Establishing abaxial-adaxial polarity is central to leaf morphogenesis and function. Groups of genes that encode different components for leaf patterning have been identified in recent years. These include transcriptional factors, small RNAs, 26S proteasome and components required for post-transcriptional gene silencing and chromatin remodeling, showing a complex regulatory network and indicating that the regulation occurs at different levels. In this work, we report the identification and characterization of asymmetric leaves1/2 enhancer5 (ae5) and ae6 mutants. These two mutants had a phenotype of abnormal leaf patterning, with the abaxial mesophyll features appearing in the adaxial mesophyll domain, and double mutants ae5 as1/2 and ae6 as1/2 producing severely abaxialized leaves. AE5 and AE6 encode the ribosomal large subunit proteins RPL28A and RPL5A, respectively, and mutations in two other ribosomal protein genes, RPL5B and RPL24B, resulted in plant phenotypes similar to those of ae5 and ae6. Because these four ribosomal proteins are located in distinct sites in the ribosomal large subunit, we propose that the conserved translational function of the ribosome may be required for regulating key components during leaf patterning. Collectively, our data indicate that specific ribosome subunit-mediated translational control is essential in leaf polarity establishment.

Genomic approaches have predicted hundreds of thousands of tissue specific cis-regulatory sequences, but the determinants critical to their function and evolutionary history are mostly unknown1–4. Here, we systematically decode a set of brain enhancers active in the zona limitans intrathalamica (zli), a signaling center essential for vertebrate forebrain development via the secreted morphogen, Sonic hedgehog (Shh)5,6. We apply a de novo motif analysis tool to identify six position-independent sequence motifs together with their cognate transcription factors that are essential for zli enhancer activity and Shh expression in the mouse embryo. Using knowledge of this regulatory lexicon, we discover novel Shh zli enhancers in mice, and a functionally equivalent element in hemichordates, indicating an ancient origin of the Shh zli regulatory network that predates the chordate phylum. These findings support a strategy for delineating functionally conserved enhancers in the absence of overt sequence homologies, and over extensive evolutionary distances.

Extracellular vesicles (EVs) are nanosized lipid bilayer-bound vesicles that are naturally secreted from most cell types as a communication mechanism to deliver proteins, lipids, and genetic material. Despite the therapeutic potential of EVs, there is limited information on EV uptake kinetics and specificity. Here, we optimized an imaging flow cytometry (IFC)-based platform to quantitatively assess dose, time, and recipient cell specificity effects on human embryonic kidney cell (HEK293T) EV internalization in a high-throughput manner. We found that HEK293T EV uptake is an active process that is dose and time dependent. Further, the selectivity of EV uptake was quantified in vitro, and we found that HEK293T EVs were internalized at higher quantities by cells of the same origin. Lastly, neural stem cells internalized significantly more HEK293T EVs relative to mature neurons, suggesting that stem cells or progenitors, which are more metabolically active than terminally differentiated cells, may have higher rates of active EV internalization. The characterization of EV uptake, notably specificity, dose and time dependence, and kinetic assays will help inform and develop targeted and efficient EV-based therapeutics.