A role for Tbx5 in proepicardial cell migration during cardiogenesis

Transcriptional regulatory cascades during epicardial and coronary vascular development from proepicardial progenitor cells remain to be defined. We have used immunohistochemistry of human embryonic tissues to demonstrate that the TBX5 transcription factor is expressed not only in the myocardium, but also throughout the embryonic epicardium and coronary vasculature. TBX5 is not expressed in other human fetal vascular beds. Furthermore, immunohistochemical analyses of human embryonic tissues reveals that unlike their epicardial counterparts, delaminating epicardial-derived cells do not express TBX5 as they migrate through the subepicardium before undergoing epithelial-mesenchymal transformation required for coronary vasculogenesis. In the chick, Tbx5 is expressed in the embryonic proepicardial organ (PEO), which is composed of the epicardial and coronary vascular progenitor cells. Retrovirus-mediated overexpression of human TBX5 inhibits cell incorporation of infected proepicardial cells into the nascent chick epicardium and coronary vasculature. TBX5 overexpression as well as antisense-mediated knockdown of chick Tbx5 produce a cell-autonomous defect in the PEO that prevents proepicardial cell migration. Thus, both increasing and decreasing Tbx5 dosage impairs development of the proepicardium. Culture of explanted PEOs demonstrates that untreated chick proepicardial cells downregulate Tbx5 expression during cell migration. Therefore, we propose that Tbx5 participates in regulation of proepicardial cell migration, a critical event in the establishment of the epicardium and coronary vasculature

Tbx5 is Required for Avian and Mammalian Epicardial Formation and Coronary Vasculogenesis.

Rationale: Holt-Oram syndrome (HOS) is an autosomal dominant heart-hand syndrome caused by mutations in the TBX5 gene. Overexpression of Tbx5 in the chick proepicardial organ (PEO) impaired coronary blood vessel formation. However, the potential activity of Tbx5 in the epicardium itself, and Tbx5\u27s role in mammalian coronary vasculogenesis, remains largely unknown. Objective: To evaluate the consequences of altered Tbx5 gene dosage during PEO and epicardial development in the embryonic chick and mouse. Methods and Results: Retroviral-mediated knockdown or upregulation of Tbx5 expression in the embryonic chick PEO as well as proepicardial-specific deletion of Tbx5 in the embryonic mouse (Tbx5(epi-/-)) impaired normal PEO cell development, inhibited epicardial and coronary blood vessel formation and altered developmental gene expression. The generation of epicardial-derived cells (EPDCs) and their migration into the myocardium was impaired between embryonic day (E) 13.5-15.5 in mutant hearts due to delayed epicardial attachment to the myocardium and subepicardial accumulation of EPDCs. This caused defective coronary vasculogenesis associated with impaired vascular smooth muscle cell recruitment, and reduced invasion of cardiac fibroblasts and endothelial cells into myocardium. In contrast to wildtype hearts that exhibited an elaborate ventricular vascular network, Tbx5(epi-/-) hearts displayed a marked decrease in vascular density that was associated with myocardial hypoxia as exemplified by HIF1α upregulation and increased binding of Hypoxyprobe-1. Tbx5(epi-/-) mice with such myocardial hypoxia exhibited reduced exercise capacity compared to wildtype mice. Conclusions: Our findings support a conserved Tbx5 dose-dependent requirement for both proepicardial and epicardial progenitor cell development in chick and mouse coronary vascular formation

Keratin gene expression profiles after digit amputation in C57BL/6 vs. regenerative MRL mice imply an early regenerative keratinocyte activated-like state

Mouse strains C57BL/6 (B6) and MRL were studied by whole mouse genome chip microarray analyses of RNA isolated from amputation sites at different times pre-and postamputation at the midsecond phalange of the middle digit. Many keratin genes were highly differentially expressed. All keratin genes were placed into three temporal response classes determined by injury/preinjury ratios. One class, containing only Krt6 and Krt16, were uniquely expressed relative to the other two classes and exhibited different temporal responses in MRL vs. B6. Immunohistochemical staining for Krt6 and Krt16 in tissue sections, including normal digit, flank skin, and small intestine, and from normal and injured ear pinna tissue exhibited staining differences in B6 (low) and MRL (high) that were consistent with the microarray results. Krt10 staining showed no injury-induced differences, consistent with microarray expression. We analyzed Krt6 and Krt16 gene association networks and observed in uninjured tissue several genes with higher expression levels in MRL, but not B6, that were associated with the keratinocyte activated state: Krt6, Krt16, S100a8, S100a9, and Il1b; these data suggest that keratinocytes in the MRL strain, but not in B6, are in an activated state prior to wounding. These expression levels decreased in MRL at all times postwounding but rose in the B6, peaking at day 3. Other keratins significantly expressed in the normal basal keratinocyte state showed no significant strain differences. These data suggest that normal MRL skin is in a keratinocyte activated state, which may provide it with superior responses to wounding. © 2013 the American Physiological Society

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

Novel Role for Perinatal Myosin in Skeletal Muscle Development

Trismus-pseudcamptodactyly (TPC) is a rare, hereditary muscle syndrome that is characterized by an inability to open the mouth fully (trismus) and involuntary contractures of interphalangeal joints (pseudocamptodactyly). These jaw, hand and foot contractures complicate dental care, feeding during infancy, intubation for anesthesia, walking and also impair manual dexterity. Occupational and social disabilities occur as a result of these complications and these contractures often require surgical correction. Our lab sought to identify the underlying molecular genetic cause for TPC through mutational analysis of its affected family members. Sequence analysis revealed an Arg674Gln missense mutation (R674Q) in the MYH8 gene encoding the perinatal isoform of myosin heavy chain, MyHCpn. The Arg674 residue is highly conserved among the myosin heavy chain family of contractile proteins and it localizes to the actin-binding domain of the perinatal myosin head close to the ATP-binding site. To define the role of MyHCpn in mammalian development, we established a genetically engineered mouse line carrying the orthologous R674Q mutant Myh8 allele. Using Cre-lox technology to express the mutant isoform of Myh8 in the mouse genome, we mated our Myh8R674Qneo/+ mice with the EIIa-Cre ubiquitous driver mice to generate Myh8 R674Q/+;EIIa-Cre mice that are heterozygous for mutant Myh8. Newborn Myh8R674Q/+;EIIa-Cre mice die within minutes after birth due to respiratory failure. Gross examination of these mice at embryonic day (E) 18.5 revealed smaller embryos with bulging abdomens compared to wildtype control embryos. Histological examination of skeletal muscles from E18.5 Myh8R674Q/+;EIIa-Cre embryos revealed variability in muscle fiber size, signs of apoptosis and increased muscle degeneration/regeneration. Electron microscopic images of skeletal muscles showed enlarged mitochondria, focal absence of myofibrils, loosely arrayed fiber bundles and regional Z-line irregularities within the sarcomeres. We also observed the presence of nemaline deposits indicative of nemaline myopathy. These findings suggest a novel and crucial role for MyHCpn in skeletal muscle development

Role of CRK and CRKL Adaptor Proteins in Vascular Endothelial Cell Biology

Introduction: Proper formation of the coronary vessels is crucial during cardiovascular development. The coronary vessels are important for delivering oxygen and nutrients to the underlying myocardium. However, the molecular and cellular mechanisms that contribute to coronary vessel formation are poorly understood. Our lab identified Reelin, an extracellular matrix glycoprotein encoded by the RELN gene, as a potential contributor to vasculogenic and angiogenic processes during mammalian heart development. The Reelin functions through a signaling cascade that leads to activation of downstream effectors. Two of these effectors are the adaptor proteins Chicken tumor virus no. 10 Regulator of Kinase (CRK) and its paralog CRK like (CRKL). We observed Reelin to be localized to the vascular endothelial cells lining nascent and mature blood vessels in the embryonic mouse heart. We demonstrated that gene silencing of RELN in primary cultures of human dermal microvascular endothelial cells (HDMECs) via small interfering RNAs (siRNA) led to changes in cell biology. These changes included a reduction in vascular endothelial cell migration, a reduction in cell membrane permeability and an increase in capillary-like tube formation. At the molecular level, silencing of RELN altered expression of the angiogenic transcripts Angiopoietin-2 (ANGPT2) and A Disintegrin And Metalloproteinase with Thrombospondin Motifs 1 (ADAMTS1). Together these findings led us to speculate how Reelin modulates vascular endothelial cells. Objectives: Based on our understanding of the Reelin signaling pathway, we established an objective to examine the role of the CRK and CRKL downstream effectors in mediating these biological functions of Reeln in vascular endothelial cells. We hypothesized that CRK and CRKL may induce changes at the molecular level to angiogenic transcripts to mediate the biological functions of Reelin in vascular endothelial cells. Methods: We conducted experiments on HDMECs because they serve as an in vitro model to examine the contribution of CRK and CRKL to expression of angiogenic markers and cell morphology in vascular endothelial cells. Gene-specific siRNAs were used to target endogenous expression of CRK and CRKL in HDMECs. We analyzed ANGPT2 and ADAMTS1 mRNA and protein expression in control and CRK/CRKL knockdown (KD) cells by Taqman qPCR and western blot analyses, respectively. Also, we analyzed the morphological characteristics of these cells. Results: siRNA-mediated knockdown of CRK/CRKL KD in HDMECs yielded almost a 90% and a 75% reduction of CRK and CRKL mRNA expression relative to negative control cells. The CRK/CRKL KD HDMECs took on an irregularly elongated morphology with reduced density in comparison to the negative control cells. ANGPT2 mRNA expression was significantly downregulated by nearly 70%, and ADAMTS1 protein expression appeared to be upregulated in CRK/CRKL KD cells versus control cells. In addition, RELN mRNA expression was unaffected by silencing of CRK and CRKL in these cells. Conclusions: We concluded that CRK and CRKL influence vascular endothelial cell morphology and the expression of the angiogenic transcripts, ANGPT2 and ADAMTS1. Knockdown of CRK/CRKL in HDMECs produced irregularly elongated cells, decreased expression of ANGPT2 mRNA and increased ADAMTS1 protein expression. We observed similar molecular findings with our previous analysis of RELN KD in HDMECs. Altogether these findings provide evidence to support a role for CRK and CRKL as angiogenic molecular mediators of Reelin in vascular endothelial cells

Specification of the cardiac conduction system by transcription factors

Diseases of the cardiovascular system that cause sudden cardiac deaths are often caused by lethal arrhythmias that originate from defects in the cardiac conduction system. Development of the cardiac conduction system is a complex biological process that can be wrought with problems. Although several genes involved in mature conduction system function have been identified, their association with development of specific subcomponents of the cardiac conduction system remains challenging. Several transcription factors, including homeodomain proteins and T-box proteins, are essential for cardiac conduction system morphogenesis and activation or repression of key regulatory genes. In addition, several transcription factors modify expression of genes encoding the ion channel proteins that contribute to the electrophysiological properties of the conduction system and govern contraction of the surrounding myocardium. Loss of transcriptional regulation during cardiac development has detrimental effects on cardiogenesis that may lead to arrhythmias. Human genetic mutations in some of these transcription factors have been identified and are known to cause congenital heart diseases that include cardiac conduction system malformations. In this review, we summarize the contributions of several key transcription factors to specification, patterning, maturation, and function of the cardiac conduction system. Further analysis of the molecular programs involved in this process should lead to improved diagnosis and therapy of conduction system disease. © 2009 American Heart Association, Inc

Taking a bite out of hypertrophic cardiomyopathy: soy diet and disease

Some forms of hypertrophic cardiomyopathy (HCM) are caused by mutations in cardiac sarcomeric genes, but environmental factors are believed to influence the hypertrophic response. A highly variable but potentially significant environmental factor is diet. Since soy-rich diets have been speculated to confer protection against cardiovascular disease, Stauffer et al. have explored the influence of a soy diet on cardiac growth and function in a transgenic mouse model of HCM. They report that mice fed a soy diet exhibited significantly worse HCM than mice fed a soy-free (milk protein) diet. This study provides the first evidence of an environmental modifier — diet — on the hypertrophic phenotype and has implications for the way in which disease phenotypes are assessed in genetically altered murine models of disease