Cardiovascular diseases are the leading cause of death in America today, and congenital heart defects remain the number one cause of death for infants in the western world during their first year of life.  By identifying and characterizing more of the genes that are involved in early heart formation, we have an opportunity to better understand not only how certain diseases, developmental problems or congenital heart defects are caused, but we will also expand our fundamental knowledge of cardiovascular development

The cardiovascular system, which is comprised of the heart and its associated network of blood vessels, forms the vertebrate embryo’s first functional organ system.  At present, however, the heart is a well studied yet little understood organ. Work over the last several years has uncovered several genes that are critical for cardiogenesis, including members of the tinman-related family.  This introduction will describe early heart development in the South African Clawed Frog, Xenopus laevis and will express the importance of the tinman-related genes in cardiogenesis.  The significance of this proposal will then be presented, stressing the value of identifying downstream targets of Tinman-related transcriptional regulation.

Early Heart Development in Xenopus laevis

Over the last century, the morphology of the developing amphibian heart has been well documented by embryologists.  Cell explantation and lineage tracing experiments have determined that the cells destined to contribute to the developing heart have their origin in two bilateral regions within the dorsal mesoderm located initially on either side of the Spemann organizer (Figure 1a).  The cells in these “presumptive heart fields” of lateral plate mesoderm are specified during gastrulation and are competent to form beating heart tissue even if excised from the embryo (Goerttler 1928; Bacon 1945; Jacobson 1960; Jacobson and Sater 1988) .  It is interesting, however, that even though the heart fields are specified at gastrulation (stage 10 in Xenopus development), differentiation does not occur until much later, after the cardiogenic cells migrate to the ventral midline (figure 1c).

At the current level of molecular detection, differentiation is initiated in Xenopus at around stage 27 (figure 1c).  At this time, the cells fated to contribute to the muscular layer of the heart, the myocardium, begin to express markers of terminal myocardial differentiation, such as cardiac troponin I (TnIc), myosin heavy chain alpha (MHCa and myosin light chain 2 (MLC-2) (Logan and Mohun 1993; O'Brien et al. 1993; Chambers et al. 1994; Drysdale et al. 1994) .  Simultaneously, the differentiating fields continue to move toward the ventral midline, delaminate from the surrounding tissues and form an epithelial layer (Davenport 1895) .  Fusion of these two differentiating patches of cells forms a single cardiogenic plate, which then rolls to create the primitive heart tube – a two layer structure with a myocardium and inner endocardium.  Rhythmic beating of the primitive heart tube occurs in Xenopus at developmental stage 32 and morphogenetic movements cause the heart tube to loop, further defining the chambers and resulting in a fully functional embryonic heart by stage 35 (figure 1e).

The tinman-related Genes and Early Heart Development

In Drosophila, the homeobox transcription factor tinman was found to be absolutely required for the formation of the dorsal vessel, the insect equivalent of the heart (Bodmer 1993) .  A search for homologues revealed the presence of a small family of tinman-related genes that are expressed in the presumptive heart fields.  In Xenopus, these include XNkx2-3, XNkx2-5 and XNkx2-10 (Tonissen et al. 1994; Evans et al. 1995; Cleaver et al. 1996; Newman and Krieg 1998) . 

Overexpression of either XNkx2-3 or XNkx2-5 in frogs leads to cardiac hyperplasia, with enlarged hearts containing twice the normal number of myocardial cells (figure 2) (Cleaver et al. 1996) .  A similar overexpression phenotype has also been reported in zebrafish (Chen and Fishman 1996) . 

Our work and that by Dr. Silvia Evans has also demonstrated that as a family, these tinman-related genes are required for heart differentiation (figure 3) (Fu et al. 1998; Grow and Krieg 1998) .  These experiments show that dominant inhibitory forms of the tinman-related factors are able to completely eliminate both myocardial differentiation and all heart morphology.

There is now mounting evidence supporting the theory that multiple transcription factors are required to regulate cardiogenesis.  For instance, experiments have shown that the transcription factors GATA-4, GATA-5 and Nkx2-5 can recognize regulatory elements in the genes encoding cardiac-specific proteins, such as ANF and b-myosin heavy-chain (Molkentin et al. 1994; Lee et al. 1998) .  Additionally, SRF has also been shown to associate with Nkx2-5 and GATA-4 to synergistically activate the cardiac a-actin promoter (Sepulveda et al. 1998; Sepulveda et al. 2002) .  Combined overexpression of members from these three families fails to produce early or ectopic hearts, however, suggesting that other regulatory mechanisms remain undiscovered.

Other Genes Involved in Early Heart Development

As mentioned previously, several other transcriptional regulators have been identified to play critical roles in the specification and differentiation of the heart.  Here, we briefly describe several of these genes, including serum response factor (SRF), myocardin, the GATA-4/5/6 subfamily, and Hop.

A MADS box transcription factor, serum response factor (SRF), has been identified to be an important regulator for proper development of all muscle cell types. (Li et al. 1997; Strobeck et al. 2001; Sepulveda et al. 2002) .  With regard to the heart, a dominant inhibitory form of SRF was shown capable of dramatically reducing heart size in mice and a dominant negative isoform has been found to be predominantly expressed in failing human hearts (Zhang et al. 2001; Davis et al. 2002) .  The ubiquitous SRF expression in many organisms, however, has led to the speculation that SRF requires cofactors for tissue-specific regulation.

Myocardin is a SAP-domain transcription factor recently identified to be an SRF cofactor expressed in both myocardial and smooth muscle cells.  The importance of Myocardin is emphasized by work showing that a dominant negative form of Myocardin is capable of blocking heart development in Xenopus (Hauschka 2001; Wang et al. 2001; Chen et al. 2002b; Wang et al. 2002) .  Interestingly, it now appears that there are several Myocardin-related genes whose functions are now being investigated (P. Krieg, personal comm.).

In vertebrates, three GATA transcription factors have been found to be expressed in the presumptive heart fields:  GATA-4, GATA-5, and GATA-6.  In mouse and Xenopus, GATA-4 and GATA-5 are expressed in both the precardiac mesoderm and adjacent endoderm.  GATA-4 expression continues in both the endocardial and myocardial layers of the developing heart, while GATA-5 is later restricted to just the endocardium (Kelley et al. 1993; Laverriere et al. 1994) .  Knockout experiments in mice have indicated that GATA-4 is required for normal heart development (Kuo et al. 1997; Molkentin et al. 1997) , but the role of GATA-5 remains largely a mystery.  The early expression of Xenopus GATA-6 is similar to that of GATA-4, where it is observed to be expressed at high levels in the heart primordia, but at the time of terminal myocardial differentiation its expression is downregulated (Laverriere et al. 1994) .  Also, differentiation of the heart can be blocked by overexpression of GATA-6, suggesting that the role of GATA-6 may be to hold the cardiogenic cells in a specified, but undifferentiated state (Gove et al. 1997) .

Recently a new cardiogenic gene, Hop, has been identified.  Encoding a homeodomain without DNA-binding ability, Hop appears to be an SRF repressor and is capable of physically interfering with SRF’s DNA-binding ability.  There is also strong evidence that Hop lies downstream from Tinman-related regulation (Chen et al. 2002a) .

Sample Nkx2-5 Perturbations

XNkx2-5 Overexpression in Xenopus Causes Myocardial Hyperplasia

Tinman-related dominant inhibition blocks heart development

To block the function of the entire family of tinman-related genes, two dominant inhibitory mutants were created.  These inhibitory constructs were created through the substitution of a conserved Leucine residue with a Proline between helix 2 and helix 3 of the homeodomain in both XNkx2-3 and 2-5, and were named XNkx2-3LP and XNkx2-5LP, respectively.  Expression of either XNkx2-3LP or XNkx2-5LP mRNA in the presumptive heart regions results in the complete loss of cardiac differentiation (figure 3) (Grow and Krieg 1998) .

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