Stage-specific markers define early steps of procambium development in Arabidopsis leaves and correlate termination of vein formation with mesophyll differentiation.

During leaf development, ground meristem cells along continuous lines undergo coordinated oriented cell divisions and differentiate to form procambial cells, the precursors of all vascular cells. The molecular genetic dissection of early procambial development suffers from the lack of easily identifiable markers, especially of cell states preceding procambium formation. In this study, we have identified and characterized three reporter gene expression markers that reflect three distinct preprocambial stages, as well as one marker whose expression seems to be perfectly congruent with the appearance of procambial cells. All four markers are invariably expressed in continuous domains connected to pre-existing vasculature and their expression profiles reveal a common spatiotemporal pattern of early vein formation. We observed progressive extension of vascular strands at the preprocambial stage, suggesting that veins are initiated as freely ending preprocambial domains and that network formation occurs through subsequent fusion of these domains. Consistent with this interpretation, we demonstrate that veins are generally not programmed to become freely ending or interconnected network elements. Instead, we found that the progressive extension of preprocambial domains can be interrupted experimentally and that this leads to less complex vein patterns consisting of fewer vein orders, in which even lower-order veins become freely ending. Mesophyll differentiation turned out to be strictly correlated with the termination of preprocambial domain extension. These findings suggest that Arabidopsis vein pattern is not inherently determinate, but arises through reiterative initiation of new preprocambial branches until this process becomes terminated by the differentiation of mesophyll.