Dynamics of the cell fate specifications during female gametophyte development in Arabidopsis

The female gametophytes of angiosperms contain cells with distinct functions, such as those that enable reproduction via pollen tube attraction and fertilization. Although the female gametophyte undergoes unique developmental processes, such as several rounds of nuclear division without cell plate formation, and the final cellularization, it remains unknown when and how the cell fate is determined during their development. Here, we visualized the living dynamics of female gametophyte development and performed transcriptome analysis of its individual cell types, to assess the cell fate specifications in Arabidopsis thaliana. We recorded time lapses of the nuclear dynamics and cell plate formation from the one-nucleate stage to the seven-cell stage after cellularization, using the in vitro ovule culture system. The movies showed that the nuclear division occurred along the micropylar-chalazal axis. During cellularization, the polar nuclei migrated while associating with forming edge of the cell plate. Then, each polar nucleus migrated to fuse linearly towards each other. We also tracked the gene expression dynamics and identified that the expression of the MYB98pro::GFP, a synergid-specific marker, was initiated before cellularization, and then restricted to the synergid cells after cellularization. This indicated that cell fates are determined immediately after cellularization. Transcriptome analysis of the female gametophyte cells of the wild type and myb98 mutant, revealed that the myb98 synergid cells had the egg cell-like gene expression profile. Although in the myb98, the egg cell-specific gene expressions were properly initiated only in the egg cells after cellularization, but subsequently expressed ectopically in one of the two synergid cells. These results, together with the various initiation timings of the egg cell-specific genes suggest the complex regulation of the individual gametophyte cells, such as cellularization-triggered fate initiation, MYB98-dependent fate maintenance, cell morphogenesis, and organelle positioning. Our system of live-cell imaging and cell-type-specific gene expression analysis provides insights into the dynamics and mechanisms of cell fate specifications in the development of female gametophytes in plants.