple time points of advancing hEB differentiation. This method, termed whole embryoid body (WEB) nucleofection was optimized using GFP-expressing DNA constructs, GFP-silencing siRNA, and also miRNA molecules within intact, whole hEB. WEB nucleofection allowed expression in 15–90% of day 4–9 hEB cells without disrupting their three-dimensional structural integrity, and with minimal toxicity. GFP-nucleofected day 5–13 hEB demonstrated peak expression levels at 48 hrs post-nucleofection, and expression was sustained for approximately one week. A FITC-labeled dsRNA oligonucleotide, was used to demonstrate that the efficiency of WEB nucleofection with RNA molecules approached ∼90%. WEB nucleofection was utilized to transiently over-express CDX2 and MLL constructs within intact, developing hEBs, and the effects on generation of hEB-derived primitive and definitive hematopoiesis were assayed by colony-forming cell (CFC), and FACS analysis. CDX2 and MLL-nucleofected hEB each produced 5-10X greater amounts of multipotent, mixed CFU, in comparison to controls. Moreover, MLL-nucleofected hEB demonstrated a bias toward development of definitive erythroid progenitors. Hematopoietic regulation by over-expression or inhibition of miRNAs implicated in HOX regulation (e.g. mir-196, mir-10) is also currently being evaluated by WEB nucleofection. Our ability to specifically control multiple combinations of transgenic DNA, siRNA or miRNA molecules, temporally and spatially during hEB differentiation, provides novel opportunities to manipulate the CDX-HOX axis for generating and expanding multi-potent hematopoietic progenitors from hESC. The role of HOX-regulating factors and miRNAs involved in regulating the earliest steps of human hematopoietic commitment can now be directly evaluated.

Transient Over-Expression of HOX-Regulating Genes CDX2 and MLL during Human Embryoid Body Differentiation Greatly Augments the Generation of Multipotent Hematopoietic Progenitors.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3745.5-3745.5
Author(s):  
Michal A. Levine ◽  
Christopher Roxbury ◽  
Elias T. Zambidis
Keyword(s):  
Stem Cell ◽  
Embryonic Development ◽  
Transient Expression ◽  
Embryoid Body ◽  
Hematopoietic Progenitors ◽  
Hematopoietic Development ◽  
Rna Molecules ◽  
Over Expression ◽  
Definitive Hematopoiesis

Abstract Homeobox (HOX) genes play critical roles in normal anterior-posterior patterning of embryonic development, and in hematopoietic stem cell (HSC) development. Conversely, dysregulated expressions of HOX-regulating factors such as CDX2 (Caudal) and MLL (Mixed Lineage Leukemia) are directly linked to development of acute leukemia. Although CDX family (e.g. cdx4) and mll factors play important roles in murine HSC development, their role in normal human embryonic blood development is obscure. The role of CDX genes (e.g., CDX1, CDX2, CDX4), expressed exclusively during embryonic development, is difficult to evaluate in human hematopoietic development, since fetal tissue is difficult to obtain. Our group has developed a human embryonic stem cell (hESC) differentiation system that recapitulates the 2nd–6th gestational weeks of human yolk sac (YS) development, and initiates from an embryonic hemangioblastic progenitor of primitive and definitive hematopoiesis. The role of HOX-regulating genes (and also HOX-regulating microRNAs (miRNAs), e.g., mir196) that regulate the earliest stages of human hematopoietic development can therefore be studied directly in vitro using our hESC model. We tested the effects of pulsatile, transient over-expression of HOX-regulating factors and miRNAs on the generation of primitive and definitive hematopoeitic progenitors during human embryoid body (hEB) differentiation. Since expression of HOX-regulating genes and miRNAs follow temporal, transient expression patterns during normal embryonic, and also hEB development, we developed a methodology that allows similar transient expression of DNA and RNA molecules at multiple time points of advancing hEB differentiation. This method, termed whole embryoid body (WEB) nucleofection was optimized using GFP-expressing DNA constructs, GFP-silencing siRNA, and also miRNA molecules within intact, whole hEB. WEB nucleofection allowed expression in 15–90% of day 4–9 hEB cells without disrupting their three-dimensional structural integrity, and with minimal toxicity. GFP-nucleofected day 5–13 hEB demonstrated peak expression levels at 48 hrs post-nucleofection, and expression was sustained for approximately one week. A FITC-labeled dsRNA oligonucleotide, was used to demonstrate that the efficiency of WEB nucleofection with RNA molecules approached ∼90%. WEB nucleofection was utilized to transiently over-express CDX2 and MLL constructs within intact, developing hEBs, and the effects on generation of hEB-derived primitive and definitive hematopoiesis were assayed by colony-forming cell (CFC), and FACS analysis. CDX2 and MLL-nucleofected hEB each produced 5-10X greater amounts of multipotent, mixed CFU, in comparison to controls. Moreover, MLL-nucleofected hEB demonstrated a bias toward development of definitive erythroid progenitors. Hematopoietic regulation by over-expression or inhibition of miRNAs implicated in HOX regulation (e.g. mir-196, mir-10) is also currently being evaluated by WEB nucleofection. Our ability to specifically control multiple combinations of transgenic DNA, siRNA or miRNA molecules, temporally and spatially during hEB differentiation, provides novel opportunities to manipulate the CDX-HOX axis for generating and expanding multi-potent hematopoietic progenitors from hESC. The role of HOX-regulating factors and miRNAs involved in regulating the earliest steps of human hematopoietic commitment can now be directly evaluated.

The Role of Shear Stress on ET-1, KLF2, and NOS-3 Expression in the Developing Cardiovascular System of Chicken Embryos in a Venous Ligation Model

Physiology ◽  
2007 ◽  
Vol 22 (6) ◽  
pp. 380-389 ◽  
Author(s):  
Bianca C. W. Groenendijk ◽  
Kim Van der Heiden ◽  
Beerend P. Hierck ◽  
Robert E. Poelmann
Keyword(s):  
Shear Stress ◽  
Heart Development ◽  
Cardiac Development ◽  
Background Information ◽  
Endothelial Shear Stress ◽  
Oxide Synthase ◽  
Endothelial Nitric Oxide ◽  
Cardiovascular Anomalies ◽  
Chicken Model

In this review, the role of wall shear stress in the chicken embryonic heart is analyzed to determine its effect on cardiac development through regulating gene expression. Therefore, background information is provided for fluid dynamics, normal chicken and human heart development, cardiac malformations, cardiac and vitelline blood flow, and a chicken model to induce cardiovascular anomalies. A set of endothelial shear stress-responsive genes coding for endothelin-1 (ET-1), lung Krüppel-like factor (LKLF/KLF2), and endothelial nitric oxide synthase (eNOS/NOS-3) are active in development and are specifically addressed.

scholarly journals The role of KMT2D and KDM6A in cardiac development: A cross-species analysis in humans, mice, and zebrafish

2020 ◽  
Author(s):  
Rwik Sen ◽  
Ezra Lencer ◽  
Elizabeth A. Geiger ◽  
Kenneth L. Jones ◽  
Tamim H. Shaikh ◽  
...  
Keyword(s):  
Gene Expression ◽  
Heart Development ◽  
Target Genes ◽  
Cardiac Development ◽  
Heart Defects ◽  
Neural Crest Cell ◽  
Kabuki Syndrome ◽  
Rna Seq ◽  
Wide Range

AbstractCongenital Heart Defects (CHDs) are the most common form of birth defects, observed in 4-10/1000 live births. CHDs result in a wide range of structural and functional abnormalities of the heart which significantly affect quality of life and mortality. CHDs are often seen in patients with mutations in epigenetic regulators of gene expression, like the genes implicated in Kabuki syndrome – KMT2D and KDM6A, which play important roles in normal heart development and function. Here, we examined the role of two epigenetic histone modifying enzymes, KMT2D and KDM6A, in the expression of genes associated with early heart and neural crest cell (NCC) development. Using CRISPR/Cas9 mediated mutagenesis of kmt2d, kdm6a and kdm6al in zebrafish, we show cardiac and NCC gene expression is reduced, which correspond to affected cardiac morphology and reduced heart rates. To translate our results to a human pathophysiological context and compare transcriptomic targets of KMT2D and KDM6A across species, we performed RNA sequencing (seq) of lymphoblastoid cells from Kabuki Syndrome patients carrying mutations in KMT2D and KDM6A. We compared the human RNA-seq datasets with RNA-seq datasets obtained from mouse and zebrafish. Our comparative interspecies analysis revealed common targets of KMT2D and KDM6A, which are shared between species, and these target genes are reduced in expression in the zebrafish mutants. Taken together, our results show that KMT2D and KDM6A regulate common and unique genes across humans, mice, and zebrafish for early cardiac and overall development that can contribute to the understanding of epigenetic dysregulation in CHDs.

The role of the neural crest in cardiac development

2018 ◽  
Author(s):  
Laura A. Dyer ◽  
Margaret L. Kirby
Keyword(s):  
Neural Crest ◽  
Neural Tube ◽  
Heart Development ◽  
Cardiac Development ◽  
Signalling Pathways ◽  
Pharyngeal Arches ◽  
Cardiac Neural Crest ◽  
Final Destination ◽  
Dorsal Neural Tube

The cardiac neural crest (CNC) plays pivotal roles in numerous steps of cardiac development. Every aspect of the CNC cell’s lifespan is highly orchestrated, from its induction in the dorsal neural tube to its migration to its differentiation at its final destination. During migration, CNC cells are affected by their environment and simultaneously modulate the extra-cellular milieu through which they migrate. In the pharyngeal arches, CNC cells repattern the originally symmetrical arch arteries, producing the great arteries. Because the cardiac neural crest is essential for many aspects of heart development, it is unsurprising that human CNC-related syndromes have severe phenotypes. This chapter describes how CNC cells are formed and contribute to their final destinations. Essential signalling pathways are presented in the context of CNC development, and CNC-related syndromes are included to highlight this population’s broad importance during development.

Vinculin knockout results in heart and brain defects during embryonic development

Development ◽  
1998 ◽  
Vol 125 (2) ◽  
pp. 327-337 ◽  
Author(s):  
W. Xu ◽  
H. Baribault ◽  
E.D. Adamson
Keyword(s):  
Embryonic Development ◽  
Focal Adhesion ◽  
Heart Development ◽  
Cardiac Development ◽  
Es Cells ◽  
Embryonic Stem ◽  
Spinal Nerve ◽  
Cell Behaviors ◽  
Migration Rates ◽  
Targeting Vector

The vinculin gene codes for a cytoskeletal protein, found in focal adhesion plaques and in cell-cell adherens junctions. Vinculin was inactivated by homologous recombination using a targeting vector in embryonic stem (ES) cells. The heterozygous ES cells were introduced into mice by established procedures to produce heterozygous animals that were normal and fertile. No homozygous vinculin−/− embryos were born and analyses during the gestational period showed that the vinculin null embryos were small and abnormal from day E8 but some survived until E10. The most prominent defect was lack of midline fusion of the rostral neural tube, producing a cranial bilobular appearance and attenuation of cranial and spinal nerve development. Heart development was curtailed at E9.5, with severely reduced and akinetic myocardial and endocardial structures. Mutant embryos were 30–40% smaller, somites and limbs were retarded and ectodermal tissues were sparse and fragile. Fibroblasts (MEF) isolated from mutant embryos were shown to have reduced adhesion to fibronectin, vitronectin, laminin and collagen compared to wild-type levels. In addition, migration rates over these substrata were two-fold higher and the level of focal adhesion kinase (FAK) activity was three-fold higher. We conclude that vinculin is necessary for normal embryonic development, probably because of its role in the regulation of cell adhesion and locomotion, cell behaviors essential for normal embryonic morphogenesis, although specific roles in neural and cardiac development cannot be ruled out.

scholarly journals Phosphodiesterases Expression during Murine Cardiac Development

2021 ◽  
Vol 22 (5) ◽  
pp. 2593
Author(s):  
Thays Maria da Conceição Silva Carvalho ◽  
Silvia Cardarelli ◽  
Mauro Giorgi ◽  
Andrea Lenzi ◽  
Andrea M. Isidori ◽  
...  
Keyword(s):  
Heart Development ◽  
Fetal Heart ◽  
Cardiac Development ◽  
Large Family ◽  
Hydrolytic Activity ◽  
Development Data ◽  
Heart Formation ◽  
Camp And Cgmp ◽  
Developing Mice

3′-5′ cyclic nucleotide phosphodiesterases (PDEs) are a large family of enzymes playing a fundamental role in the control of intracellular levels of cAMP and cGMP. Emerging evidence suggested an important role of phosphodiesterases in heart formation, but little is known about the expression of phosphodiesterases during cardiac development. In the present study, the pattern of expression and enzymatic activity of phosphodiesterases was investigated at different stages of heart formation. C57BL/6 mice were mated and embryos were collected from 14.5 to 18.5 days of development. Data obtained by qRT-PCR and Western blot analysis showed that seven different isoforms are expressed during heart development, and PDE1C, PDE2A, PDE4D, PDE5A and PDE8A are modulated from E14.5 to E18.5. In heart homogenates, the total cAMP and cGMP hydrolytic activity is constant at the evaluated times, and PDE4 accounts for the majority of the cAMP hydrolyzing ability and PDE2A accounts for cGMP hydrolysis. This study showed that a subset of PDEs is expressed in developing mice heart and some of them are modulated to maintain constant nucleotide phosphodiesterase activity in embryonic and fetal heart.

scholarly journals Cardiac Organoids and Gastruloids to Study Physio-Pathological Heart Development

2021 ◽  
Vol 8 (12) ◽  
pp. 178
Author(s):  
Marisa E. Jaconi ◽  
Michel Puceat
Keyword(s):  
Embryonic Development ◽  
Heart Development ◽  
Ethical Issues ◽  
Cardiac Development ◽  
In Vitro Models ◽  
Beating Heart ◽  
Human Embryos ◽  
Functional Studies ◽  
Review Reports

Ethical issues restrict research on human embryos, therefore calling for in vitro models to study human embryonic development including the formation of the first functional organ, the heart. For the last five years, two major models have been under development, namely the human gastruloids and the cardiac organoids. While the first one mainly recapitulates the gastrulation and is still limited to investigate cardiac development, the second one is becoming more and more helpful to mimic a functional beating heart. The review reports and discusses seminal works in the fields of human gastruloids and cardiac organoids. It further describes technologies which improve the formation of cardiac organoids. Finally, we propose some lines of research towards the building of beating mini-hearts in vitro for more relevant functional studies.

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