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HOME PUBLICATIONS PROJECTS PEOPLE CONTACT	The Papaioannou Lab
Current Projects Tbx2 and Tbx3 in the cardiac conduction system The cardiac conduction system (CCS) is essential for the coordinated and rhythmic beating action of the heart. Functional from well before birth to the moment of death, it represents one of the most important organ systems in the body and its proper development is essential. The T-box transcription factors Tbx2 and Tbx3 demarcate the CCS from the earliest stages of its development until close to birth. Understanding how these two genes function both in tandem and individually in the development of the CCS will be important to shed light on congenital arrythmias as well as other heart defects. Using genetic studies including RNA assays as well as protein immunofluorescence in conjunction with murine disease models will allow us to shed light on the development of this critical system. Tbx4 Work on Tbx4 is largely focused on two organ systems: the allantois and the hindlimb. The allantois, which will later form the umbilical cord, expresses Tbx4 from its earliest inception at 7.5 dpc. Mutation of Tbx4 performed in this lab has shown that Tbx4 is not critical for the initial outgrowth of the allantois, but is required for ongoing survival of allantois cells and for proper differentiation within the allantois. The Tbx4 mutant allantois fails to fuse with the chorion, resulting in embryonic death at 10.5 dpc due to vascular insufficiency. Additionally, vasculogenesis in the developing allantois is arrested after the formation of endothelial cells and blood vessels are not formed. Currently research has indicated that Tbx4 may regulate a lateral inhibition process that decides which cells in the allantois assume endothelial fates. We are engaged in an ongoing project to elucidate this and other roles of Tbx4 in the allantoic vasculature. We are also exploring the role of Tbx4 in the hindlimb. Multiple labs had previously postulated that Tbx4 and the closely related gene Tbx5 regulate hindlimb and forelimb identities, respectively. Our mutation of Tbx4 and the corresponding mutation of Tbx5 by the Bruneau lab have demonstrated conclusively that these genes are required for outgrowth of the limbs, but recent work by the Logan lab has shown that they do not determine hind- or forelimb fate. Comparison of the mutations of Tbx4 and Tbx5 has shown subtle but important differences early limb roles of these genes. Ablation of Tbx5 results in a total block in forelimb outgrowth before the initial expression of Fgf10, whereas loss of Tbx4 allows the hindlimb bud to form, but Fgf10 is quickly downregulated and the hindlimb bud fails to grow. We are currently using a conditional allele of Tbx4 to examine later roles of this gene in the ongoing outgrowth of the hindlimb. Tbx6 Tbx6 is a T-box gene involved in paraxial mesoderm specification in the mouse embryo. In normal embryos Tbx6 is expressed in the early primitive streak, the presomitic mesoderm and the tail bud upon somite formation untill 12.5 dpc. Tbx6 null embryos die by 13.5 dpc. They form acanonical anterior somites and, in place of posterior somites, bilateral neural tubes form that branch in the abnormally expanded tail bud. The current hypothesis states that in Tbx6 null embryos, cells normally expressing Tbx6 fail to migrate into the region of developing somites resulting in an enlarged tail bud. In the case where mutant cells reach the site of somite formation, they differentiate to neural structures. In order to test this hypothesis, imaging of cells expressing Tbx6 in the presomitic mesoderm will be achieved using a vital nuclear marker that has recently been developed. Since Tbx6 is also involved in somite patterning and morphogenesis as shown by studies on embryos with a hypomorphic Tbx6 allele, the process of somitogenesis will also be investigated in these mutants. In the quest of rationalizing the normality of tissues in the Tbx6 null embryos that are thought to derive from Tbx6-expressing cells, a binary transgenic system has been adopted where Tbx6-expressing cells are conditionally fate mapped. We will also test whether Tbx6 acts in a positive manner by promoting mesoderm specification or a negative manner by repressing neural-specific genes. This will be achieved by making use of a knock-in strain that misexpresses Tbx6 only in tissues that express Cre recombinase. Embryonic pancreatic stem cells in the treatment of type 1 diabetes Type 1 diabetes mellitus is a chronic autoimmune disease caused by the pathogenic action of T lymphocytes on insulin producing beta cells. This is one of the most common chronic diseases of children and adolescents in USA. Currently, there is no permanent cure and all patients with type 1 diabetes require daily insulin shots to survive. The ultimate goal of our project is therapeutic intervention to improve metabolic control of the disease. Diabetic NOD (non-obese diabetic) mice can be rendered tolerant to the autoimmune process by treatment with anti CD3 antibody, although they do not recover full beta cell function. In this model, we will use transplantation approaches to test the efficacy of embryonic pancreatic stem cell therapy to correct beta cell deficiency. Pancreatic anlagen represents an unquestionable source of islet stem cells with the potential capability to restore normal beta cell mass. We are on the way of determining whether pancreatic anlagen can regenerate sufficient islet mass to reverse hyperglycemia in a tolerant NOD mouse. These studies have direct relevance to potential therapeutic treatments in humans, as anti CD3 antibody treatment is already in clinical trials in human type 1 diabetes patients. Derivation of human stem cell lines from nonviable embryos Human embryonic stem cells (hESCs) hold great promise for treatment and cure of patients suffering from degenerative diseases. However, this enormous potential in employing hESCs as a therapeutic tool is hampered by ethical and political issues. In the United States federal policy forbids the derivation of hESC from donated surplus embryos after in vitro fertilization (IVF) treatment. Thus, there is a need to provide a paradigm that would encompass derivation of hESCs, protect nascent human life, be consistent with federal policy and yet nurture investigations in the field of therapeutic innovation. In a recent retrospective study, hypocellular early morulae were determined as criteria that identified the subset of nonviable embryos in irreversible arrest. Such arrest corresponds to an irreversible loss of integrated organic function and thus death of the organism. Furthermore, it was shown that these developmentally arrested/dead embryos could be used for stem cell derivation. and to date, 12 hESC line were derived. Our goal is to establish the firm prospective criteria for classifying an embryo as irreversibly arrested, so that ethical norms of essential organ donation could be applied to stem cell derivation. Furthermore, our aim is to successfully isolate hESCs from nonviable/arrested embryos by establishing the protocol for derivation. Ultimately, we plan to generate a depository of hESCs under complete xeno-free conditions in feeder-independent culture.

Tbx2 and Tbx3 in the cardiac conduction system

The cardiac conduction system (CCS) is essential for the coordinated and rhythmic beating action of the heart. Functional from well before birth to the moment of death, it represents one of the most important organ systems in the body and its proper development is essential. The T-box transcription factors Tbx2 and Tbx3 demarcate the CCS from the earliest stages of its development until close to birth. Understanding how these two genes function both in tandem and individually in the development of the CCS will be important to shed light on congenital arrythmias as well as other heart defects. Using genetic studies including RNA assays as well as protein immunofluorescence in conjunction with murine disease models will allow us to shed light on the development of this critical system.

Tbx4

Tbx4 Picture Work on Tbx4 is largely focused on two organ systems: the allantois and the hindlimb. The allantois, which will later form the umbilical cord, expresses Tbx4 from its earliest inception at 7.5 dpc. Mutation of Tbx4 performed in this lab has shown that Tbx4 is not critical for the initial outgrowth of the allantois, but is required for ongoing survival of allantois cells and for proper differentiation within the allantois. The Tbx4 mutant allantois fails to fuse with the chorion, resulting in embryonic death at 10.5 dpc due to vascular insufficiency. Additionally, vasculogenesis in the developing allantois is arrested after the formation of endothelial cells and blood vessels are not formed. Currently research has indicated that Tbx4 may regulate a lateral inhibition process that decides which cells in the allantois assume endothelial fates. We are engaged in an ongoing project to elucidate this and other roles of Tbx4 in the allantoic vasculature.

We are also exploring the role of Tbx4 in the hindlimb. Multiple labs had previously postulated that Tbx4 and the closely related gene Tbx5 regulate hindlimb and forelimb identities, respectively. Our mutation of Tbx4 and the corresponding mutation of Tbx5 by the Bruneau lab have demonstrated conclusively that these genes are required for outgrowth of the limbs, but recent work by the Logan lab has shown that they do not determine hind- or forelimb fate. Comparison of the mutations of Tbx4 and Tbx5 has shown subtle but important differences early limb roles of these genes. Ablation of Tbx5 results in a total block in forelimb outgrowth before the initial expression of Fgf10, whereas loss of Tbx4 allows the hindlimb bud to form, but Fgf10 is quickly downregulated and the hindlimb bud fails to grow. We are currently using a conditional allele of Tbx4 to examine later roles of this gene in the ongoing outgrowth of the hindlimb.

Tbx6

tbx6 image

Tbx6 is a T-box gene involved in paraxial mesoderm specification in the mouse embryo. In normal embryos Tbx6 is expressed in the early primitive streak, the presomitic mesoderm and the tail bud upon somite formation untill 12.5 dpc. Tbx6 null embryos die by 13.5 dpc. They form acanonical anterior somites and, in place of posterior somites, bilateral neural tubes form that branch in the abnormally expanded tail bud. The current hypothesis states that in Tbx6 null embryos, cells normally expressing Tbx6 fail to migrate into the region of developing somites resulting in an enlarged tail bud. In the case where mutant cells reach the site of somite formation, they differentiate to neural structures. In order to test this hypothesis, imaging of cells expressing Tbx6 in the presomitic mesoderm will be achieved using a vital nuclear marker that has recently been developed. Since Tbx6 is also involved in somite patterning and morphogenesis as shown by studies on embryos with a hypomorphic Tbx6 allele, the process of somitogenesis will also be investigated in these mutants. In the quest of rationalizing the normality of tissues in the Tbx6 null embryos that are thought to derive from Tbx6-expressing cells, a binary transgenic system has been adopted where Tbx6-expressing cells are conditionally fate mapped. We will also test whether Tbx6 acts in a positive manner by promoting mesoderm specification or a negative manner by repressing neural-specific genes. This will be achieved by making use of a knock-in strain that misexpresses Tbx6 only in tissues that express Cre recombinase.

Embryonic pancreatic stem cells in the treatment of type 1 diabetes

Kidney Type 1 diabetes mellitus is a chronic autoimmune disease caused by the pathogenic action of T lymphocytes on insulin producing beta cells. This is one of the most common chronic diseases of children and adolescents in USA. Currently, there is no permanent cure and all patients with type 1 diabetes require daily insulin shots to survive. The ultimate goal of our project is therapeutic intervention to improve metabolic control of the disease. Diabetic NOD (non-obese diabetic) mice can be rendered tolerant to the autoimmune process by treatment with anti CD3 antibody, although they do not recover full beta cell function. In this model, we will use transplantation approaches to test the efficacy of embryonic pancreatic stem cell therapy to correct beta cell deficiency.

Pancreatic anlagen represents an unquestionable source of islet stem cells with the potential capability to restore normal beta cell mass. We are on the way of determining whether pancreatic anlagen can regenerate sufficient islet mass to reverse hyperglycemia in a tolerant NOD mouse. These studies have direct relevance to potential therapeutic treatments in humans, as anti CD3 antibody treatment is already in clinical trials in human type 1 diabetes patients.

Derivation of human stem cell lines from nonviable embryos

Human embryonic stem cells (hESCs) hold great promise for treatment and cure of patients suffering from degenerative diseases. However, this enormous potential in employing hESCs as a therapeutic tool is hampered by ethical and political issues. In the United States federal policy forbids the derivation of hESC from donated surplus embryos after in vitro fertilization (IVF) treatment. Thus, there is a need to provide a paradigm that would encompass derivation of hESCs, protect nascent human life, be consistent with federal policy and yet nurture investigations in the field of therapeutic innovation.

In a recent retrospective study, hypocellular early morulae were determined as criteria that identified the subset of nonviable embryos in irreversible arrest. Such arrest corresponds to an irreversible loss of integrated organic function and thus death of the organism. Furthermore, it was shown that these developmentally arrested/dead embryos could be used for stem cell derivation. and to date, 12 hESC line were derived.

Our goal is to establish the firm prospective criteria for classifying an embryo as irreversibly arrested, so that ethical norms of essential organ donation could be applied to stem cell derivation. Furthermore, our aim is to successfully isolate hESCs from nonviable/arrested embryos by establishing the protocol for derivation. Ultimately, we plan to generate a depository of hESCs under complete xeno-free conditions in feeder-independent culture.

Current Projects

Tbx4

Embryonic pancreatic stem cells in the treatment of type 1 diabetes