A census of the lung: CellCards from LungMAP.

The human lung plays vital roles in respiration, host defense, and basic physiology. Recent technological advancements such as single-cell RNA sequencing and genetic lineage tracing have revealed novel cell types and enriched functional properties of existing cell types in lung. The time has come to take a new census. Initiated by members of the NHLBI-funded LungMAP Consortium and aided by experts in the lung biology community, we synthesized current data into a comprehensive and practical cellular census of the lung. Identities of cell types in the normal lung are captured in individual cell cards with delineation of function, markers, developmental lineages, heterogeneity, regenerative potential, disease links, and key experimental tools. This publication will serve as the starting point of a live, up-to-date guide for lung research at https://www.lungmap.net/cell-cards/. We hope that Lung CellCards will promote the community-wide effort to establish, maintain, and restore respiratory health.

POU4F3 pioneer activity enables ATOH1 to drive diverse mechanoreceptor differentiation through a feed-forward epigenetic mechanism.

During embryonic development, hierarchical cascades of transcription factors interact with lineage-specific chromatin structures to control the sequential steps in the differentiation of specialized cell types. While examples of transcription factor cascades have been well documented, the mechanisms underlying developmental changes in accessibility of cell type-specific enhancers remain poorly understood. Here, we show that the transcriptional "master regulator" ATOH1-which is necessary for the differentiation of two distinct mechanoreceptor cell types, hair cells in the inner ear and Merkel cells of the epidermis-is unable to access much of its target enhancer network in the progenitor populations of either cell type when it first appears, imposing a block to further differentiation. This block is overcome by a feed-forward mechanism in which ATOH1 first stimulates expression of POU4F3, which subsequently acts as a pioneer factor to provide access to closed ATOH1 enhancers, allowing hair cell and Merkel cell differentiation to proceed. Our analysis also indicates the presence of both shared and divergent ATOH1/POU4F3-dependent enhancer networks in hair cells and Merkel cells. These cells share a deep developmental lineage relationship, deriving from their common epidermal origin, and suggesting that this feed-forward mechanism preceded the evolutionary divergence of these very different mechanoreceptive cell types.

Enhancer decommissioning imposes an epigenetic barrier to sensory hair cell regeneration.

Adult mammalian tissues such as heart, brain, retina, and the sensory structures of the inner ear do not effectively regenerate, although a latent capacity for regeneration exists at embryonic and perinatal times. We explored the epigenetic basis for this latent regenerative potential in the mouse inner ear and its rapid loss during maturation. In perinatal supporting cells, whose fate is maintained by Notch-mediated lateral inhibition, the hair cell enhancer network is epigenetically primed (H3K4me1) but silenced (active H3K27 de-acetylation and trimethylation). Blocking Notch signaling during the perinatal period of plasticity rapidly eliminates epigenetic silencing and allows supporting cells to transdifferentiate into hair cells. Importantly, H3K4me1 priming of the hair cell enhancers in supporting cells is removed during the first post-natal week, coinciding with the loss of transdifferentiation potential. We hypothesize that enhancer decommissioning during cochlear maturation contributes to the failure of hair cell regeneration in the mature organ of Corti.

Foxc1 establishes enhancer accessibility for craniofacial cartilage differentiation.

The specification of cartilage requires Sox9, a transcription factor with broad roles for organogenesis outside the skeletal system. How Sox9 and other factors gain access to cartilage-specific cis-regulatory regions during skeletal development was unknown. By analyzing chromatin accessibility during the differentiation of neural crest cells into chondrocytes of the zebrafish head, we find that cartilage-associated chromatin accessibility is dynamically established. Cartilage-associated regions that become accessible after neural crest migration are co-enriched for Sox9 and Fox transcription factor binding motifs. In zebrafish lacking Foxc1 paralogs, we find a global decrease in chromatin accessibility in chondrocytes, consistent with a later loss of dorsal facial cartilages. Zebrafish transgenesis assays confirm that many of these Foxc1-dependent elements function as enhancers with region- and stage-specific activity in facial cartilages. These results show that Foxc1 promotes chondrogenesis in the face by establishing chromatin accessibility at a number of cartilage-associated gene enhancers.

Animals with backbones (or vertebrates) have body shape determined, in part, by their skeletons. These emerge in the embryo in the form of cartilage structures that will then get replaced by bone during development. The neural crest is a group of embryonic cells that can become different tissues. In the head, it forms the cartilage scaffold for some of the facial bones and the base of the skull. During this process, a protein called Sox9 is required for neural crest cells to morph into cartilage. This transcription factor binds to regulatory sequences in the genome to turn cartilage genes on. But Sox9 is also required to form non-cartilage tissues in organs such as the liver, lung, and kidneys. How, then, does Sox9 only turn on the genes required for cartilage formation in the embryonic face? This specificity can be controlled by which regulatory sequences Sox9 can physically access in a cell: controlling which regulatory sequences Sox9 can access determines which genes it can activate, and which type of tissue a cell will become. Xu, Yu et al. wanted to understand exactly how Sox9 switches on the genes that turn neural crest cells into facial cartilage. They studied the genomes of zebrafish embryos, which have a cartilaginous skeleton similar to other vertebrates, and found out which areas were accessible to transcription factors in the neural crest cells that became facial cartilage. Analyzing these regions suggested that sites where Sox9 could bind were often close to binding sites for another protein, called Foxc1. When zebrafish embryos were genetically modified to inactivate Foxc1 proteins, many of the regulatory sequences in cartilage failed to become accessible, and the cartilaginous skeleton did not form properly. These results support a model in which Foxc1 opens up the genomic regions that Sox9 needs to bind for cartilage to form, as opposed to the regions that Sox9 would bind to make different organ cell types. The findings of Xu, Yu et al. uncover the stepwise process by which cartilage cells are made during development. Further research based on these results could allow scientists to develop new ways of replacing cartilage in degenerative conditions such as arthritis.

Generation of inner ear hair cells by direct lineage conversion of primary somatic cells.

The mechanoreceptive sensory hair cells in the inner ear are selectively vulnerable to numerous genetic and environmental insults. In mammals, hair cells lack regenerative capacity, and their death leads to permanent hearing loss and vestibular dysfunction. Their paucity and inaccessibility has limited the search for otoprotective and regenerative strategies. Growing hair cells in vitro would provide a route to overcome this experimental bottleneck. We report a combination of four transcription factors (Six1, Atoh1, Pou4f3, and Gfi1) that can convert mouse embryonic fibroblasts, adult tail-tip fibroblasts and postnatal supporting cells into induced hair cell-like cells (iHCs). iHCs exhibit hair cell-like morphology, transcriptomic and epigenetic profiles, electrophysiological properties, mechanosensory channel expression, and vulnerability to ototoxin in a high-content phenotypic screening system. Thus, direct reprogramming provides a platform to identify causes and treatments for hair cell loss, and may help identify future gene therapy approaches for restoring hearing.

Worldwide, hearing loss is the most common loss of sensation. Most cases of hearing loss are due to the death of specialized hair cells found deep inside the ear. These hair cells convert sounds into nerve impulses which can be understood by the brain. Hair cells naturally degrade as part of aging and can be damaged by other factors including loud noises, and otherwise therapeutic drugs, such as those used in chemotherapy for cancer. In humans and other mammals, once hair cells are lost they cannot be replaced. Hair cells have often been studied using mice, but the small number of hair cells in their ears, and their location deep inside the skull, makes it particularly difficult to study them in this way. Scientists are seeking ways to grow hair cells in the laboratory to make it easier to understand how they work and the factors that contribute to their damage and loss. Different cell types in the body are formed in response to specific combinations of biological signals. Currently, scientists do not have an efficient way to grow hair cells in the laboratory, because the correct signals needed to create them are not known. Menendez et al. have now identified four proteins which, when activated, convert fibroblasts, a common type of cell, into hair cells similar to those in the ear. These proteins are called Six1, Atoh1, Pou4f3 and Gfi1. Menendez et al. termed the resulting cells induced hair cells, or iHCs for short, and analyzed these cells to identify those characteristics that are similar to normal hair cells, as well as their differences. Importantly, the iHCs were found to be damaged by the same chemicals that specifically harm normal hair cells, suggesting they are useful test subjects. The ability to create hair cells in the laboratory using more easily available cells has many uses. These cells can help to understand the normal function of hair cells and how they become damaged. They can also be used to test new drugs to assess their success in preventing or reversing hearing loss. These findings may also lead to genetic solutions to curing hearing loss.

Transcriptomic and epigenetic regulation of hair cell regeneration in the mouse utricle and its potentiation by Atoh1.

The mammalian cochlea loses its ability to regenerate new hair cells prior to the onset of hearing. In contrast, the adult vestibular system can produce new hair cells in response to damage, or by reprogramming of supporting cells with the hair cell transcription factor Atoh1. We used RNA-seq and ATAC-seq to probe the transcriptional and epigenetic responses of utricle supporting cells to damage and Atoh1 transduction. We show that the regenerative response of the utricle correlates with a more accessible chromatin structure in utricle supporting cells compared to their cochlear counterparts. We also provide evidence that Atoh1 transduction of supporting cells is able to promote increased transcriptional accessibility of some hair cell genes. Our study offers a possible explanation for regenerative differences between sensory organs of the inner ear, but shows that additional factors to Atoh1 may be required for optimal reprogramming of hair cell fate.