No description available.

Identifying the molecular and genetic pathways important for heart development and deciphering the causes of CHD are still a challenging puzzle. A newly identified piece of this puzzle is KLF13, a member of the Krüppel-like family of zinc-finger proteins, was found to be important for atrial septation and ventricular trabeculation of Xenopus embryos. The protein is expressed predominantly in the heart, binds evolutionarily-conserved regulatory elements on cardiac promoters, and activates cardiac transcription. In this study we examined KLF13 mechanism of action by investigating its transcriptional activity on the ANF promoter using a deletion/mutagenesis approach. We reported the identification of a new synergistic partnership between KLF13 and the individual cardiac transcription factors TBX5, NKX2.5, PEX1, and CATF1. Also, we localized KLF13’s transcriptional activation domain, the nuclear localization region/zinc-fingers, and the DNA binding zinc-fingers. This study will provide insight into the contribution of KLF13 to the development of CHDs.

Identifying the molecular and genetic pathways important for heart development and deciphering the causes of CHD are still a challenging puzzle. A newly identified piece of this puzzle is KLF13, a member of the Krüppel-like family of zinc-finger proteins, was found to be important for atrial septation and ventricular trabeculation of Xenopus embryos. The protein is expressed predominantly in the heart, binds evolutionarily-conserved regulatory elements on cardiac promoters, and activates cardiac transcription. In this study we examined KLF13 mechanism of action by investigating its transcriptional activity on the ANF promoter using a deletion/mutagenesis approach. We reported the identification of a new synergistic partnership between KLF13 and the individual cardiac transcription factors TBX5, NKX2.5, PEX1, and CATF1. Also, we localized KLF13’s transcriptional activation domain, the nuclear localization region/zinc-fingers, and the DNA binding zinc-fingers. This study will provide insight into the contribution of KLF13 to the development of CHDs.

Identifying the molecular and genetic pathways important for heart development and deciphering the causes of CHD are still a challenging puzzle. A newly identified piece of this puzzle is KLF13, a member of the Krüppel-like family of zinc-finger proteins, was found to be important for atrial septation and ventricular trabeculation of Xenopus embryos. The protein is expressed predominantly in the heart, binds evolutionarily-conserved regulatory elements on cardiac promoters, and activates cardiac transcription. In this study we examined KLF13 mechanism of action by investigating its transcriptional activity on the ANF promoter using a deletion/mutagenesis approach. We reported the identification of a new synergistic partnership between KLF13 and the individual cardiac transcription factors TBX5, NKX2.5, PEX1, and CATF1. Also, we localized KLF13’s transcriptional activation domain, the nuclear localization region/zinc-fingers, and the DNA binding zinc-fingers. This study will provide insight into the contribution of KLF13 to the development of CHDs.

Identifying the molecular and genetic pathways important for heart development and deciphering the causes of CHD are still a challenging puzzle. A newly identified piece of this puzzle is KLF13, a member of the Krüppel-like family of zinc-finger proteins, was found to be important for atrial septation and ventricular trabeculation of Xenopus embryos. The protein is expressed predominantly in the heart, binds evolutionarily-conserved regulatory elements on cardiac promoters, and activates cardiac transcription. In this study we examined KLF13 mechanism of action by investigating its transcriptional activity on the ANF promoter using a deletion/mutagenesis approach. We reported the identification of a new synergistic partnership between KLF13 and the individual cardiac transcription factors TBX5, NKX2.5, PEX1, and CATF1. Also, we localized KLF13’s transcriptional activation domain, the nuclear localization region/zinc-fingers, and the DNA binding zinc-fingers. This study will provide insight into the contribution of KLF13 to the development of CHDs.

Mospd3, a gene located on mouse chromosome 5, was identified in a gene trap screen in ES cells. The gene trap vector integration in multiple copies into the putative promoter of the gene, resulted in a loss of expression of Mospd3 at the trapped allele. In mice generated from ES cells carrying the vector integration it was found that the lack of Mospd3 expression resulted in the death of a proportion of the homozygote mutants within the first day after birth. Homozygote neonates exhibited a thinning of the right ventricular free heart wall which resembles other mouse mutant phenotypes as well as human congenital heart defects caused by a loss of desmosome and adherens junction mediated cell adhesion between cardiomyocytes. The protein encoded by Mospd3, contains an N-terminal Major Sperm Protein (MSP) domain implicated as a mediator of protein- protein interactions, as well as a two C-terminal transmembrane domains. Both, protein structure and phenotypic similarities with defects in desmosomal and adherens junction proteins suggests that Mospd proteins might play a role in cell adhesion and maintaining the structural integrity of the heart. The phenotype of Mospd3 mutants was highly dependent on genetic background, which led us to speculate that there might be genetic redundancy between Mospd3 and its closest family member the X-linked Mospd1. The aims of this thesis were to generate tools to better understand the function of the Mospd gene family in cardiac development as well as assessing genetic redundancy between Mospd1 and Mospd3. A conditional gene targeting strategy was designed for both Mospd genes. Large genomic regions of the Mospd1 and Mospd3 loci were subcloned from bacterial artificial chromosomes (BACs) and using a recombineering approach, loxP sites and a drug selection cassette (neomycin) were placed in precise locations surrounding the MSP domain of both genes. The conditional targeting vectors were electroporated into both CGR8 and E14 ES cells and homologous recombinant clones were identified at a frequency of 2% and 1.3% for Mospd1 and Mospd3 respectively. Five euploid targeted clones for both Mospd1 and Mospd3 have been generated. Transient expression of Cre recombinase in ES cells carrying the conditional Mospd1 allele was used to delete the one copy of this X-linked gene. Phenotypic characterisation of this null ES cell line revealed that Mospd1 is neither essential for ES cell viability and self-renewal, nor for the early differentiation of these cells towards a cardiac fate. In order to investigate the mechanism of action of Mospd proteins, specific polyclonal antibodies were generated to detect either Mospd1 or Mospd3. These antibodies were purified and tested by western blotting using COS7 cells overexpressing either Mospd protein as well as mouse tissue lysates. Whilst the antibodies were found to detect the proteins and differentiate between Mospd1 and Mospd3, they showed insufficient purification to be used in co-localisation and co-immunoprecipitation experiments to identify interacting proteins and determine whether Mospd proteins are involved in cell adhesion complexes. Monoclonal antibodies were subsequently generated and initial western blotting experiments showed promising results, indicating that these antibodies may be better suited for immunohistochemical analysis of Mospd proteins.

No description available.

The incidence of malformed newborns is higher in human pregnancies complicated by diabetes mellitus, as compared to non-diabetic pregnancies. Neural tube and cardiac defects predominate among the fetal malformations induced by hyperglycemia. Non-specific maternal immune stimulation is protective in mice against birth malformations caused by chemical or physical teratogens, or by maternal diabetes mellitus. Insulin dependent diabetes was induced in ICR females to study the late gestation fetal heart by morphometric analysis. Diabetic females treated with Freund's compete adjuvant (FCA) or interferon-gamma (IFNγ) were also generated to elucidate potential positive effects of maternal immune stimulation during the diabetic pregnancy by morphometric analysis and pathologic scoring. Insulin-dependent CD1 females were generated to analyze late gestation fetal myocardial apoptosis by flow cytometric analysis and by real time-polymerase chain reaction (RT-PCR) analysis of a panel of 5 genes involved in apoptosis/proliferation (Bcl-2, P53, Caspase3, Caspase9 and PkC-e). The morphometric analysis of fetal hearts revealed visibly obvious dilation of ventricular chambers and outflow channel of the left ventricle, and reduction of total myocardial ventricular area in late gestation fetuses, as predominant changes seen in the offspring of diabetic dams. Pathologic scoring revealed that maternal immune stimulation, particularly with FCA, in part alleviated fetal heart changes of cavitary dilation and myocardial reduction. Increased rate of apoptosis/necrosis in the fetal myocardium in late gestation during the diabetic pregnancy was evidenced by flow cytometric analysis. Particularly there was a significant increase in percentage of early apoptotic cells in the fetal myocardium detected by cell markers annexin V and propidium iodide. There was also a significant increase in percentage of late apoptotic/necrotic fetal myocardial cells in the diabetic group compared to the control group. These results suggest that maternal treatment with FCA may in part protect the heart from high hyperglycemia by reducing the number of myocardial cells undergoing apoptosis and necrosis. The RT-PCR analysis revealed subtle changes in gene expression for all the genes except Bcl-2. A paradoxical and dramatic up-regulation of this anti-apoptotic gene was observed in late gestation fetal myocardium from the insulin-dependent hyperglycemic groups. Possibly, this could be a mechanism to protect the fetal myocardial cell from the chronic exposure to a severe hyperglycemic insult and consequent apoptosis. In conclusion, maternal insulin-dependent diabetes caused morphological changes in the late gestation fetal heart. Such changes were in part related to dysregulation of myocardial apoptosis. Maternal immune stimulation with FCA improved fetal heart morphology, by a mechanism that may in part relate to normalizing fetal myocardial apoptosis. / Ph. D.

Thisbe (Ths) and Pyramus (Pyr) are the ligands for the Fibroblast-Growth-Factor (FGF)receptor Heartless (Htl), which is expressed in all mesodermal cells during gastrulation. To understand how these two FGFs orchestrate mesoderm spreading in gastrulation and mesoderm differentiation during organogenesis, loss and gain of function studies were performed. In an approach of functional analysis, a single mutant allele of ths was generated, ths759, for comparison of the single mutant conditions of ths and the null mesodermal cells to migrate and differentiate in a precise pattern.

Iroquois homeobox (Irx) family members, a group of highly conserved homeodomain containing transcription factors, are involved in the patterning and the proper functions of vertebrate organs. They can act as transcriptional activators or repressors in a context-dependent manner. Preliminary data indicated that Irx3 and Irx5 are functionally redundant during cardiac morphogenesis, and they physically interact with other cardiac transcription factors. At E14.5, outflow tract septation failure and ventricular septation failure were observed in Irx3/5DKO mouse hearts. Loss of Irx3/5 in neural crest and endothelial cell lineages led to outflow tract septation failure and ventricular septal defect. In adult mice, Irx3 is expressed in the atrioventricular conduction system, and loss of Irx3 leads to slower ventricular conduction velocity. qRT-PCR analysis and immunofluorescence staining revealed that the expression of gap junction proteins, Cx40 and Cx43, are affected by the loss of Irx3. Over-expression of Irx3 and a dominant repressor form of Irx3, Irx3-EnR, resulted in Cx40 upregulation, indicating that Irx3 acts as an indirect positive regulator of Cx40. Irx3-EnR over-expression in vivo resulted in postnatal onset of atrial enlargement, ventricular hypertrophy, and conduction failure. Taken together, this study demonstrates the significance of Irx3/5 in both cardiovascular development and cardiac electrophysiology.