Following fertilization, human embryos undergo a dramatic self-assembly process, culminating in a reproducibly structured animal with trillions of cells. An enormous number of decisions are required for this process, each burdened with a significant rate of error. It is thus remarkable that human zygotes so frequently give rise to a proper individual. While quality control systems that continually correct these errors must be pervasive and are critically important for human embryogenesis, virtually nothing is known about how such systems operate. We propose to investigate how such high-fidelity development is achieved by investigating the highly reproducible development of C. elegans embryos in response to discordant conditions imposed by temperature (T) gradients. The hypothesis driving the proposed studies is that cell division rates are coordinated across disparate lineages by correcting or compensating for deviations from the norm, thereby ensuring a reproducible order of cell division events and a stereotyped cellular geometry at key stages.
In Aim 1, we will challenge embryos to discordant conditions by subjecting them to a steep thermal gradient with a v1.0 device that we have manufactured and validated, and will examine the outcome on cell division rates, embryo geometry, and viability. We will investigate whether checkpoints operating at particular stages in embryogenesis or with a particular polarity are used to monitor and correct for discordance between disparate lineages, as has been suggested by our preliminary data.
In Aim 2, we will refine our v2.0 microfluidic device, which allows high-throughput processing of embryos in thermal gradients and high-resolution imaging in real time. We will develop ratiometric thermometry techniques that allow us to measure regional T differences in living embryos.
In Aim 3, we will test the hypothesis that polarization of the embryo at the two-cell stage is required for compensation to discordant conditions and will investigate the role of gap junction activity in this process. Defects in cellular processes that ensure fidelity in embryos underlie wide-ranging pathologies including birth defects and dysregulated cell proliferation in the genesis of tumors. By revealing these previously unexplored mechanisms that ensure proper coordination between cells, our studies may reveal new insights into cancer biology and a more complete understanding of how high-fidelity human development is achieved, which is crucially important for the formation and maintenance of healthy tissues and organs.
Essential to the genesis of all healthy humans is the faithful execution of a very large number of developmental decisions. Errors in such decisions, caused by natural molecular and environmental variations, can lead to devastating consequences, including birth defects and abortive development. Embryos are capable of correcting errors during development, resulting in a normal healthy individual, but how such error-correcting processes occur is entirely unknown. The proposed studies will examine mechanisms used to adjust for the inevitable mistakes made during gestation and development. These studies will provide important information about how defects in development can arise and will also prove informative regarding the mechanisms that control proper cellular restraints that can, for example, prevent the formation of cancers.
