Interspersed expression of CUP-SHAPED COTYLEDON2 and REDUCED COMPLEXITY shapes Cardamine hirsuta complex leaf form.

How genetically regulated growth shapes organ form is a key problem in developmental biology. Here, we investigate this problem using the leaflet-bearing complex leaves of Cardamine hirsuta as a model. Leaflet development requires the action of two growth-repressing transcription factors: REDUCED COMPLEXITY (RCO), a homeodomain protein, and CUP-SHAPED COTYLEDON2 (CUC2), a NAC-domain protein. However, how their respective growth-repressive actions are integrated in space and time to generate complex leaf forms remains unknown. By using live imaging, we show that CUC2 and RCO are expressed in an interspersed fashion along the leaf margin, creating a distinctive striped pattern. We find that this pattern is functionally important because forcing RCO expression in the CUC2 domain disrupts auxin-based marginal patterning and can abolish leaflet formation. By combining genetic perturbations with time-lapse imaging and cellular growth quantifications, we provide evidence that RCO -mediated growth repression occurs after auxin-based leaflet patterning and in association with the repression of cell proliferation. Additionally, through the use of genetic mosaics, we show that RCO is sufficient to repress both cellular growth and proliferation in a cell-autonomous manner. This mechanism of growth repression is different to that of CUC2 , which occurs in proliferating cells. Our findings clarify how the two growth repressors RCO and CUC2 coordinate to subdivide developing leaf primordia into distinct leaflets and generate the complex leaf form. They also indicate different relationships between growth repression and cell proliferation in the patterning and post-patterning stages of organogenesis. [Display omitted] • CUC2 and RCO are expressed in an interspersed pattern in C. hirsuta complex leaves • This interspersed expression is required for complex leaf development • RCO limits both cell proliferation and cellular growth in a cell-autonomous manner • The cellular parameters for growth repression by CUC2 and RCO are distinct Bhatia et al. use time-lapse imaging, genetic perturbations, and cellular growth analysis to show that spatially separated growth-repressive activities of CUC2 and RCO shape C. hirsuta complex leaf form. They find different relationships between cell proliferation and growth repression during patterning and post-patterning stages of organogenesis. [ABSTRACT FROM AUTHOR]