David A. Elliott

Cardiac T-box factor Tbx20 directly interacts with Nkx2-5, GATA4, and GATA5 in regulation of gene expression in the developing heart

Tbx20 is a member of the T-box transcription factor family expressed in the forming hearts of vertebrate and invertebrate embryos. We report here analysis of Tbx20 expression during murine cardiac development and assessment of DNA-binding and transcriptional properties of Tbx20 isoforms. Tbx20 was expressed in myocardium and endocardium, including high levels in endocardial cushions. cDNAs generated by alternative splicing encode at least four Tbx20 isoforms, and Tbx20a uniquely carried strong transactivation and transrepression domains in its C terminus. Isoforms with an intact T-box bound specifically to DNA sites resembling the consensus brachyury half site, although with less avidity compared with the related factor, Tbx5. Tbx20 physically interacted with cardiac transcription factors Nkx2-5, GATA4, and GATA5, collaborating to synergistically activate cardiac gene expression. Among cardiac GATA factors, there was preferential synergy with GATA5, implicated in endocardial differentiation. In Xenopus embryos, enforced expression of Tbx20a, but not Tbx20b, led to induction of mesodermal and endodermal lineage markers as well as cell migration, indicating that the long Tbx20a isoform uniquely bears functional domains that can alter gene expression and developmental behaviour in an in vivo context. We propose that Tbx20 plays an integrated role in the ancient myogenic program of the heart, and has been additionally coopted during evolution of vertebrates for endocardial cushion development.

Cardiac homeobox gene NKX2-5 mutations and congenital heart disease: associations with atrial septal defect and hypoplastic left heart syndrome.

OBJECTIVES We sought to examine the importance of mutations in the cardiac transcription factor gene NKX2-5 in patients with an atrial septal defect (ASD), patent foramen ovale (PFO), or hypoplastic left heart syndrome (HLHS). BACKGROUND Mutations in NKX2-5 have been found in families showing secundum ASD and atrioventricular (AV) conduction block and in some individuals with tetralogy of Fallot. The prevalence of NKX2-5 mutations in sporadic cases of ASD/PFO and other forms of congenital heart disease is unknown. METHODS A cohort of 146 individuals with secundum ASD, PFO complicated by paradoxical embolism, or HLHS were evaluated. Patients with ASD or PFO were ascertained irrespective of family history or associated cardiac abnormalities. The coding region of the NKX2-5 locus was amplified by polymerase chain reaction and sequenced. RESULTS Among 102 ASD and 25 PFO patients screened, 13 patients (10%) had a positive family history and 5 patients (4%) had AV conduction block. We found one previously documented NKX2-5 missense mutation, T178M, in members of a family with ASD without AV conduction block. One NKX2-5 mutation-positive child from this family had HLHS, although no mutations were subsequently found in 18 patients with sporadic or familial HLHS. In a second ASD family without AV conduction block, we found a missense change, E21Q, previously reported as pathogenic. Because this change did not segregate with disease status, we propose that it is a non-disease-causing polymorphism. CONCLUSIONS Our findings suggest that NKX2-5 mutations are a relatively infrequent cause of sporadic ASD and HLHS. Screening for NKX2-5 mutations may be warranted in individuals with ASD and a positive family history, irrespective of the presence or absence of AV conduction block.

Murine T-box transcription factor Tbx20 acts as a repressor during heart development, and is essential for adult heart integrity, function and adaptation

The genetic hierarchies guiding lineage specification and morphogenesis of the mammalian embryonic heart are poorly understood. We now show by gene targeting that murine T-box transcription factor Tbx20 plays a central role in these pathways, and has important activities in both cardiac development and adult function. Loss of Tbx20 results in death of embryos at mid-gestation with grossly abnormal heart morphogenesis. Underlying these disturbances was a severely compromised cardiac transcriptional program, defects in the molecular pre-pattern, reduced expansion of cardiac progenitors and a block to chamber differentiation. Notably, Tbx20-null embryos showed ectopic activation of Tbx2 across the whole heart myogenic field. Tbx2 encodes a transcriptional repressor normally expressed in non-chamber myocardium, and in the atrioventricular canal it has been proposed to inhibit chamber-specific gene expression through competition with positive factor Tbx5. Our data demonstrate a repressive activity for Tbx20 and place it upstream of Tbx2 in the cardiac genetic program. Thus, hierarchical, repressive interactions between Tbx20 and other T-box genes and factors underlie the primary lineage split into chamber and non-chamber myocardium in the forming heart, an early event upon which all subsequent morphogenesis depends. Additional roles for Tbx20 in adult heart integrity and contractile function were revealed by in-vivo cardiac functional analysis of Tbx20 heterozygous mutant mice. These data suggest that mutations in human cardiac transcription factor genes, possibly including TBX20, underlie both congenital heart disease and adult cardiomyopathies.

A Targeted NKX2.1 Human Embryonic Stem Cell Reporter Line Enables Identification of Human Basal Forebrain Derivatives

We have used homologous recombination in human embryonic stem cells (hESCs) to insert sequences encoding green fluorescent protein (GFP) into the NKX2.1 locus, a gene required for normal development of the basal forebrain. Generation of NKX2.1‐GFP+ cells was dependent on the concentration, timing, and duration of retinoic acid treatment during differentiation. NKX2.1‐GFP+ progenitors expressed genes characteristic of the basal forebrain, including SHH, DLX1, LHX6, and OLIG2. Time course analysis revealed that NKX2.1‐GFP+ cells could upregulate FOXG1 expression, implying the existence of a novel pathway for the generation of telencephalic neural derivatives. Further maturation of NKX2.1‐GFP+ cells gave rise to γ‐aminobutyric acid‐, tyrosine hydroxylase‐, and somatostatin‐expressing neurons as well as to platelet‐derived growth factor receptor α‐positive oligodendrocyte precursors. These studies highlight the diversity of cell types that can be generated from human NKX2.1+ progenitors and demonstrate the utility of NKX2.1GFP/w hESCs for investigating human forebrain development and neuronal differentiation. STEM CELLS 2011;29:462–473

Congenital Asplenia in Mice and Humans with Mutations in a Pbx/Nkx2-5/p15 Module

The molecular determinants of spleen organogenesis and the etiology of isolated congenital asplenia (ICA), a life-threatening human condition, are unknown. We previously reported that Pbx1 deficiency causes organ growth defects including asplenia. Here, we show that mice with splenic mesenchyme-specific Pbx1 inactivation exhibit hyposplenia. Moreover, the loss of Pbx causes downregulation of Nkx2-5 and derepression of p15Ink4b in spleen mesenchymal progenitors, perturbing the cell cycle. Removal of p15Ink4b in Pbx1 spleen-specific mutants partially rescues spleen growth. By whole-exome sequencing of a multiplex kindred with ICA, we identify a heterozygous missense mutation (P236H) in NKX2-5 showing reduced transactivation in vitro. This study establishes that a Pbx/Nkx2-5/p15 regulatory module is essential for spleen development.

Reconstructing Rangea: New Discoveries from the Ediacaran of Southern Namibia

Abstract Rangea is the type genus of the Rangeomorpha, an extinct clade near the base of the evolutionary tree of large, complex organisms which prospered during the late Neoproterozoic. It represents an iconic Ediacaran taxon, but the relatively few specimens previously known significantly hindered an accurate reconstruction. Discovery of more than 100 specimens of Rangea in two gutter casts recovered from Farm Aar in southern Namibia significantly expands this data set, and the well preserved internal and external features on these specimens permit new interpretations of Rangea morphology and lifestyle. Internal structures of Rangea consist of a hexaradial axial bulb that passes into an axial stalk extending the length of the fossil. The axial bulb is typically filled with sediment, which becomes increasingly loosely packed and porous distally, with the end of the stalk typically preserved as an empty, cylindrical cone. This length of the axial structure forms the structural foundation for six vanes arranged radially around the axis, with each vane consisting of a bilaminar sheet composed of a repetitive pattern of elements exhibiting at least three orders of self-similar branching. Rangea was probably an epibenthic frond that rested upright on the sea bottom, and all known fossil specimens were transported prior to their final burial in storm deposits.

Elastomeric nanocomposites as cell delivery vehicles and cardiac support devices

A new family of elastomeric nanocomposites has been developed from a soft elastomer poly(glycerol sebacate, PGS) and nanoparticles of Bioglass®. The new nanocomposites have been characterised in terms of materials science and evaluated for their potential clinical applications as cell delivery vehicles and cardiac support devices in the heart patch strategy. The addition of alkaline Bioglass® effectively counteracts the acidity caused by the degradation of PGS without severely compromising the compliance of PGS. As a result, the newly developed PGS–nanoBioglass (<5 wt%) composites have a greatly improved biocompatibility, compared to PGS, and remain mechanically compatible with heart muscle. The interaction between PGS and Bioglass® and the reinforcement of the PGS polymer network by the nanoBioglass® particles have also been explored in depth.

Analysis of Mitochondrial Function and Localisation during Human Embryonic Stem Cell Differentiation In Vitro

Human embryonic stem cell (hESC) derivatives show promise as viable cell therapy options for multiple disorders in different tissues. Recent advances in stem cell biology have lead to the reliable production and detailed molecular characterisation of a range of cell-types. However, the role of mitochondria during differentiation has yet to be fully elucidated. Mitochondria mediate a cells response to altered energy requirements (e.g. cardiomyocyte contraction) and, as such, the mitochondrial phenotype is likely to change during the dynamic process of hESC differentiation. We demonstrate that manipulating mitochondrial biogenesis alters mesendoderm commitment. To investigate mitochondrial localisation during early lineage specification of hESCs we developed a mitochondrial reporter line, KMEL2, in which sequences encoding the green fluorescent protein (GFP) are targeted to the mitochondria. Differentiation of KMEL2 lines into the three germ layers showed that the mitochondria in these differentiated progeny are GFP positive. Therefore, KMEL2 hESCs facilitate the study of mitochondria in a range of cell types and, importantly, permit real-time analysis of mitochondria via the GFP tag.

A tyrosine-rich domain within homeodomain transcription factor Nkx2-5 is an essential element in the early cardiac transcriptional regulatory machinery

Homeodomain factor Nkx2-5 is a central component of the transcription factor network that guides cardiac development; in humans, mutations in NKX2.5 lead to congenital heart disease (CHD). We have genetically defined a novel conserved tyrosine-rich domain (YRD) within Nkx2-5 that has co-evolved with its homeodomain. Mutation of the YRD did not affect DNA binding and only slightly diminished transcriptional activity of Nkx2-5 in a context-specific manner in vitro. However, the YRD was absolutely essential for the function of Nkx2-5 in cardiogenesis during ES cell differentiation and in the developing embryo. Furthermore, heterozygous mutation of all nine tyrosines to alanine created an allele with a strong dominant-negative-like activity in vivo: ES cell↔embryo chimaeras bearing the heterozygous mutation died before term with cardiac malformations similar to the more severe anomalies seen in NKX2.5 mutant families. These studies suggest a functional interdependence between the NK2 class homeodomain and YRD in cardiac development and evolution, and establish a new model for analysis of Nkx2-5 function in CHD.

Transcriptional Control and Pattern Formation in the Developing Vertebrate Heart: Studies on NK-2 Class Homeodomain Factors

Publisher Summary This chapter summarizes what is known about one specific gene family acting in transcriptional control and pattern formation in vertebrate and invertebrate hearts: the NK-2 class of homeodomain factors. It is found that frog Nkx2.5 is expressed in a region much broader than that occupied by the definitive heart progenitors, in a pattern resembling that of the morphogenetic field mapped in other amphibia. Thus, the regulatory ability of the field may be limited to the zone of Nkx2.5 expression. On the other hand, loss of regulatory ability in the field occurs in advance of the loss of the Nkx2.5 expression. In the fish, two NK-2 genes—nkx2.5 and nkx2. 7—are expressed in different but overlapping domains in a region encompassing the heart field. nkx2.5 expression occurs in those cells definitively fated to the heart but also extends more caudally beyond the heart field into cells that will never participate in heart development, even if definitive progenitors are killed by laser ablation. The notochord may provide inhibitory signals, which keep cells in the caudal region from differentiating as heart. Cells that participate in regulation have been mapped to the cranial region of the heart field, and these cells express nkx2.7 but not nkx2.5. The findings are consistent with the view that heart potency in the morphogenetic field requires the presence of a cardiac Nkx2 gene, but that other positive and negative interactions determine the actual extent of the definitive progenitors and of the field itself. Ectopic expression of Nkx2.5 in frog and fish embryos, achieved by microinjection of Nkx2.5 mRNA into fertilized eggs, leads to larger hearts and myocardial hyperplasia, without ectopic activation of the myogenic program.