Heart Development – A Paradigm of Cell Differentiation and Organogenesis

Stems cells from the embryo are derived from the inner cell mass (ICM), embryonic ectoderm, and primordial germ cells of the fetal genital ridge and represent pluripotent undifferentiated cells capable of proliferation, cell-renewal and the generation of large numbers of differentiated cell progeny. As development proceeds and a stem cell become committed to a specific lineage or decreases its proliferative potential, it usually describes as progenitor cell. Progenitor cells and stem cells are capable of serial frog forming multiple cell types but are believed to have a more limited potential than embryonic cells. Within the developing embryonic bodies, cardiomyocytes are located between an epithelial layer and a basal layer of mesenchymal cells. Embryonic stem cells derive cardiomyocytes express cardiac gene products in developmentally controlled manner. The heart development pathway begins with external inductive signals from overlaying tissues that instruct a specific subset of transcription factors within the signaled cells to coordinate a complex array of gene expression. A variety of transcription factor combine to specify the cardiomyocyes, endocardium, pericardium and cells of cardiac conduction system. This process of heart development is termed as cardiogenesis which involves a series of events that consists of
– Specification of mesodermal and neural crest-derived cells to become programmed to the cardiac lineage.
– The growth and differentiation of these cells to cardiomyocytes and
– Their migration and morphogenenic patterning in to the mature heart.

The subsequent enlargement of the embryonic heart is largely dependent on an increase in myocyte number, which continues until shortly after birth, when cardiac myocytes loose their proliferative capacity and acquire the terminal differentiated phenotype of adult cardiac muscle cells.

Signaling molecules in Cardiogenesis involve bone morphogenetic protein-2 (BMP-2) and Wnt family of morphogens. In vertebrates, BMP signaling is conveyed through a complex interaction of the ligand with cellular receptors that activate a family of proteins termed Smads. The signaling molecules such as XWNT-8, Sonic hedghog and nodel have also been shown to mediate morphological patterning of the developing heart in Xenopus and Chicken. Based on several finding on these signaling molecule, a model was derived that involves a delicate interplay of morphogenic signals that, when present at right time and the right concentration, convey this specific signal to the nucleus of a predestined cardiomyocytes.

Transcriptional control of cardiogenesis:
Tubular heart is capable of rhythmic contraction and contains at least three distinct cell types viz. endocardial-endothelia, ventricular myocyte and atrial myocytes. Drosophila heart is a simple tubular structure consisting of two types of cells and is known as dorsal vessel. Since vertebrate heart is also formed as a tubular structure in the beginning, dorsal vessel in Drosophila can be perceived as a primitive form of vertebrate heart. Furthermore, both Drosophila and the vertebrate hearts originate from similar region of the mesoderm. Molecular genetics of Drosophila development has thus significantly contributed to the general understanding of the molecular basis of heart muscle development.

In spite of the biochemical and structural similarities between the cardiac and skeletal muscle cells, the basic difference between these two-muscle types is the absence of MyoD family of master regulators in cardiac myocytes. Based on the genetic analysis of the Drosophila and Zebrafish development as well as analysis of the regulatory regions of various muscle specific promoters, a number of transcription factor such as Nkx2.5, GATA-4/5/6, MEF2 and e/dHAND have been implicated to cardiac muscle development. It is functional redundancy and combinatorial interaction of these transcription factors that reflect the complex network of gene expression that orchestrate heart development.

Discovery of tinman gene in Drosophila, led to the isolation of its vertebrate homologues viz Nkx2.3, Nkx2.5, Nkx2.6, Nkx2.7, Nkx2.8 and Nkx2.9 from a number of species such as mouse, chicken, zebrafish and frogs. Among these Nkx family members, only Nkx2.5 is consistently expressed in all species and the cardiac expression of others is either restricted or they are expressed only at a certain stage of development. Nkx2.5 (also known as Csx) is an evolutionarily conserved cardiac transcription factor. Tinman, Nkx2.5 and other members of this family act as transcription factors that bind to the DNA element AAGTG as monomers. Targeted interruption of Nkx2.5 function in transgenic mouse embryos indicates that it is not necessary for myocyte commitment. It is hypothesized that Nkx family harbors a common “Nkx code” and it is their unique combinatorial expression that directs various cell fates.

Recently a bioinformatics-based screen for unknown cardiac-specific genes identified a novel and highly potent SAP domain family member, myocardin which is expressed in cardiac cells and activates cardiac muscle promoters by associating with serum response factor, SRF. Myocardin expression is regulated by Nkx2.5.

A second multigene family involved in cardiac commitment and differentiation is the GATA family of zinc finger proteins. Six members of GATA family (GATA1-6) have been identified. GATA4/5/6 are expressed in heart, gut epithelium and lung. During mouse embryogenesis, GATA 4/6 mRNAs are present in precardiac mesoderm and visceral endoderm. However, GATA5 restricted to the atrial endocardium. Embryos homozygous null for GATA4 form cardiac myocyte expressing contractile protein genes but fail to migrate them to form heart tube. The transcription factor GATA6 is first expressed at the blastocyst stage in part of the inner cell mass and in the trophectoderm. The second wave of expression is in parietal endoderm, mesoderm and endoderm that form the heart and gut. Inactivation of GATA6 leads to lethality shortly after implantation.

A third set of nuclear factor important in vertebrate heart development is members of the MEF2 family. Among various cis-regulatory elements frequently found in the regulatory region of muscle specific genes is C/TTA(A/T) 4, target site for the MEF2 (Myocyte Enhancer Factor-2) family of transcription factors. Two members of this family, MEF2C and MEF2B are expressed in mouse cardiac primordia and throughout heart development, suggesting that they play an important role in cardiac myocyte differentiation. Targeted disruption of MEF2C results in defects in initiation in rightward looping and right ventricle formation.

A fourth set of nuclear proteins involved in cardiogenesis is basic helix-loop-helix (bHLH) proteins HAND. Two related bHLH proteins eHAND and dHAND are expressed in heart. dHAND is expressed in all cardiac myocytes whereas eHAND shows a transient pattern of expression predominantly in one ventricle; suggesting it may be involved in segment-specific gene expression during development. Recently two highly related bHLH gene, MesP1 and MesP2 are found to be expressed in heart. MesP1 expression is reported to be the earliest molecular marker expressed in heart precursor.
Recently, six homeodomain transcription factors, which have identity to the genes of the Drosophila Iroquoris complex, were isolated in mice. Irx homeobox genes are involved in establishing cardiac structural identity. Another paired homeodomain transcription factor that is involved in heart development is PAX3. One recently identified homeodomain protein is Hop that lacks certain conserved residues required for DNA binding. Hop gene expression initiates early in cardiogenesis and continues in cardiomyocytes throughout embryonic and postnatal development. Hop modulates SRF-dependent cardiac-specific gene expression and cardiac development. Genetic and biochemical data indicate that Hop functions directly downstream of Nkx2.5. Another set of proteins, the T-box transcription factors (Tbx5 and Tbx20) play critical roles in embryonic development. In vertebrate embryos, tbx5 is expressed in the developing heart, forelimb, eye, and liver. Tbx20 physically interact with cardiac transcription factors Nkx2.5, GATA4, and GATA5, collaborating to synergistically activate cardiac gene expression.

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