Physiological cues that include electrical and ionic impulses or metabolic intermediates have been long implicated in the orchestration of organ development during early embryonic patterning. While much is known about genetically encoded developmental pathways, the mechanisms by which these small molecules interact during embryogenesis are poorly understood. We have developed technologies to explore the role of electrochemical and other physiologic stimuli during development in the zebrafish. Recently, we have demonstrated that Wnt11 non-canonical signaling, a major developmental pathway regulating tissue morphogenesis and organ formation, patterns intercellular electrical coupling in the myocardial epithelium through effects on transmembrane calcium conductance mediated via the L-type calcium channel. This finding offers a new entry point into an aspect of Wnt non-canonical signaling that has been proven difficult to explore. We focus on studying the Wnt/calcium pathway in the context of the developing embryo utilizing various techniques ranging from electrophysiology to developmental biology. Our immediate interest is to describe the molecular mechanisms that lead to the Wnt-mediated attenuation of L-type calcium channels, to characterize how non-canonical Wnt signals compartmentalize and affect different calcium domains in various tissues, and to determine how electrochemical signals modulated by Wnt11 regulate cardiac morphogenesis and function.
Wnt11 regulates Ca2+ transient amplitudes in cardiomyocytes: a,b Colour maps of Ca2+ transient amplitudes from control hearts (a) and Wnt11 morphants (b). Colour code depicts Ca2+ transient amplitudes in fluorescence ratio units (F340/F380). Squares indicate automated ROIs for measurements averaged in c. (c) Averaged Ca2+ transients from ROIs in a,b.
Project 1: Molecular bases of interactions between Wnt non-canonical pathway and L-type Ca2+ channel.
While we have shown that Wnt11 non-canonical signaling modulates the electrical coupling of cardiomyocytes via negative regulation of the L-type Ca2+ channel (LTCC) conductance, there are many questions that remain to be answered. How does Wnt11 signaling modulate LTCC function? Is the interaction between Wnt11 and LTCC direct or indirect? What known downstream components of Wnt11 signaling pathways are involved in the LTCC attenuation? In order to define the molecular mechanism by which Wnt11 regulates LTCC at a subcellular resolution, we have moved from zebrafish embryonic hearts to cell-based systems. This allows us to perform not only immunological experiments, but also more importantly detailed biochemistry.
Project 2: Characterization of the components of the Wnt non-canonical transduction pathway involved in the L-type Ca2+ channel attenuation.
The specificity and a wide range of diverse biological effects of Wnt signaling are mediated via binding and affinity of distinct Wnts to their corresponding receptors, Frizzleds (Fzd). It has been shown that Wnt11 interacts with Fzd7 genetically as well as physically. A crucial step in Wnt signaling is the translocation of Dishevelled (Dsh) from the cytoplasm to the plasma membrane, relaying the Wnt signal to various downstream effectors. Dsh has been implicated so far in all known aspects of Wnt signaling, canonical and non-canonical. We aim to define the requirement of the Wnt transduction machinery in the process of attenuation of L-type Ca2+ channel.
Project 3: Role of Wnt11/Ca2+ signaling in cardiac development.
We have demonstrated that Wnt11 patterns electrical coupling through regulation of LTCC conductance. However, we do not know what cellular responses precede this patterning. It is feasible to envisage the development of electrical coupling as a process of junction formation. Indeed, Wnt non-canonical signaling has been implicated in regulating cell adhesion and junctional remodeling in many cell biological and developmental contexts. Hence, we have begun addressing the question whether Wnt/calcium signaling is involved in junction formation.
Project 4: Quantitative analysis of Ca2+ compartmentalization by non-canonical Wnt-dependent mechanisms.
To further understand the role of Wnt11 signaling in regulating Ca2+ fluxes, we decided to take a systematic approach. We are taking advantage of the ratiometric high-resolution Ca2+ imaging with Fura-2, that we have developed, to perform quantitative analysis of Ca2+ fluxes in both excitable and non-excitable tissues. The final aim is to create a comprehensive quantitative atlas of [Ca2+]i in Wnt/Ca2+ signaling in distinct cell types.
Project 5: The role of ionic cues in angiogenesis.
It is well established that during their development, vessels and nerouns share common molecular mechanism. Besides genetically encoded signals, functional cues have been shown to play an important role in regulating neurogenic processes. However, very little is known about such cues during angiogenesis. The central idea of this project is to determine the role of ionic fluxes during vessel outgrowth.