Genetic and cytological evidence that heterocyst patterning is regulated by inhibitor gradients that promote activator decay

Abstract

The formation of a pattern of differentiated cells from a group of seemingly equivalent, undifferentiated cells is a central paradigm of developmental biology. Several species of filamentous cyanobacteria differentiate nitrogen-fixing heterocysts at regular intervals along unbranched filaments to form a periodic pattern of two distinct cell types. This patterning has been used to exemplify application of the activator-inhibitor model to periodic patterns in biology. The activator-inhibitor model proposes that activators and inhibitors of differentiation diffuse from source cells to form concentration gradients that in turn mediate patterning, but direct visualization of concentration gradients of activators and inhibitors has been difficult. Here we show that the periodic pattern of heterocysts produced by cyanobacteria relies on two inhibitors of heterocyst differentiation, PatS and HetN, in a manner consistent with the predictions of the activator-inhibitor model. Concentration gradients of the activator, HetR, were observed adjacent to heterocysts, the natural source of PatS and HetN, as well as adjacent to vegetative cells that were manipulated to overexpress a gene encoding either of the inhibitors. Gradients of HetR relied on posttranslational decay of HetR. Deletion of both patS and hetN genes prevented the formation of gradients of HetR, and a derivative of the inhibitors was shown to promote decay of HetR in a concentration-dependent manner. Our results provide strong support for application of the activator-inhibitor model to heterocyst patterning and, more generally, the formation of periodic patterns in biological systems.activator-inhibitor ͉ Anabaena ͉ development

Structures of complexes comprised of Fischerella transcription factor HetR with Anabaena DNA targets

“…4). G36 is in a short 3 10 helix near the N terminus of H3; its mutation may interfere with the orientation of this helix and with the conformations of Arg34 and His35, which provide solvent-mediated interactions with the phosphate backbone. Another DNA bindingdeficient mutation, D17E, results in a Het − phenotype in Anabaena (35).…”

“…However, what creates the gradient of HetR, assuming that its activity is the critical variable in establishing the pattern? The answer seems to be a gradient of the PatS peptide RGSGR (9) early in the process, and later, a gradient of the protein HetN, which also contains the sequence RGSGR (9,10). Both of these "regulators" interact with HetR, preventing it from binding to specific DNA targets, by a mechanism that is still not fully understood.…”

“…Constitutively expressed HetR is rapidly degraded by cellular protease(s). Therefore, the gradient of HetR is due to a gradient of degradation caused by proteins or peptides produced in the heterocyst and diffused along the filament (10).…”

Biased inheritance of the protein PatN frees vegetative cells to initiate patterned heterocyst differentiation

“…Competitive resolution in the context of lateral inhibition is proposed to be attributable to the production of HetR and PatS in a differentiating heterocyst, resistance of that cell to the effects of PatS, and diffusion of PatS-5 to adjacent vegetative cells (7). Because time is also a factor, such a model may account for the delay in heterocyst formation in the ΔpatN strain.…”

Restricted cellular differentiation in cyanobacterial filaments

“…The patS and hetN genes encode polypeptides containing a conserved amino acid sequence (RGSGR) that inhibits heterocyst differentiation (10,11). A PatS-derived peptide produced by some cells early in differentiation and HetN or a HetN-related compound produced by mature heterocysts inhibit the differentiation of nearby cells in a process that can be described as lateral inhibition, which likely involves an interaction with HetR (12,13). The interplay between positive and negative regulators appears to be widely used in the development of spatial patterns in metazoans (14), and it also plays a key role in the development of the diazotrophic cyanobacterial filament.…”