ß-Catenin maintains lung epithelial progenitors after lung specification.

The entire lung epithelium arises from SRY box 9 (SOX9)-expressing progenitors that form the respiratory tree and differentiate into airway and alveolar cells. Despite progress in understanding their initial specification within the embryonic foregut, how these progenitors are subsequently maintained is less clear. Using inducible, progenitor-specific genetic mosaic mouse models, we showed that ß-catenin (CTNNB1) maintains lung progenitors by promoting a hierarchical lung progenitor gene signature, suppressing gastrointestinal (GI) genes, and regulating NK2 homeobox 1 (NKX2.1) and SRY box 2 (SOX2) in a developmental stage-dependent manner. At the early, but not later, stage post-lung specification, CTNNB1 cell-autonomously maintained normal NKX2.1 expression levels and suppressed ectopic SOX2 expression. Genetic epistasis analyses revealed that CTNNB1 is required for fibroblast growth factor (Fgf)/Kirsten rat sarcoma viral oncogene homolog (Kras)-mediated promotion of the progenitors. In silico screening of Eurexpress and translating ribosome affinity purification (TRAP)-RNAseq identified a progenitor gene signature, a subset of which depends on CTNNB1. Wnt signaling also maintained NKX2.1 expression and suppressed GI genes in cultured human lung progenitors derived from embryonic stem cells.

Supramolecular Click Product Interactions Induce Dynamic Stiffening of Extracellular Matrix-Mimetic Hydrogels.

Progressive stiffening of the extracellular matrix (ECM) is observed in tissue development as well as in pathologies such as cancer, cardiovascular disease, and fibrotic disease. However, methods to recapitulate this phenomenon in vitro face critical limitations. Here, we present a poly(ethylene glycol)-based peptide-functionalized ECM-mimetic hydrogel platform capable of facile, user-controlled dynamic stiffening. This platform leverages supramolecular interactions between inverse-electron demand Diels-Alder tetrazine-norbornene click products (TNCP) to create pendant moieties that undergo non-covalent crosslinking, stiffening a pre-existing network formed via thiol-ene click chemistry over the course of 6 h. Pendant TNCP moieties have a concentration-dependent effect on gel stiffness while still being cytocompatible and permissive of cell-mediated gel degradation. The robustness of this approach as well as its simplicity and ease of translation give it broad potential utility.

Hydrogel Synthesis and Stabilization via Tetrazine Click-Induced Secondary Interactions.

The discovery of tetrazine click-induced secondary interactions is reported as a promising new tool for polymeric biomaterial synthesis. This phenomenon is first demonstrated as a tool for poly(ethylene glycol) (PEG) hydrogel assembly via purely non-covalent interactions and is shown to yield robust gels with storage moduli one to two orders of magnitude higher than other non-covalent crosslinking methods. In addition, tetrazine click-induced secondary interactions also enhance the properties of covalently crosslinked hydrogels. A head-to-head comparison of PEG hydrogels crosslinked with tetrazine-norbornene and thiol-norbornene click chemistry reveals an approximately sixfold increase in storage modulus and unprecedented resistance to hydrolytic degradation in tetrazine click-crosslinked gels without substantial differences in gel fraction. Molecular dynamic simulations attribute these differences to the presence of secondary interactions between the tetrazine-norbornene cycloaddition products, which are absent in the thiol-norbornene crosslinked gels.