Research

Our lab is interested in the developmental, molecular and cellular underpinnings of cardiac and skeletal muscle progenitors. Thus far, our studies have primarily been recognized for their novel insights into the link between head muscle and heart development during vertebrate embryogenesis. In essence, our lab forms a bridge between developmental biology and the emerging field of regenerative medicine, with particular relevance to adult heart diseases and cancer. Current projects underway in the lab are briefly summarized below:

Embryogenesis

1. Signaling mechanisms
Our research into myogenesis and cardiogenesis demonstrates that the FGF-ERK signaling pathway plays an inhibitory role in myogenic differentiation. We now seek to understand the molecular and cellular underpinnings of this phenomenon, in both chick and mouse embryos and adult model systems. In particular, we focus on the crosstalk between mesoderm and neural crest cells during embryogenesis. Along the same line, the molecular and cellular nature of interactions which take place between organs during development is a fundamental and mostly unaddressed embryological question. We take a systems physiology view of embryonic development, and examine the hypothesis of a coordinated interaction in the morphogenesis of several organ systems, including: heart, liver, and blood.

2. Cardiac and craniofacial development
We are interested in learning how the heart is formed during embryogenesis and, more specifically, the cellular origins of distinct heart progenitor populations. We use both chick and mouse embryonic models to address these questions. In the process, we have revealed novel developmental roles of vascular endothelial cells during heart and craniofacial development (Fig. 1). Our search for the developmental mechanisms driving organogenesis led to identification of a gene regulatory network that orchestrates heart and craniofacial morphogenesis, findings that could pave the way toward a deeper understanding of certain birth defects (e.g., DiGeorge syndrome; Fig. 2).

Regeneration

3. Cardiomyocyte cell-cycle control and dedifferentiation
Ischemic heart disease is the most common postnatal cardiac ailment, and the leading cause of death in the Western world today. We combine novel approaches to study cardiomyocyte (CM) renewal and dedifferentiation. We focus on various signaling strategies: genetic and epigenetic modifications combined with attention to the microenvironment (Fig. 3). Indeed, our results demonstrate that CMs grown on soft substrates, as well as those grown in the presence of the extracellular matrix, proliferate more extensively, suggesting an essential role for the microenvironment in the control of CM proliferation (Fig. 4).

In an attempt to stimulate the re-entry of adult CMs into the cell cycle, we use mitogens and oncogenes (e.g., the NRG-ErbB2 signaling pathway) to test whether CMs can lose their terminally differentiated properties, proliferate, and then re-differentiate. These experiments are aimed to maximize the heart’s regenerative response.

4. CM reprogramming
Using the ‘defined factors’ reprogramming approach we were able to reprogram a CM cell line to induced pluripotent stem (iPS) cells (Fig. 5). Understanding how mature adult CMs can disassemble their sarcomeric architecture and re-enter the cell cycle, combined with the development of novel procedures that can facilitate CM dedifferention and proliferation, are major challenges facing current biomedical research.

Cancer

5. Manipulating tumorigenic processes to stimulate heart regeneration
Perhaps one of the most surprising and interesting aspects of the adult heart is the fact that cardiac tumors don’t exist. This implies that some factor/s within the heart, which we are keen to identify, resists tumorigenesis. If we could create tumors in the heart, we could more readily understand its resistance to cancer. Unleashing cell cycle control is a hallmark of cancer; on the other hand, these same mechanisms could be very useful in promoting heart regeneration. Hence, another angle of our research focus on the pros and cons of cell-cycle re-entry, finding just the right balance between these two processes (regeneration and tumorigenesis). In addition, we have found that reprogramming of tumor cells somehow resulted in nearly normal iPS cells. Paradoxically, it appears that the dedifferentiation processes can ‘cure’ the cancerous cell state, and we wish to understand how.

Fig.1:

 Fig.1: Vascular endothelial cells give rise to the heart endocardium and play a key role in cranial and cardiac morphogenesis. (A) Control E9.5 mouse embryo with normal pharyngeal arches and heart structures. (D) Section of control E9.5 embryo showing normal formation of endothelial cells (red). (B) Conditional mutant for Flk1 at E9.5 with deformation of heart and pharyngeal arches. (E) Section of mutant embryo showing loss of endothelial cells (red). (C) Model showing the distinction of endothelial / endocardial progenitors from myocardial progenitors as apposed to the diversification of multipotent cardiovascular progenitors.

Fig. 2:

Fig.2: The search for developmental mechanisms driving vertebrate organogenesis has paved the way toward a deeper understanding of birth defects. During embryogenesis, parts of the heart and craniofacial muscles arise from pharyngeal mesoderm (PM) progenitors. We are able to reveal a hierarchical regulatory network of a set of transcription factors expressed in the PM that initiates heart and craniofacial organogenesis; Lhx2 and Tcf21 genetically interact with Tbx1, the major determinant in the etiology of DiGeorge/velo-cardio-facial/22q11.2 deletion syndrome. Furthermore, knockout of these genes in the mouse recapitulates specific cardiac features of this syndrome. These findings shed new light on the developmental underpinnings of congenital defects.

Fig. 3:

Fig.3: Characterization of sorted populations for the separation of pure CM and non-CM populations derived from 1d and 7d hearts. (a) 1d and 7d MHC-Cre X dTomato-lox mice were sacrificed, hearts were removed and cells were isolated (b) Single cells were sorted by FACS according to dTomato fluorescence levels to separate CM from non-CM populations. Graphs represent RT-qPCR analysis of mature MHC, cTnt Nkx2.5, Gata4, Runx1, Vimentin.

Fig. 4:

Fig.4: The role played by the extracellular matrix in cardiac regeneration. Cells derived from the heart of 1 day old mice were seeded and grown for 4 days followed by 3 days of growth with fragments of extracted cardiac ECM from day 1 and 7 old mice. Cells were stained for cTNT (red) Ki67 (green) and Dapi (blue) and counted for cTNT+/Ki67+ vs cTNT+/Ki67 (graph).

Fig. 5:

Fig.5: Cardiomyocytes reprogramming to iPS cells. Reprogrammed CMs adapted ES morphology (A) and expression profile (B), while losing the expression of cardiac markers (C).

Acknowledgment:
This work was supported by research grants to E.T. from the European Research Council – FP7 program; the Association Francaise Contre les Myopathies; the Israel Science Foundation; the German-Israeli Foundation; and the United States – Israel Binational Science Foundation.

Keywords:
Cardiogenesis, Head muscles, organogenesis, cardiac regeneration