Several groups have successfully adapted imaging or other assays to multiwell plates and undertaken screens for genetic or chemical modifiers with varying degrees of success. For example, in the cardiovascular arena, sophisticated assays of cardiac rate, rhythm, contractility, blood flow, vasomotion, and rheology are available, and can often be multiplexed using different fluorescent reporters and refined image analysis techniques.43,45,57
reporters have been used to screen for mutants affecting the developmental maturation of cardiac physiology. Combining screening technologies in series to optimize sensitivity and specificity in turn, highlights the utility of counter-screening. This type of staged or combination screen is readily feasible with higher throughput assays, and allows sophisticated screen logic to be applied.59
Assays for many other physiologic functions have been developed (see examples in ), and as the zebrafish is becoming increasingly popular as a model for physiology and disease, the range of these assays will continue to grow exponentially. Investigators can exploit not only the integrative physiology of the zebrafish, but also the ontologic sequence that the organism offers. Different stages of integration can be defined for many of the processes being studied, enabling evaluation of the role of early function in the patterning of whole organism responses. Perhaps the most powerful strategies will incorporate aspects of cell biology, physiology, and drug challenges, all in the context of validated zebrafish models of disease. The potential for data collection on a scale matching that of functional genomics is close to being realized, but will require collaboration among geneticists, cell and computational biologists, engineers, and physiologists.
Examples of Scalable Modeling in Zebrafish
Where higher throughput assays have been developed, it has been possible to exploit the zebrafish for the annotation of small molecule function, for in vivo
approaches to the study of drug toxicity, empiric pathway discovery or the testing of large numbers of variables in genetically faithful disease models.41,60
This approach has been used to explore phenotypes including the pharmacogenetics of specific drug responses and the development of repolarization. Similarly, behaviors ranging from individual reflexes through midbrain responses to social interaction have been characterized in the zebrafish, and large-scale screens for fundamental neurological responses are underway.
A recent study by Mitchison et al. depicts the power of the zebrafish model for studying dynamic physiologic processes.61
The authors focused on the problem of wound detection and healing—in particular the mechanism by which, in the initial minutes after injury, leukocytes from hundreds of microns away sense and migrate toward damaged tissue. Given its biochemical properties, they hypothesized that hydrogen peroxide (H2
) may contribute toward the required spatial signaling.
An in vivo
evaluation of the hypothesis required that the authors genetically and pharmacologically manipulate a living biological system, while simultaneously allowing visualization of the dynamics of a chemical gradient and the resulting migration of leukocytes—a task ideally suited for study in the zebrafish embryo.61
The authors made use of genetically encoded H2
sensor, HyPer, which consists of a circularly permuted yellow fluorescent protein (YFP) covalently attached to OxyR, a bacterial transcription factor that changes conformation in response to H2
. The resulting change is then transmitted to YFP, altering its fluorescent properties, and allowing specific H2
quantification in vivo
. Using this probe in the context of an embryonic zebrafish tail injury model, the authors were able to detect a rapid increase in H2
at the wound margin which extended outwards several hundred microns. Leukocyte influx was tracked using leukocyte-specific fluorescent tags and found to temporally follow rather than precede H2
production, further supporting H2
’s role as a spatial chemotactic signal. Finally, the authors elucidated the molecular basis for the peroxide gradient through chemical and genetic inhibition, thus establishing dual oxidase
(Duox1) as the enzyme responsible for generating H2
at the wound margin.
Small model organisms not only permit the opportunity to model intercellular
crosstalk in vivo
, but such approaches can potentially lead to quantitative, dynamic models of higher-order processes such as cell-fate specification, which rely on the integration of local signals to determine cellular identity. Although there has been little use of zebrafish for this purpose to date, similar investigations in other small organisms can suggest potential applications of the zebrafish system toward defining mechanisms of intercellular communication for vertebrate-specific processes such as glomerular filtration or arrhythmias.62
One of the most elegant examples of cell-fate specification is that of C. elegans
vulval development, originally described in detail in 1989 by Sternberg and Horwitz.63
The C. elegans
vulva arises from three vulval precursor cells, which integrate signals from a gonadal anchor cell, the surrounding hypodermal syncytium and one another to attain one of three cellular fates. The genes important in sending and receiving signals have been identified, and a systematic series of mutations has resulted in genotype–phenotype descriptions for over 48 mutation combinations. The wealth of data has led to the parallel development of dynamic mathematical64
and computational (executable) models65
to describe the intercellular communication—with the ability to parameterize models on the basis of empiric data. In the case of computational models, a prediction regarding the requirement of a time-delay between signals were verified with the use of fluorescent reporters of cell-fate in vivo
Multicellular processes in zebrafish, which may have greater relevance to vertebrates, should lend themselves to similar modeling approaches. One area of significant interest is cardiac regeneration, which has recently been shown in zebrafish to involve dedifferentiation of mature cardiomyocytes.66
Genetic and chemical screens could be used to define mediators of this process, and fluorescent-based reporters could then be used to assist the development and refinement of quantitative models describing the timing of intercellular communication. Such models in fact may have clinical relevance for healing postmyocardial infarction.