Electrical pattern formation and function in organogenesis and tissue growth

Abstract

Monoatomic ions are among the most abundant chemical species in the biological environment. Cells maintain complex protein machinery and expend a large fraction of their energy budget to control the relative distribution of different ions between themselves and their surroundings, as well as between cellular compartments in eukaryotes. We have a deep understanding of ion physiology in the electrical action potentials of excitable cells like neurons and myocytes. However, the universality of ion regulation across all cells suggests that much more can be learned about how cells sense their ionic state in diverse contexts and use it to represent important information for other biological decisions. Here, I present studies that advance our understanding of the organization and function of cellular ion fluxes at three different spatial scales: 1) a contiguous tissue of genetically similar cells; 2) a physiological functional unit consisting of multiple cell types across multiple organs; and 3) individual molecular machines within single cells. Across these works I exploit all-optical approaches combining light-activated, spatiotemporally localized perturbations with sensors of physiological dynamics in living cells. These tools allow us to dissect causal relationships between biological parameters that have historically been understood to be important to physiology but challenging to directly visualize and control in their native context. First, we study the earliest spiking activity in the zebrafish heart, imaging the transition from silent to electrically excitable to spontaneously spiking during cardiac development and developing mathematical descriptions for the timing and spatial organization of the earliest heartbeats. In the adult heart, rhythmic activity is generated by specialized pacemaker cells, whereas most other cardiomyocytes have much less spontaneous electrical activity\cite{bartos_ion_2015,liu_electrophysiological_2016}. In general, immature cardiomyocytes have more spontaneous activity and share more molecular similarities with pacemaker cells\cite{liu_electrophysiological_2016}. We observe that the initiation of periodic spiking during embryonic heart development is not a direct consequence of pacemaker cell differentiation and show that it does not involve deterministic encoding of periodicity. Second, we consider ion physiology in the body vasculature. The vertebrate circulatory system selectively and transiently adjusts blood flow to meet the local demand for specific tissues, for example, in neurovascular coupling and exercise. Several of the mechanisms that enable these responses involve ion flows, including calcium entry and membrane potential hyperpolarization downstream of physiological inputs . One such input is change in shear stress exerted by blood flow on vessels, which is assumed to be driven by a change in heart rate . However, changes in heart rate should systemically adjust flow, and additional mechanisms are required to explain local changes in vascular tone. We use the larval zebrafish to observe multiple different processes executed by different cell types, driving the physiological cascade to a vascular response. We show that, rather than sensing changes in cardiac output, the mechanosensitive ion channel Piezo1 triggers intracellular calcium elevation by directly sensing compression on blood vessels. This constitutes a novel mechanism for converting routine organismal behavior into a local cellular response. Third, we examine how cellular ion physiology might influence proliferation and differentiation. We develop approaches to read out the effects of ionic perturbations on ERK/MAPK signaling, a pathway critical for cell cycle progression and various differentiation processes during development. ERK/MAPK signaling has been reported to be sensitive to calcium elevation , transmembrane potential and externally applied electric fields. We isolate physiological events immediately downstream of these perturbations and show that ionic triggers of ERK signaling act through the structural dynamics of specific potassium channels, rather than by universal sensing of any physiological parameter. This may confer different response behaviors on different cell types and could allow spatio-temporal coupling of divergent behaviors in complex developmental processes. Together, these studies extend our boundaries of where electrophysiological function can occur, of what the systemic function of such activity can be, and of the nature of the biological information it can carry. These efforts provide a framework for exploring new functions of ion fluxes in contexts beyond classical excitability, and offer a glimpse of future work that could further integrate ion physiology into our global view of how cells and tissues sense and adapt to their environment and internal state.