Spatial Fluctuations in Expression of the Heterocyst Differentiation Regulatory Gene hetR in Anabaena Filaments

Under nitrogen deprivation, filaments of the cyanobacterium Anabaena undergo a process of development, resulting in a one-dimensional pattern of nitrogen-fixing heterocysts separated by about ten photosynthetic vegetative cells. Many aspects of gene expression before nitrogen deprivation and during the developmental process remain to be elucidated. Furthermore, the coupling of gene expression fluctuations between cells along a multicellular filament is unknown. We studied the statistics of fluctuations of gene expression of HetR, a transcription factor essential for heterocyst differentiation, both under steady-state growth in nitrogen-rich conditions and at different times following nitrogen deprivation, using a chromosomally-encoded translational hetR-gfp fusion. Statistical analysis of fluorescence at the individual cell level in wild-type and mutant filaments demonstrates that expression fluctuations of hetR in nearby cells are coupled, with a characteristic spatial range of circa two to three cells, setting the scale for cellular interactions along a filament. Correlations between cells predominantly arise from intercellular molecular transfer and less from cell division. Fluctuations after nitrogen step-down can build up on those under nitrogen-replete conditions. We found that under nitrogen-rich conditions, basal, steady-state expression of the HetR inhibitor PatS, cell-cell communication influenced by the septal protein SepJ and positive HetR auto-regulation are essential determinants of fluctuations in hetR expression and its distribution along filaments. A comparison between the expression of hetR-gfp under nitrogen-rich and nitrogen-poor conditions highlights the differences between the two HetR inhibitors PatS and HetN, as well as the differences in specificity between the septal proteins SepJ and FraC/FraD. Activation, inhibition and cell-cell communication lie at the heart of developmental processes. Our results show that proteins involved in these basic ingredients combine together in the presence of inevitable stochasticity in gene expression, to control the coupled fluctuations of gene expression that give rise to a one-dimensional developmental pattern in this organism.Author Summary: Under prolonged nitrogen deprivation, one-dimensional filaments of the multicellular cyanobacterium Anabaena undergo a process of development, forming a pattern consisting of cells specialized for nitrogen fixation-heterocysts-, separated by a chain of about ten photosynthetic vegetative cells. The developmental program uses activation, inhibition, and transport to create spatial and temporal patterns of gene expression, in the presence of unavoidable stochastic fluctuations in gene expression among cells. Using a chromosomally-encoded fluorescent marker, we followed the expression of the important regulator HetR in individual cells along filaments, both under abundant nitrogen conditions as well as at different times after nitrogen deprivation. The results of our statistical analysis of these fluctuations illuminate the fundamental role that positive feedback, lateral inhibition and cell-cell communication play in the developmental program, not only after exposure to the external cue that triggers differentiation but also under non-inducing conditions. Furthermore our results establish the spatial extent to which gene expression is correlated along filaments.