Cells demonstrate plasticity following injury, but the extent of this phenomenon and the cellular mechanisms involved remain underexplored. Using single-cell RNA sequencing (scRNA-seq) and lineage tracing, we uncover that myoepithelial cells (MECs) of the submucosal glands (SMGs) proliferate and migrate to repopulate the airway surface epithelium (SE) in multiple injury models. Specifically, SMG-derived cells display multipotency and contribute to basal and luminal cell types of the SMGs and SE. Ex vivo expanded MECs have the potential to repopulate and differentiate into SE cells when grafted onto denuded airway scaffolds. Significantly, we find that SMG-like cells appear on the SE of both extra- and intra-lobular airways of large animal lungs following severe injury. We find that the transcription factor SOX9 is necessary for MEC plasticity in airway regeneration. Because SMGs are abundant and present deep within airways, they may serve as a reserve cell source for enhancing human airway regeneration.

Using scRNA-seq coupled with computational approaches, we studied transcriptional changes in cell states of sea urchin embryos during development to the larval stage. Eighteen closely spaced time points were taken during the first 24 hours of development of Lytechinus variegatus (Lv). Developmental trajectories were constructed using Waddington-OT, a computational approach to "stitch" together developmental timepoints. Skeletogenic and primordial germ cell trajectories diverged early in cleavage. Ectodermal progenitors were distinct from other lineages by sixth cleavage, though a small percentage of ectoderm cells briefly co-expressed endoderm markers indicating an early ecto-endoderm cell state, likely in cells originating from the equatorial region of the egg. Endomesoderm cells originated at 6th cleavage also and this state persisted for more than two cleavages, then diverged into distinct endoderm and mesoderm fates asynchronously, with some cells retaining an intermediate specification status until gastrulation. 79 of 80 genes (99%) examined, and included in published developmental gene regulatory networks (dGRNs), are present in the Lv-scRNA-seq dataset, and expressed in the correct lineages in which the dGRN circuits operate.

Changes in developmental gene regulatory networks (dGRNs) underlie much of the diversity of life 1 , but the evolutionary mechanisms that operate on interactions with these networks remain poorly understood. Closely related species with extreme phenotypic divergence provide a valuable window into the genetic and molecular basis for changes in dGRNs and their relationship to adaptive changes in organismal traits. Here we analyze genomes, epigenomes, and transcriptomes during early development in two sea urchin species in the genus Heliocidaris that exhibit highly divergent life histories and in an outgroup species. Signatures of positive selection and changes in chromatin status within putative gene regulatory elements are both enriched on the branch leading to the derived life history, and particularly so near core dGRN genes; in contrast, positive selection within protein-coding regions have at most a modest enrichment in branch and function. Singlecell transcriptomes reveal a dramatic delay in cell fate specification in the derived state, which also has far fewer open chromatin regions, especially near dGRN genes with conserved roles in cell fate specification. Experimentally perturbing the function of three key transcription factors reveals profound evolutionary changes in the earliest events that

Changes in developmental gene regulatory networks (dGRNs) underlie much of the diversity of life1, but the evolutionary mechanisms that operate on interactions with these networks remain poorly understood. Closely related species with extreme phenotypic divergence provide a valuable window into the genetic and molecular basis for changes in dGRNs and their relationship to adaptive changes in organismal traits. Here we analyze genomes, epigenomes, and transcriptomes during early development in two sea urchin species in the genus Heliocidaris that exhibit highly divergent life histories and in an outgroup species. Signatures of positive selection and changes in chromatin status within putative gene regulatory elements are both enriched on the branch leading to the derived life history, and particularly so near core dGRN genes; in contrast, positive selection within protein-coding regions have at most a modest enrichment in branch and function. Single-cell transcriptomes reveal a dramatic delay in cell fate specification in the derived state, which also has far fewer open chromatin regions, especially near dGRN genes with conserved roles in cell fate specification. Experimentally perturbing the function of three key transcription factors reveals profound evolutionary changes in the earliest events that pattern the embryo, disrupting regulatory interactions previously conserved for ∼225 million years. Together, these results demonstrate that natural selection can rapidly reshape developmental gene expression on a broad scale when selective regimes abruptly change and that even highly conserved dGRNs and patterning mechanisms in the early embryo remain evolvable under appropriate ecological circumstances.

Here we employed scRNA-seq coupled with computational approaches to examine molecular changes in cells during specification and differentiation. We examined the first 24 hours of development of the sea urchin Lytechinus variegatus (Lv) with 18 time points during which the embryo develops to the larval stage. Using Waddington-OT, the time points were computationally “stitched” together to calculate developmental trajectories. Skeletogenic cells displayed the expected immediate early divergence while other lineages diverged asynchronously, with many cells retaining an intermediate specification status until late in gastrulation. The Lv-scRNA-seq dataset was compared to the developmental Gene Regulatory Network (dGRN) model of specification in Strongylocentrotus purpuratus (Sp). 79 of 80 genes (98%) in that dGRN are present in the Lv-scRNA-seq dataset, and expressed in the correct lineages in which the dGRN circuits operate. Surprisingly, however, many heterochronies in timing of first expression of dGRN genes have evolved between the two species. Replotting the two dGRNs with precise attention to time of expression revealed a number of feedback inputs that likely buffer the dGRNs, allowing them to maintain function in the face of accumulating heterochronies.Summary statementThe early development of the sea urchin embryo was followed using scRNA-seq plus computational methods to trace lineage diversifications. These were matched to gene regulatory network changes over time.