UC Santa Barbara

Animal development is a complex process, shepherded by systems of robustness to ensure its success. To date, such systems that have been experimentally identified, largely have been found to ensure specific and correct cell identities in terms of gene and protein expression and spatial position. Little is known about systems that supervise, guide, and compensate for variability in the timing of events within development. The developing embryo of the nematode C. elegans follows a highly-stereotyped sequence of events starting at the two-cell stage where an asymmetric first division results in two cells differing in size, genetic and proteomic identity, and lineage dependent rates of division. These first two cells stereotypically divide at different times, with the larger dividing before the smaller. This sequence will later result in a number of key cell-cell interactions necessary for successful development. The rigid stereotype of this sequence and the critical nature of the dependent later events, suggests that the sequence itself may be under the influence of a system of robustness. At this stage such a system would necessarily include communication between the two cells to ensure their coordination in time. The work presented here establishes a method of challenging this system, by placing the C. elegans embryo in a temperature gradient sufficient to push the temperature dependent rate of division of the two cells away from their stereotyped temporal relationship. To achieve this we built and characterized a novel microfluidic temperature gradient device that can establish a 7.5 OC temperature gradient across the ~ 50 μm long developing embryo within biologically permissive temperatures. This temperature gradient establishes a condition that would be considered aberrant by the embryo at this stage, if and only if the two cells are monitoring each other's behavior. We have found that within a temperature gradient, the two cells of the embryo identify the existence of an aberrant condition and compensate for it by slowing their division rates. We find that the fold change in division timing of the two cells is dependent on their orientation in the temperature gradient, and that embryos that survive this condition to hatching generally slow down more than those that do not. We are able to reverse the sequence of divisions between the two cells and although they do not hatch, a surprising percentage undergo morphogenesis and result in a product that looks “wormlike” suggesting even later checkpoints and compensation. We also found that in a fraction of the embryos loaded into the gradient after the first division, the cleavage between the two cells reverses and the two nuclei of the two cells migrate back toward each other.

The behavior of the two-cell embryos in the temperature gradient: 1) surviving a high percentage of time in lower gradients, and even a fraction of the time in higher gradients, 2) the slowing down of each cell relative to its expected behavior at the temperature it is experiencing with greater slowing resulting in a greater likelihood of survival, and 3) the entry of embryos into morphogenesis even after violation of the stereotypical sequence of division at the two-cell stage, constitutes evidence for one and possibly two previously unidentified compensation and coordination mechanisms that act to ensure robustness against variation in the timing of events in the development of the early C. elegans embryo.