Contrary to previous understanding, recent evidence indicates that synonymous codon changes may sometimes face strong selection. However, it remains difficult to generalize the nature, strength, and mechanism(s) of such selection. Previously, we showed that synonymous variants of a key enzyme-coding gene (fae) of Methylobacterium extorquens AM1 decreased enzyme production and reduced fitness dramatically. We now show that during laboratory evolution, these variants rapidly regained fitness via parallel yet variant-specific, highly beneficial point mutations in the N-terminal region of fae. These mutations (including four synonymous mutations) had weak but consistently positive impacts on transcript levels, enzyme production, or enzyme activity. However, none of the proposed mechanisms (including internal ribosome pause sites or mRNA structure) predicted the fitness impact of evolved or additional, engineered point mutations. This study shows that synonymous mutations can be fixed through strong positive selection, but the mechanism for their benefit varies depending on the local sequence context.

Highlights d Cx3cr1 CreER -driven neonatal Cre induction adversely affects microglial development d Cre induction causes DNA damage and IFN-1 signaling, affecting microglial functions d Adverse effects of Cx3cr1 CreER induction are limited to neonatal stages d Vehicle-versus TAM-treated Cre + animal comparison is a critical control

Inflammation is known to adversely affect adult neurogenesis, wherein the source of inflammation is largely thought to be extraneous to the neurogenic niche. Here, we demonstrate that the adult hippocampal neural progenitors harbor an inflammatory potential that is proactively suppressed by transcription factor 4 (Tcf4). Deletion of Tcf4 in hippocampal nestin-expressing progenitors causes loss of proliferative capacity and acquisition of myeloid inflammatory properties. This transformation abolishes their differentiation potential and causes production of detrimental factors that adversely affect niche cells, causing inflammation in the dentate gyrus. Thus, on one hand, Tcf4 deletion causes abrogation of proliferative progenitors leading to reduction of adult neurogenesis, while on the other, their accompanying inflammatory transformation inflicts inflammation in the niche. Taken together, we provide the first evidence for a latent inflammatory potential of adult hippocampal neural progenitors and identify Tcf4 as a critical regulator that facilitates adult neurogenesis via proactive suppression of this detrimental potential.