An important aspect of development biology is to understand how the thousands of genes predicted from the genome sequence are controlling the spatial and temporal events that are critical for the development and maturation of organism. Homeobox genes encode transcription factors of critical importance for development. Systematic studies of dynamic, spacio-temporal gene expression patterns of developmental control genes have been rare so far in C. elegans, even though C. elegans is predestined for such an analysis. We have started to investigate the expression patterns of particular homeobox genes during development. The PCR-based cloning strategy adapted from Oliver Hobert allows the production of transcriptional fusions in which the upstream regulatory regions of each gene is placed in frame with the translational initiation ATG of the reporter gene GFP. The homeobox::GFP reporter constructs are being used to investigate the expression pattern in larval stages, and more importantly, also to examine the embryonic expression pattern using live GFP time-lapse recordings. We have developed a 2-channel 4D recording system based on Jim Waddle's original concepts. We can now successfully record GPF through embryogenesis without deleterious effects using Openlab ( Improvision) Automations that we developed. Normal DIC 3D stacks are taken every 30 to 40 seconds for lineage and consequently cell identification purposes. Interspersed within the DIC stacks, e.g. after every 2. DIC stack, a GFP stack is recorded to monitor the expression pattern. Presently, we have been recording 4D stacks for the following genes
ceh-5,
ceh-33,
ceh-34,
ceh-41,
ceh-44, and ZK993.1. More genes, such as
ceh-14,
ceh-6,
ceh-32,
ceh-26, and other, presently uncharacterized homeobox genes will be investigated. Analysis of our 4D recordings has revealed in many instances early dynamic expression that fades again in later development, whose functional significance is currently unclear. However, it suggests that there may be events going on in early development to set up patterns, regions and cell fates that are not reflected by the expression seen later in larval stages.