The ability to regenerate a heart after ablation of cardiogenic mesoderm has been demonstrated in early stage fish and amphibian embryos but this type of regulation of the heart field has not been seen in avians or mammals. The regulative potential of the cardiogenic mesoderm was examined in avian embryos and related to the spatial expression of genes implicated in early cardiogenesis. With the identification of early cardiac regulators such as bmp-2 and nkx-2.5, it is now possible to reconcile classical embryological studies with molecular mechanisms of cardiac lineage determination in vivo. The most anterior lateral embryonic cells have been identified as the region that becomes the heart; removal of all or any subset of these cells results in the loss of corresponding cardiac structures. In addition, removal of the lateral heart forming mesoderm while leaving the lateral endoderm intact also results in loss of cardiac structures. Thus the medial anterior mesoderm cannot be recruited into the heart lineage in vivo even in the presence of potential cardiac inducing endoderm. In situ analysis demonstrates that genes involved in early events of cardiogenesis such as bone morphogenetic protein 2 (bmp-2) and nkx-2.5 are expressed coincidentally with the mapped far lateral heart forming region. The activin type IIa receptor (actR-IIa) is a potential mediator of BMP signaling since it is expressed throughout the anterior mesoderm with the highest level of expression occurring in the lateral prospective heart cells. The posterior boundary of actR-IIa is consistent with the posterior boundary of nkx-2.5 expression, supporting a model whereby ActR-IIa is involved in restricting the heart forming region to an anterior subset of lateral cells exposed to BMP-2. Analysis of the cardiogenic potential of the lateral plate mesoderm posterior to nkx-2.5 and actR-IIa expression demonstrates that these cells are not cardiogenic in vitro and that removal of these cells from the embryo does not result in loss of heart tissue in vivo. Thus, the region of the avian embryo that will become the heart is defined medially, laterally, and posteriorly by nkx-2.5 gene expression. Removal of all or part of the nkx-2.5 expressing region results in the loss of corresponding heart structures, demonstrating the inability of the chick embryo to regenerate cardiac tissue in vivo at stages after nkx-2.5 expression is initiated (Ehrman, 1999).

In a study of the mechanisms by which cells become committed to the cardiac myocyte lineage during avian development, chick tissues from outside the fate map of the heart were combined with potential inducing tissues from quail embryos, and cultured in vitro. Species-specific RT-PCR was employed to detect the appearance of the cardiac muscle markers chick Nkx-2.5 (cNkx-2.5), cardiac troponin C and ventricular myosin heavy chain in the chick responder tissues. Stage 4-5 anterior lateral (AL) endoderm and anterior central (AC) mesendoderm, but not AL mesoderm or posterior lateral mesendoderm, induce cells to differentiate as cardiac myocytes. Induction of cardiogenesis is accompanied by a marked decrease in the expression of rho-globin, implying that non cardiac cells are being induced by anterior endoderm to become cardiac myocytes instead of blood-forming tissue. These results suggest that anterior endoderm contains signaling molecules that can induce cardiac myocyte specification of early primitive streak cells. One of the cardiac muscle markers induced by anterior endoderm, cNkx-2.5, is described in this paper for the first time. cNkx-2.5 is a chick homeobox-containing gene that shares extensive sequence similarity with the Drosophila gene tinman, which is required for Drosophila heart formation. The mesodermal component of cNkx-2.5 expression from stage 5 onward matches the fate map of the avian heart. By the time the myocardium and endocardium form distinct layers, cNkx-2.5 is found only in the myocardium. cNkx-2.5 thus appears to be the earliest described marker of avian mesoderm fated to give rise to cardiac muscle (Schultheiss, 1995).

BMP signals act in concert with FGF8, WNT11 and WNT antagonists to induce the formation of cardiac tissue in the vertebrate embryo. In an effort to understand how these signaling pathways control the expression of key cardiac regulators, the cis-regulatory elements of the chick tinman homolog chick Nkx2.5 have been characterized. At least three distinct cardiac activating regions (CARs) of chick Nkx2.5 cooperate to regulate early expression in the cardiac crescent and later segmental expression in the developing heart. In this report, attention was focused on a 3' BMP-responsive enhancer, termed CAR3, which directs robust cardiac transgene expression. By systematic mutagenesis and gel shift analysis of this enhancer, it has been demonstrated that GATA4/5/6, YY1 and SMAD1/4 are all necessary for BMP-mediated induction and heart-specific expression of CAR3. Adjacent YY1 and SMAD-binding sites within CAR3 constitute a minimal BMP response element, and interaction of SMAD1/4 with the N terminus of YY1 is required for BMP-mediated induction of CAR3. These data suggest that BMP-mediated activation of this regulatory region reflects both the induction of GATA genes by BMP signals, as well as modulation of the transcriptional activity of YY1 by direct interaction of this transcription factor with BMP-activated SMADs (Lee, 2004).

How might the interaction of SMADs with YY1 modulate the activity of this transcription factor when bound to CAR3? Because YY1 can function as either a transcriptional activator or repressor, SMAD association with YY1 may serve to recruit co-activators that modulate the activity of this transcription factor to become an efficient transcriptional activator. Indeed, recruitment of co-activators such as p300 by TGFß activated SMADs is a well-characterized mechanism for SMAD target gene activation. Similarly, known interacting partners of YY1 also include several members of the histone deacetylase family as well as a histone H4 methylase, which have been implicated in either transcriptional repression or activation of YY1 regulated target genes, respectively. It will be interesting to determine if SMAD association with YY1 alters the interaction of this transcription factor with either of these families of histone modifying enzymes, and to what extent chromatin modification is responsible for appropriate regulation of Nkx2.5 (Lee, 2004).

SMAD-mediated modulation of YY1 activity adds an interesting new facet to the repertoire of functions of YY1 during heart development, which also includes direct recruitment of transcriptional co-activators to promote the expression of cardiac B-type natriuretic peptide, inhibition of the expression of the cardiac {alpha}-actin gene, and both activation and inhibition of the expression of the cardiac-specific Mlc2 gene. Clearly, the context within which YY1 functions is of great importance, and it is likely that transcription factors such as GATA and SMAD proteins, when bound to neighboring cognate binding sites, modulate either the association of co-factors with adjacently bound YY1 or the activity of such co-factors. In addition to the GATA, YY1- and SMAD-binding sites, linker scanning mutational analysis of the chick Nkx2.5 CAR3 BMPRE has revealed other sites yet to be characterized that also have a significant impact on the BMP response of this regulatory element. A complete understanding of complex enhancers such as Nkx2.5 CAR3 will require not only the identification of the transcription factors that regulate their expression but also elucidation of the transcriptional co-factors that are recruited to such regulatory elements in a combinatorial fashion (Lee, 2004).

Csx/Nkx2.5 serves critical developmental functions in heart formation in vertebrates and nonvertebrates. The putative nuclear localization signal (NLS) of Csx/Nkx2.5 has been identified by site-directed mutagenesis to the amino terminus of the homeodomain, which is conserved in almost all homeodomain proteins. When the putative NLS of Csx/Nkx2.5 is mutated, a significant amount of the cytoplasmically localized Csx/Nkx2.5 is unphosphorylated. This is in contrast to the Csx/Nkx2.5 that is localized to the nucleus; that is serine- and threonine-phosphorylated, and suggests that Csx/Nkx2.5 phosphorylation is regulated, at least in part, by intracellular localization. Tryptic phosphopeptide mapping indicates that Csx/Nkx2.5 has at least five phosphorylation sites. Using in-gel kinase assays, a Csx/Nkx2.5 kinase has been identified whose molecular mass is approximately 40 kDa in both cytoplasmic and nuclear extracts. Mutational analysis and in vitro kinase assays suggest that this 40-kDa Csx/Nkx2.5 kinase is a catalytic subunit of casein kinase II (CKII) that phosphorylates the serine residue between the first and second helix of the homeodomain. This CKII site is phosphorylated in vivo. CKII-dependent phosphorylation of the homeodomain increases the DNA binding activity of Csx/Nkx2.5. Serine-to-alanine mutation at the CKII phosphorylation site reduces transcriptional activity when the carboxyl-terminal repressor domain is deleted. Although the precise biological function of Csx/Nkx2.5 phosphorylation by CKII remains to be determined, it may play an important role, since this CKII phosphorylation site within the homeodomain is fully conserved in all known members of the NK2 family of the homeobox genes (Kasahara, 1999).

A murine cardiac-specific homeodomain gene named csx or Nkx2-5 is a potential vertebrate homolog of Drosophila tinman.

High affinity Nkx-2.5 DNA binding sites, 5'-TNNAGTG-3', represented novel homeodomain binding sequences, whereas intermediate and weaker affinity sites, 5'-C(A/T)TTAATTN-3', contained the typical 5'-TAAT-3' core required by most homeodomain factors for DNA binding. Nkx-2.5 served as a modest transcription activator. Functional dissection of Nkx-2.5 reveals a COOH-terminal inhibitory domain composed mainly of clusters of alanines and prolines, which appeared to mask a potent activation domain composed of hydrophobic and highly charged amino acids (Chen, 1995).

The Drosophila homeobox gene tinman and its vertebrate homologs Nkx-2.5 and Nkx-2.3 are critical determinants of cardiac development. A new tinman-related gene, nkx2.7 is described, as well as orthologs of Nkx-2.5 and Nkx-2.3 in the zebrafish. Analysis of their expression in the developing zebrafish embryo reveals that nkx2.7 transcripts are the first to appear in cardiac mesodermal and pharyngeal endodermal precursors of the anterior hypoblast, anticipating both temporally and spatially the later expression of nkx2.5 and nkx2.3 in these lineages. The preeminence of nkx2.7 in these embryonic lineages is consistent with a key role in cell fate determination, perhaps in part through the induction of nkx2.5 and nkx2.3. The findings provide the first molecular clues as to the spatial organization of endodermal and cardiac mesodermal precursors in the zebrafish hypoblast immediately following gastrulation. They suggest a coordinate role for these three tinman-related genes in the development of the heart and pharyngeal arches, and reinforce the paradigm of gene duplication and subspecialization between Drosophila and vertebrate species. The results provide a framework in which to analyze potential changes in tinman-related gene expression during abnormal zebrafish development (Lee, 1996).

Mutations in the gene encoding the homeobox transcription factor NKX2-5 were found to cause nonsyndromic, human congenital heart disease. A dominant disease locus associated with cardiac malformations and atrioventricular conduction abnormalities maps to chromosome 5q35, the same location as NKX2-5, a Drosophila tinman homolog. Three different NKX2-5 mutations were identified. Two are predicted to impair binding of NKX2-5 to target DNA, resulting in haploinsufficiency, and a third might augment target-DNA binding. These data indicate that NKX2-5 is important for regulation of septation during cardiac morphogenesis and for maturation and maintenance of atrioventricular node function throughout life (Schott, 1998).

In Drosophila, the tinman gene is absolutely required for development of the dorsal vessel, the insect equivalent of the heart. In vertebrates, the tinman gene is represented by a small family of tinman-related sequences, some of which are expressed during embryonic heart development. At present however, the precise importance of this gene family for vertebrate heart development is unclear. Using the Xenopus embryo, a dominant inhibitory strategy was employed to interfere with the function of the endogenous tinman-related genes. In these experiments, suppression of tinman gene function can result in the complete elimination of myocardial gene expression and the absence of cell movements associated with embryonic heart development. This inhibition can be rescued by expression of wild-type tinman sequences. These experiments indicate that function of tinman family genes is essential for the development of the vertebrate heart (Grow, 1998).

In many vertebrates, removal of early embryonic heart precursors can be repaired, leaving the heart and embryo without visible deficit. One possibility is that this 'regulation' involves a cell fate switch whereby cells, perhaps in regions surrounding normal progenitors, are redirected to the heart cell fate. However, the lineage and spatial relationships between cells that are normal heart progenitors and those that can assume that role after injury are not known, nor are their molecular distinctions. A laser-activated technique was adapted to label single or small patches of cells in the lateral plate mesoderm of the zebrafish and to track their subsequent lineage. The heart precursor cells are found clustered in a region adjacent to the prechordal plate, just anterior to the notochord tip. Complete unilateral ablation of all heart precursors with a laser does not disrupt heart development, if performed before the 18-somite stage. By combining extirpation of the heart precursors with cell labeling, it is found that cells anterior to the normal cardiogenic compartments constitute the source of regulatory cells that compensate for the loss of the progenitors. One of the earliest embryonic markers of the premyocardial cells is the divergent homeodomain gene, Nkx2.5. Interestingly, normal cardiogenic progenitors derive from only the anterior half of the Nkx2.5-expressing region in the lateral plate mesoderm. The posterior half, adjacent to the notochord, does not include cardiac progenitors and the posterior Nkx2.5-expressing cells do not contribute to the heart, even after ablation of the normal cardiogenic region. The cells that can acquire a cardiac cell fate after injury to the normal progenitors also reside near the prechordal plate, but anterior to the Nkx2.5-expressing domain. These cells express Nkx2.7. The overlap between GATA4 and Nkx2.7 expressing cells may explain why Nkx2.5 mutation in mice does not prevent assembly of a heart tube. Normally these anterior cells give rise to head mesenchyme. In common with cardiac progenitors, they share early expression of GATA 4. The location of the different elements of the cardiac field, and their response to injury, suggests that the prechordal plate supports and/or the notochord suppresses the cardiac fate (Serbedzija, 1998).

Nkx-2.5 transcripts are first detected at early headfold stages in myocardiogenic progenitor cells. Expression precedes the onset of myogenic differentiation, and continues in cardiomyocytes of embryonic, foetal and adult hearts. Transcripts are also detected in future pharyngeal endoderm, the tissue believed to produce the heart inducer. Expression in endoderm is only found laterally, where it is in direct apposition to promyocardium, suggesting an interaction between the two tissues. After foregut closure, Nkx-2.5 expression in endoderm is limited to the pharyngeal floor, dorsal to the developing heart tube. The thyroid primordium, a derivative of the pharyngeal floor, continues to express Nkx-2.5 after transcript levels diminish in the rest of the pharynx. Nkx-2.5 transcripts are also detected in lingual muscle, spleen and stomach. The expression data implicate Nkx-2.5 in commitment to and/or differentiation of the myocardial lineage. The data further demonstrate that cardiogenic progenitors can be distinguished at a molecular level by late gastrulation. Nkx-2.5 expression will be a valuable marker in the analysis of mesoderm development and an early entry point for dissection of the molecular basis of myogenesis in the heart (Lints, 1993).

The Nkx2-5 homeodomain protein plays a key role in cardiomyogenesis. Ectopic expression in frog and zebrafish embryos results in an enlarged myocardium; however, expression of Nkx2-5 in fibroblasts is not able to trigger the development of beating cardiac muscle. In order to examine the ability of Nkx2-5 to modulate endogenous cardiac specific gene expression in cells undergoing early stages of differentiation, P19 cell lines overexpressing Nkx2-5 were differentiated in the absence of Me2SO. Nkx2-5 expression induces cardiomyogenesis in these cultures aggregated without Me2SO. During differentiation into cardiac muscle, Nkx2-5 expression results in the activation of myocyte enhancer factor 2C (MEF2C), but not MEF2A, -B, or -D. In order to compare the abilities of Nkx2-5 and MEF2C to induce cellular differentiation, P19 cells overexpressing MEF2C were aggregated in the absence of Me2SO. Similar to Nkx2-5, MEF2C expression initiates cardiomyogenesis, resulting in the up-regulation of Brachyury T, bone morphogenetic protein-4, Nkx2-5, GATA-4, cardiac alpha-actin, and myosin heavy chain expression. These findings indicate the presence of a positive regulatory network between Nkx2-5 and MEF2C and show that both factors can direct early stages of cell differentiation into a cardiomyogenic pathway (Skerjanc, 1998). Nkx2-5 is expressed in precardiac mesoderm and in the myocardium of embryonic and fetal hearts. Targeted interruption of Nkx2-5 results in abnormal heart morphogenesis, growth retardation and embryonic lethality. Heart tube formation occurs normally in mutant embryos, but looping morphogenesis, a critical determinant of heart form, is not initiated at the linear heart tube stage. Commitment to the cardiac muscle lineage, expression of most myofilament genes and myofibrillogenesis are not normal. However, the myosin light-chain 2V gene, earliest known molecular marker of ventricular differentiation, is not expressed in mutant hearts. The regional expression in mutant hearts of two other ventricular markers, myosin heavy-chain beta and cyclin D2, indicates that not all ventricle-specific gene expression is dependent on Nkx2-5 (Lyons, 1995).

Tinman is a Drosophila homeodomain protein that is required for formation of both visceral and cardiac mesoderm, including formation of the dorsal vessel, a heart-like organ. Although several vertebrate Tinman homologs have been characterized, their requirement at the earliest stages of heart formation has been an open question, perhaps complicated by the potential functional redundancy of tinman homologs. A novel approach was used to investigate functional redundancy within a gene family, by coinjecting DNA encoding dominantly acting repressor derivatives specific for each family member into developing Xenopus embryos. These results provide the first evidence that vertebrate Tinman homologs are required for earliest stages of heart formation, and that they are required in a functionally redundant manner. Coinjection of dominant repressor constructs for both XNkx2-3 and XNkx2-5 is synergistic, resulting in a much higher frequency of mutant phenotypes than that obtained with injection of either dominant repressor construct alone. Rescue of mutant phenotypes can be effected by coinjection of either wild-type Tinman homolog. The most extreme mutant phenotype is a complete absence of expression of XNkx2-5 in cardiogenic mesoderm, an absence of markers of differentiated myocardium, and absence of morphologically distinguishable heart on the EnNkxHD-injected side of the embryo. This phenotype represents the most severe cardiac phenotype of any vertebrate mutant yet described, and underscores the importance of the Tinman family for heart development. These results provide the first in vivo evidence that XNkx2-3 and XNkx2-5 are required as transcriptional activators for the earliest stages of heart formation. Furthermore, these results suggest an intriguing mechanism by which functional redundancy operates within a gene family during development. These experiments have been performed utilizing a recently developed transgenic strategy, and attest to the efficacy of this strategy for enabling transgene expression in limited cell populations within the developing Xenopus embryo (Fu, 1998).

The murine homeobox gene Csx/Nkx2.5 is an evolutionarily highly conserved gene related to the Drosophila tinman gene, which specifies cardiac and visceral mesoderm. Since Csx/Nkx2.5 plays an essential role in heart development, studying its regulation is essential for the better understanding of molecular mechanisms of cardiogenesis and the pathogenesis of congenital heart disease in humans. The murine Csx/Nkx2.5 gene has been characterized and two novel untranslated exons, 1a, and 1b, have been identified which give rise to three different Csx/Nkx2.5 transcripts. To examine the tissue-specific transcriptional regulation in vivo, a total of 23 kb of Csx/Nkx2.5 upstream and downstream sequences were analyzed by generating transgenic embryos carrying lacZ reporter constructs containing various lengths of flanking sequence. With 14 kb of 5' flanking sequence, lacZ expression is observed in the cardiac crescent at E7.5, and in the outflow tract, the interatrial groove, the atrioventricular canal and right and left ventricles, as well as in pharyngeal floor, thyroid primordia, and stomach at E10.5. In adult animals, lacZ expression of the transgene is limited to the atrioventricular junction and the subendocardium of the ventricular septum. Reducing the size of flanking sequence to 3.3 kb of intron 2 restricts lacZ expression to the outflow tract and the basal part of the right ventricle in E10.5 embryos. In contrast, the addition of 6 kb of 3' flanking sequence causes strong expression of the reporter gene in the entire right ventricle. Interestingly, Csx/Nkx2.5 seems to be negatively regulated by its own gene product, because when lacZ is "knocked-in" to replace all coding exons, lacZ expression is much higher in the heart of homozygous embryos than that in the heterozygote. These results indicate that the transcriptional regulatory elements of Csx/Nkx 2.5 seems unexpectedly highly modular, and is temporally regulated in a dynamic manner by different enhancer regions. Since Csx/Nkx2.5-like genes are expressed in all species having a heart, their complex modular organization with multiple enhancers probably reflects progressive addition of regulatory elements during the evolution from a simple heart tube to a complex four-chambered organ (Tanaka, 1999b).

Nkx2-5 marks the earliest recognizable cardiac progenitor cells, and is activated in response to inductive signals involved in lineage specification. Nkx2-5 is also expressed in the developing foregut, thyroid, spleen, stomach and tongue. One approach to elucidate the signals involved in cardiogenesis was to examine the transcriptional regulation of early lineage markers such as Nkx2-5. F0 transgenic mice, which carry Nkx2-5 flanking sequences linked to a lacZ reporter gene, were generated. Multiple regulatory regions located within the proximal 10.7 kb of the Nkx2-5 gene were identified. In addition to a proximal promoter, a second promoter and a novel upstream exon were identified that could participate in the regulation of Nkx2-5 transcription. Although used rarely in normal development, this novel exon could be spliced into the Nkx2-5 coding region in several ways, thereby potentially creating novel Nkx2-5 protein isoforms, whose transcriptional activity is greatly diminished as compared to wild-type Nkx2-5. An enhancer that directs expression in pharynx, spleen, thyroid and stomach was identified within 3.5 kb of exon 1 between the coding exon 1 and the novel upstream exon 1a. Two or more enhancers upstream of exon 1a are capable of driving expression in the cardiac crescent, throughout the myocardium of the early heart tube, then in the outflow tract and right ventricle of the looped heart tube. A negative element was also located upstream of exon1a, that interacts in complex ways with enhancers to direct correct spatial expression. In addition, potential autoregulatory elements can be cooperatively stimulated by Nkx2-5 and GATA-4. These results demonstrate that a complex suite of interacting regulatory domains regulate Nkx2-5 transcription. Dissection of these elements should reveal essential features of cardiac induction and positive and negative signaling within the cardiac field (Reecy, 1999).

Cardiomyocyte proliferation is high in early development and decreases progressively with gestation, resulting in the lack of a robust cardiomyocyte proliferative response in the adult heart after injury. Little is understood about how both cell-autonomous and nonautonomous signals are integrated to regulate the balance of cardiomyocyte proliferation during development. This study shows that a single transcription factor, Foxp1, can control the balance of cardiomyocyte proliferation during development by targeting different pathways in the endocardium and myocardium. Endocardial loss of Foxp1 results in decreased Fgf3/Fgf16/Fgf17/Fgf20 expression in the heart, leading to reduced cardiomyocyte proliferation. This loss of myocardial proliferation can be rescued by exogenous Fgf20, and is mediated, in part, by Foxp1 repression of Sox17. In contrast, myocardial-specific loss of Foxp1 results in increased cardiomyocyte proliferation and decreased differentiation, leading to increased myocardial mass and neonatal demise. Nkx2.5 is a direct target of Foxp1 repression, and Nkx2.5 expression is increased in Foxp1-deficient myocardium. Moreover, transgenic overexpression of Nkx2.5 leads to increased cardiomyocyte proliferation and increased ventricular mass, similar to the myocardial-specific loss of Foxp1. These data show that Foxp1 coordinates the balance of cardiomyocyte proliferation and differentiation through cell lineage-specific regulation of Fgf ligand and Nkx2.5 expression (Zhang, 2010).

Establishment of specific characteristics of each embryonic cardiac chamber is crucial for development of a fully functional adult heart. Despite the importance of defining and maintaining unique features in ventricular and atrial cardiomyocytes, the regulatory mechanisms guiding these processes are poorly understood. This study shows that the homeodomain transcription factors Nkx2.5 and Nkx2.7 are necessary to sustain ventricular chamber attributes through repression of atrial chamber identity. Mutation of nkx2.5 in zebrafish yields embryos with diminutive ventricular and bulbous atrial chambers. Removal of nkx2.7 function from nkx2.5 mutants exacerbates the loss of ventricular cells and the gain of atrial cells. Moreover, in these Nkx-deficient embryos, expression of vmhc, a ventricular gene, fades, whereas expression of amhc, an atrial gene, expands. Cell-labeling experiments suggest that ventricular cardiomyocytes can transform into atrial cardiomyocytes in the absence of Nkx gene function. Through suggestion of transdifferentiation from ventricular to atrial fate, these data reveal a pivotal role for Nkx genes in maintaining ventricular identity and highlight remarkable plasticity in differentiated myocardium (Targoff, 2013).

The vertebrate heart develops from mesoderm and requires inductive signals secreted from early endoderm. During embryogenesis, Nkx2.5 acts as a key transcription factor and plays essential roles for heart formation from Drosophila to human. In mice, Nkx2.5 is expressed in the early first heart field, second heart field pharyngeal mesoderm, as well as pharyngeal endodermal cells underlying the second heart field. Currently, the specific requirements for Nkx2.5 in the endoderm versus mesoderm with regard to early heart formation are incompletely understood. This study performed tissue-specific deletion in mice to dissect the roles of Nkx2.5 in the pharyngeal endoderm and mesoderm. It was found that heart development appeared normal after endodermal deletion of Nkx2.5 whereas mesodermal deletion engendered cardiac defects almost identical to those observed on Nkx2.5 null embryos (Nkx2.5-/-). Furthermore, re-expression of Nkx2.5 in the mesoderm rescued Nkx2.5-/- heart defects.These findings reveal that Nkx2.5 in the mesoderm is essential while endodermal expression is dispensable for early heart formation in mammals (Zhang, 2014).

A complex regulatory network of morphogens and transcription factors is essential for normal cardiac development. Nkx2-5 is among the earliest known markers of cardiac mesoderm that is central to the regulatory pathways mediating second heart field (SHF) development. This study has examined the specific requirements for Nkx2-5 in the SHF progenitors. Nkx2-5 was found to potentiate Wnt signaling by regulating the expression of the R-spondin3 (Rspo3) gene during cardiogenesis. R-spondins are secreted factors and potent Wnt agonists that in part regulate stem cell proliferation. The data show that Rspo3 is markedly downregulated in Nkx2-5 mutants and that Rspo3 expression is regulated by Nkx2-5. Conditional inactivation of Rspo3 in the Isl1 lineage resulted in embryonic lethality secondary to impaired development of SHF. More importantly, it was found that Wnt signaling is significantly attenuated in Nkx2-5 mutants and that enhancing Wnt/beta-catenin signaling by pharmacological treatment or by transgenic expression of Rspo3 rescues the SHF defects in the conditional Nkx2-5(+/-) mutants. A previously unrecognized genetic link between Nkx2-5 and Wnt signaling was uncovered that supports continued cardiac growth and proliferation during development. Identification of Rspo3 in cardiac development provides a new paradigm in temporal regulation of Wnt signaling by cardiac-specific transcription factors (Cambier, 2014).