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 Calum A. MacRae |
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Science
High speed digital microscopy enables functional assessment of the zebrafish heart as seen in this M-mode image of the zebrafish heart.
Cell fate in the cardiovascular system
Multiple cell types are required for the normal function of the vertebrate cardiovascular system. Recent work suggests that working cardiomyocytes display subtle functional differences, while others display electrical or endocrine functions. Similarly, the discovery of local addressing molecules and transport functions suggests that endocardial or endothelial cells are highly specialized with unique functions in each venous or arterial vascular bed.
By exploiting the tractability and transparency of the embryonic zebrafish investigators are able to follow the focal expression of genes in individual cells within the cardiovascular system, and study the effects of modifying gene function. We have developed novel tools to allow the characterization of cellular physiology in the embryonic fish, allowing us to define cell fate, for the first time not just in terms of gene expression, but also in terms of cell function. Examples include transgenic reporters of critical developmental signaling events, in vivo and ex vivo optical mapping of cardiac electrophysiology, and regional vascular transport functions using labeled nanoparticles
Combining these tools in longitudinal studies of zebrafish development, we are beginning to dissect the pathways required for the specification of specific myocardial cell types, such as the central conduction system. In addition to defining the genes required for the particular physiologic phenotypes, we are able to study the role of epigenetic factors that may supply unique positional information or other developmental cues determining and maintaining specific cell fates. We are exploring the role of flow, mechanical stimuli and electrical activity (in collaboration with the laboratory of Dr. David Milan at the CVRC) on the structural and functional patterning of the entire cardiovascular system. An early goal of this work is the generation of a functional fate map of the developing zebrafish heart.
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Even within the tiny zebrafish heart
there is cellular heterogeneity.
In this instance notch gene expression
is restricted to only a few cells in the
endocardium of the AV ring. |
Genetic Basis of Common Cardiovascular Disease
Genetic studies of common human diseases have to date been rather disappointing. One major reason for this lack of success is the fact that many common or 'complex' traits are in reality aggregates of several superficially similar entities. This 'phenotypic complexity' is considerably more tractable than true genetic complexity, where small gene effects modifying the final phenotype across the entire population often obscure large causal gene effects in discrete subsets.
We are combining traditional proband-based family collections (so-called "kin-cohort" study design) with novel phenotyping technologies to overcome many of the difficulties intrinsic to the study of common disorders. In this way we are able to detect gene effects of various magnitudes in a single systematic ascertainment, define the genetic epidemiology of a disorder, and prioritize genetic investigation based on the magnitude of the genetic effect. Monogenic forms of each disorder are identified, while populations suitable for other genetic approaches, including non-parametric mapping and association studies, also can be collected. A hallmark of this approach is that high-resolution clinical investigation often offers insight into the underlying condition long before the genes are discovered. An example is the evidence of endocrine links between arrhythmia and congestive heart failure emerging from studies of the genetic basis of lone atrial fibrillation (in collaboration with the laboratory of Dr. Patrick T. Ellinor). These new phenotypes can be applied immediately to risk assessment in patient cohorts, while also facilitating the study of families with poorly penetrant inherited forms of atrial fibrillation. Currently genetic studies are also underway to uncover the genetic basis of left ventricular remodeling and premature coronary artery disease.
As the causal genes are identified in human studies we are generating zebrafish and mouse models. These mechanistically faithful models are permitting investigation of the pathogenesis of a range of diseases including atrial fibrillation, cardiomyopathies and vascular disorders. Using the zebrafish models we have generated it will be feasible to undertake automated secondary genetic screens for other genes in the disease pathways, in addition to performing screens for chemical suppressors to act as probes in pathway analysis or as potential therapies for the cognate human disease (ongoing work in collaboration with the laboratory of Dr. Randall Peterson at the CVRC).
High-Throughput Physiology
The zebrafish occupies a unique niche between tractable invertebrate organisms such as C. elegans and Drosophila, and higher vertebrates with closer homology to humans, such as rodents. We have established that even by 48 hours post fertilization there is remarkable physiologic and pharmacologic homology between the larval zebrafish and adult humans. At this stage of development the zebrafish can be maintained in 96 or 384 multi-well plates. We have developed a number of automated quantitative assays for zebrafish physiologic variables including heart rate, myocardial contractility, and blood flow. These assays are capable of being automated, allowing high-throughput quantitative studies of the role of genes, environment and their interactions in integrative vertebrate physiology.
There are numerous applications of these technologies including genetic screens for physiologic rather than morphologic endpoints, systematic pharmacogenetics and chemical suppressor screens. Currently, we are undertaking a dominant and recessive screen for modifiers of drug-induced cardiotoxicity. This screen will define the genes responsible for sensitivity and resistance to IKr-mediated repolarization toxicity (the most common cause of drug withdrawal from the market), which then will be studied as candidates for human susceptibility to the same drugs as well as illuminating the basis of normal repolarization. Similar cross talk between human and zebrafish studies might be imagined in systematic approaches to a host of other biological problems.
Collaborative Relationships
Calum MacRae's group is part of the Developmental Biology Program and works closely with the labs of Randall Peterson, David Milan, and Patrick T. Ellinor.
Publications
Click here to view Calum MacRae's publications.
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