Coordination between cell division and differentiation is essential for mammalian development and tissue homeostasis. Early in embryonic development cell division and differentiation undergo a major transition during gastrulation. Prior to gastrulation, the gap phases of the cell cycle of the rapidly dividing pluripotent cells are extremely short.. During gastrulation, the gap phases lengthen and the rate of cell division decreases. Furthermore, genes that show periodic expression during the cell cycle phases in adult somatic cells, do not oscillate in pluripotent cells. However, very little is known about when and how during early mammalian development, these genes begin to oscillate with cell cycle phases and the relationship of this oscillation to extension of gap phases.. Given the intimate connection between cell division and differentiation, the establishment of periodic cell cycle expression is likely to play a central role in cell fate decisions. The objective for this exploratory grant is to identify genes induced to periodically oscillate during mammalian gastrulation as well as differences in the genes that oscillate between lineages following gastrulation. The central hypothesis is that the number of oscillating genes markedly increases coincident with the emergence of somatic cell fates during gastrulation and that the identity of many of these oscillating genes differs between post-gastrulation cell lineages. To address the hypothesis, two aims are proposed: 1) Identify transcript oscillation induced during gastrulation and 2) identify lineage-specific oscillating transcripts following gastrulation.
For aim 1, single-cell transcriptomes of pre-gastrulation epiblast cells and gastrulating somatic cells will be captured from cells sorted based on DNA content. The identity of periodically expressed genes from these two developmental stages will be compared with a collection of bioinformatics tools. Differentially oscillating genes will be validated by single molecule fluorescence in situ hybridization (FISH).
For aim 2, post-gastrulation embryos will be dissociated, sorted based on DNA content and their transcriptomes single-cell sequenced. The gene expression signature of these cells will be used to separate cells into distinct embryonic lineages. Transcript oscillation will be identified as differential expression across phases within each lineage and compared between lineages to find lineage-specific oscillation programs. These will be validated by single molecule FISH. This proposal is highly significant as it will elucidate cell cycle programs that emerge during, and therefore likely regulate, cell fate decisions. Future studies where these programs are targeted or functionally manipulated could redirect cell fate or uniquely alter proliferation in select lineages.
The proposed research is relevant to public health as it addresses the fundamental question of how cell cycle and cell differentiation are interrelated during formation of mammalian somatic cell types. New knowledge gained through this application will advance the fields of directed and redirected differentiation of cells for regenerative medicine purposes. It should also give insight into potential therapeutic targets for cell type specific growth. Therefore the research is relevant to NIH?s mission to foster fundamental creative discoveries that increase the nation's capacity to protect and improve human health.
