WHOLISTIC framework

by Virginia MS Ruetten on June 1, 2023

All cells in an animal collectively ensure, moment-to-moment, the survival of the whole organism in the face of environmental stressors^1,². Physiology seeks to elucidate the intricate network of interactions that sustain life, which often span multiple organs, cell types, and timescales, but a major challenge lies in the inability to simultaneously record time-varying cellular activity throughout the entire body.

We developed WHOLISTIC, a method to image second-timescale, time-varying intracellular dynamics across cell-types of the vertebrate body. By advancing and integrating volumetric fluorescence microscopy, machine learning, and pancellular transgenic expression of calcium sensors in transparent young Danio rerio (zebrafish) and adult Danionella, the method enables real-time recording of cellular dynamics across the organism. Calcium is a universal intracellular messenger, with a large array of cellular processes depending on changes in calcium concentration across varying time-scales, making it an ideal proxy of cellular activity³.

Using this platform to screen the dynamics of all cells in the body, we discovered unexpected responses of specific cell types to stimuli, such as chondrocyte reactions to cold, meningeal responses to ketamine, and state-dependent activity, such as oscillatory ependymal-cell activity during periods of extended motor quiescence. At the organ scale, the method uncovered pulsating traveling waves along the kidney nephron. At the multi-organ scale, we uncovered muscle synergies and independencies, as well as muscle-organ interactions. Integration with optogenetics allowed us to all-optically determine the causal direction of brain-body interactions. At the whole-organism scale, the method captured the rapid brainstem-controlled redistribution of blood flow across the body.

Finally, we advanced Whole-Body Expansion Microscopy⁴ to provide ground-truth molecular and ultrastructural anatomical context, explaining the spatiotemporal structure of activity captured by WHOLISTIC. Together, these innovations establish a new paradigm for systems biology, bridging cellular and organismal physiology, with broad implications for both fundamental research and drug discovery.

Background

Cells across an organism must continuously coordinate with one another to sustain life and adapt to changing external environments and alterations within the body. This is achieved through a complex network of dynamic interactions that maintains homeostasis and underpins the organism’s resilience to stress⁵. Disease can arise from breakdowns in these intercellular feedback mechanisms^6,^7,^8,^9,^10,¹¹. Although biomedicine has made strides in uncovering key interactions, such as stress responses mediated by neuroendocrine signaling¹² or neural pathways between the brain and the gut^13,^14,^15,¹⁶, a vast number of mechanisms of whole-organism function remain elusive^1,^17,². Our understanding is limited by the challenges of accessing body-wide cellular dynamics in both health and disease, highlighting the need for advancements in technologies to measure, analyze, and model body-wide control mechanisms at the cellular level.

Modern synergies between imaging technology and protein engineering allow for time-varying molecular signals to be recorded in tissues. This has caused revolutions in fields like neuroscience through the imaging of calcium — a fast, universal intracellular messenger involved in a wide range of cellular processes, including neuronal action potentials³ — and other signals including voltage and neuromodulators across many neurons simultaneously^18,^19,^20,^21,²². However, time-varying activity patterns of most cell types in the body have not yet been recorded.

For most vertebrate models, optical access to large, opaque tissues poses a currently insurmountable challenge to whole-body imaging. Transparent vertebrate animals such as young zebrafish and adult Danionella^23,^24,²⁵ overcome this barrier, making them uniquely suited as models for in vivo studies of cellular dynamics across the body²⁶, offering unparalleled access to the inner workings of evolutionarily conserved organs such as the liver, pancreas, gut, brain, and the immune and cardiovascular systems^27,²⁸.

This study introduces WHOLISTIC (WHole Organism Live Imaging System for recording Tissue and IntraCellular activity), a platform that enables in vivo imaging of cellular calcium dynamics, generalizable to other molecular dynamics, across nearly all cells of transparent vertebrates, such as the young zebrafish. By extending and integrating an array of technical advances, including pancellular transgenic lines expressing genetically encoded calcium indicators in almost all cells in the body, high-speed volumetric fluorescence imaging, and a suite of computational methods for registration and cell population analysis, along with Whole-Body Expansion Microscopy, we capture, analyze, and interpret cellular activity across tissues and organ systems.

Together, these techniques provide a discovery platform that allows for the comprehensive investigation of coordinated cellular activity within an intact and awake vertebrate.

Looking ahead

The organism has long been acknowledged as a cohesive entity, comprised of a network of cells that communicate and coordinate across multiple scales²⁹. The elaborate feedback loops embedded within this network enable animals to respond to, predict, and prepare for both internal and external challenges. While many regulatory pathways—such as those governing hunger, glucose balance, and immune responses—are well characterized³⁰, the complete picture of organism-wide control systems remains a formidable challenge. Given the interdependence of these cellular interactions, considering the organism as an integrated whole is essential for full comprehension^2,^17,¹. Despite this acknowledged complexity, methodologies that probe vertebrate organisms in their entirety while retaining cellular-level access have remained elusive. This study introduces a novel approach to elucidating whole-body cellular dynamics through the imaging and analysis of genetically encoded sensors expressed ubiquitously across all cells, thereby offering unprecedented insights into the integration of cellular interactions and organism-wide control systems.

Future implementations of WHOLISTIC in freely behaving animals will enable the study of body-wide cellular control systems in naturalistic settings. This advancement will facilitate the investigation of the dynamic interplay between complex behavioral states and strategies, such as goal-driven navigation and sleep-wake transitions, and organism-wide physiology. A parallel route to such studies will occur through the creation of experimental systems that incorporate multimodal virtual-reality environments³¹ composed of, for example, visual, temperature, chemical, mechanical, and other types of stimuli which respond to an animal's behavioral output. Combined with the further improvements in the ability to monitor and activate cellular signals across the entire body in closed-loop through advances in protein engineering and microscopy¹⁸, this will open up fundamentally new avenues of scientific inquiry.

The use of calcium sensors^32,³³ serves as an effective proxy for cellular activity due to calcium's ubiquitous role in cellular processes³⁴. The finding that most cells have calcium-activity dynamics within the range of standard sensors has enabled the extraction of a plethora of cellular dynamics, but this is still a limited view of the full dynamics within and between cells, which occur through signaling via a multitude of molecular, voltage, and mechanical pathways. Further measurements of additional molecules and physical cellular properties will provide deeper insights. Indeed, the WHOLISTIC methodology is extendable to co-imaging with other signals within the rapidly expanding repertoire of molecular, voltage, and mechanical sensors^35,^36,^37,^38,^39,⁴⁰, allowing access to a variety of additional information channels such as metabolic states and molecules like hormones, ATP, and neuromodulators that convey communication signals between cells across the organism.

Such additional measurements will enrich our data-driven understanding of organism-wide physiology, but due to the size and complexity of multicellular interactions will necessitate advanced analytical and modeling frameworks that should leverage advances in modern AI algorithms including Graph Neural Networks⁴¹, possibly in combination with large-scale biophysical models and model-free methods for predicting dynamics, such as Empirical Dynamic Modeling⁴². Furthermore, platform automation⁴³ AI-in-the-loop systems that combine measurements of whole-body cellular dynamics with online cell-targeted perturbation will be useful not only for reaching fundamental biological insights but also for prototyping future real-time interventions for medical conditions[@hovorka_closed-loop_2011]⁴³[@Pardo-Martin2013].

The fact that all findings presented here — pertaining to different cell types, in different parts of the body, under different physiological assays and screens — initially derived from the same WHOLISTIC methodology underscores its wide-ranging applicability to fundamental biology and to medical research. The methodology can be used as a discovery tool, while validation and deeper insight can be gained using more specific molecular techniques, as performed here to understand phenomena like the brainstem's control of blood flow redistribution during physiological stress and the molecular identification of the quiescence-related signals as originating from ependymal cells.

Cellular function is determined by a cell's molecular and biophysical properties, its tissue environment, and connectivity to other cells^44,⁴⁵. The combination of organism-wide cellular activity imaging, the concept of `functional tissue compartments' which merges function and anatomy, and whole body tissue expansion methods offers the integration of whole-body cellular activity measurements and the molecular, cellular, and connectivity properties of cells across the body. Developing accurate, body-wide registration methods to match the in-vivo to the ex-vivo expanded data cell-by-cell will enable analyses and insights at an enhanced level of depth and detail, including, for instance, matching sets of hormone-secreting neurons with neurons expressing the corresponding receptors[@ripoll-sanchez_neuropeptidergic_2023]. Much information throughout the body flows through the nervous system, the central and peripheral nervous systems, the connectivity of which is becoming more readily accessible through light-based connectomics⁴⁶ improved machine learning algorithms for connectome reconstruction[@januszewski_high-precision_2018]. The current work will seed the creation of an atlas encompassing the mechanistic, molecular basis for the observed functional coupling at cellular detail across the whole organism. %body-wide multimodal body model to enable a body-wide dynamic, cellular, molecular, and connectivity-based understanding of whole-organismal function.

Beyond enabling progress in fundamental biology, WHOLISTIC enables screens in disease models that allow for tracking the effects of pathologies and potential treatments at multiple spatial scales from cells to the whole body and at temporal scales from seconds to days. The majority of drug-screening workflows depend on observing a subset of drug-induced phenomenology such as molecular binding properties⁴⁷, effects on individual cells or subset of tissues⁴⁸, or influence on behavior^49,⁵⁰. The approach presented here offers a complementary lens through which to observe the impact of drugs and genetic interventions on the entire body, enabling the identification of unanticipated off-target consequences in organ systems not under direct study, as well as the discovery of unexpected benefits in tissues not previously considered. It would also reveal outcomes arising from system-wide interactions that could be missed when studying cells, tissues, or organs individually, rather than collectively within their native whole-body environment.

In conclusion, WHOLISTIC constitutes a bridge between cellular and organismal physiology, systems neuroscience and behavior, ushering in a new frontier in systems biology. By embracing the complexity of organisms while retaining the individual cell as a fundamental unit of analysis, this work affords a holistic yet mechanistic approach to unravel the complete cellular interplay governing health and disease in organism-wide networks.

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