Spatiotemporal dynamics exhibited by horizontal basal cells reveal a pro-neurogenic pathway during injury-induced olfactory epithelium regeneration.

Horizontal basal cells (HBCs) mediate olfactory epithelium (OE) regeneration following severe tissue injury. The dynamism of the post-injury environment is well illustrated by in silico modeling of RNA sequencing data that demonstrate an evolving HBC transcriptome. Unfortunately, spatiotemporally dynamic processes occurring within HBCs in situ remain poorly understood. Here, we show that HBCs at 24 h post-OE injury spatially redistribute a constellation of proteins, which, in turn, helped to nominate Rac1 as a regulator of HBC differentiation during OE regeneration. Using our primary culture model to activate HBCs pharmacologically, we demonstrate that concurrent Rac1 inhibition attenuates HBC differentiation potential. This in vitro functional impairment manifested in vivo as decreased HBC differentiation into olfactory sensory neurons following HBC-specific Rac1 conditional knockout. Taken together, our data potentiate the design of hyposmia-alleviating therapies and highlight aspects of in situ HBC spatiotemporal dynamics that deserve further investigation.

Horizontal basal cells self-govern their neurogenic potential during injury-induced regeneration of the olfactory epithelium.

Horizontal basal cells (HBCs) residing within severely damaged olfactory epithelium (OE) mediate OE regeneration by differentiating into odorant-detecting olfactory sensory neurons (OSNs) and other tissue supporting non-neuronal cell types. Depending on both tissue type and integrity, the Notch signaling pathway can either positively or negatively regulate resident stem cell activity. Although Notch1 specifies HBC dormancy in the uninjured OE, little is known about how HBCs are influenced by the Notch pathway following OE injury. Here, we show that HBCs depend on a functional inversion of the Notch pathway to appropriately mediate OE regeneration. At 24 h post-injury, HBCs enhance Notch1-mediated signaling. Moreover, at 3 days post-injury when the regenerating OE is composed of multiple cell layers, HBCs enrich both Notch1 and the Notch ligand, Dll1. Notably, HBC-specific Notch1 knockout increases HBC quiescence and impairs HBC differentiation into neuronal progenitors and OSNs. Interestingly, complete HBC knockout of Dll1 only decreases differentiation of HBC-derived OSNs. These data underscore the context-dependent nature of Notch signaling. Furthermore, they reveal that HBCs regulate their own neurogenic potential after OE injury.

Molecular Evidence for Olfactory Neuroblastoma as a Tumor of Malignant Globose Basal Cells.

Olfactory neuroblastoma (ONB, esthesioneuroblastoma) is a sinonasal cancer with an underdeveloped diagnostic toolkit, and is the subject of many incidents of tumor misclassification throughout the literature. Despite its name, connections between the cancer and normal cells of the olfactory epithelium have not been systematically explored and markers of olfactory epithelial cell types are not deployed in clinical practice. Here, we utilize an integrated human-mouse single-cell atlas of the nasal mucosa, including the olfactory epithelium, to identify transcriptomic programs that link ONB to a specific population of stem/progenitor cells known as olfactory epithelial globose basal cells (GBCs). Expression of a GBC transcription factor NEUROD1 distinguishes both low- and high-grade ONB from sinonasal undifferentiated carcinoma, a potential histologic mimic with a distinctly unfavorable prognosis. Furthermore, we identify a reproducible subpopulation of highly proliferative ONB cells expressing the GBC stemness marker EZH2, suggesting that EZH2 inhibition may play a role in the targeted treatment of ONB. Finally, we study the cellular states comprising ONB parenchyma using single-cell transcriptomics and identify evidence of a conserved GBC transcriptional regulatory circuit that governs divergent neuronal-versus-sustentacular differentiation. These results link ONB to a specific cell type for the first time and identify conserved developmental pathways within ONB that inform diagnostic, prognostic, and mechanistic investigation.

An in vitro model of acute horizontal basal cell activation reveals dynamic gene regulatory networks underlying the acute activation phase.

Horizontal basal cells (HBCs) activate only in response to severe olfactory epithelium (OE) injury. This activation is mediated by the loss of the transcription factor TP63. Using the compound phorbol 12-myristate 13-acetate (PMA), we find that we can model the process of acute HBC activation. First, we find that PMA treatment induces a rapid loss in TP63 protein and induces the expression of HOPX and the nuclear translocation of RELA, previously identified to mediate HBC activation. Using bulk RNA sequencing, we find that PMA-treated HBCs pass through various stages of acute activation identifiable by transcriptional regulatory signatures that mimic stages identified in vivo . These temporal stages are associated with varying degrees of engraftment and differentiation potential in transplantation assays. Together, this data shows that our model can model physiologically relevant features of HBC activation and identifies new candidates for mechanistic testing.

Spatial Determination of Neuronal Diversification in the Olfactory Epithelium.

Neurons in the murine olfactory epithelium (OE) differ by the olfactory receptor they express as well as other molecular phenotypes that are regionally restricted. These patterns can be precisely regenerated following epithelial injury, suggesting that spatial cues within the tissue can direct neuronal diversification. Nonetheless, the permanency and mechanism of this spatial patterning remain subject to debate. Via transplantation of stem and progenitor cells from dorsal OE into ventral OE, we demonstrate that, in mice of both sexes, nonautonomous spatial cues can direct the spatially circumscribed differentiation of olfactory sensory neurons. The vast majority of dorsal transplant-derived neurons express the ventral marker OCAM (NCAM2) and lose expression of NQO1 to match their new location. Single-cell analysis also demonstrates that OSNs adopt a fate defined by their new position following progenitor cell transplant, such that a ventral olfactory receptor is expressed after stem and progenitor cell engraftment. Thus, spatially constrained differentiation of olfactory sensory neurons is plastic, and any bias toward an epigenetic memory of place can be overcome.SIGNIFICANCE STATEMENT Spatially restricted differentiation of olfactory sensory neurons is both key to normal olfactory function and a challenging example of biological specificity. That the stem cells of the olfactory epithelium reproduce the organization of the olfactory periphery to a very close approximation during lesion-induced regeneration begs the question of whether stem cell-autonomous genomic architecture or environmental cues are responsible. The plasticity demonstrated after transfer to a novel location suggests that cues external to the transplanted stem and progenitor cells confer neuronal identity. Thus, a necessary prerequisite is satisfied for using engraftment of olfactory stem and progenitor cells as a cellular therapeutic intervention to reinvigorate neurogenesis whose exhaustion contributes to the waning of olfaction with age.

Dynamic Cellular Integration Drives Functional Assembly of the Heart's Pacemaker Complex.

Impulses generated by a multicellular, bioelectric signaling center termed the sinoatrial node (SAN) stimulate the rhythmic contraction of the heart. The SAN consists of a network of electrochemically oscillating pacemaker cells encased in a heterogeneous connective tissue microenvironment. Although the cellular composition of the SAN has been a point of interest for more than a century, the biological processes that drive the tissue-level assembly of the cells within the SAN are unknown. Here, we demonstrate that the SAN's structural features result from a developmental process during which mesenchymal cells derived from a multipotent progenitor structure, the proepicardium, integrate with and surround pacemaker myocardium. This process actively remodels the forming SAN and is necessary for sustained electrogenic signal generation and propagation. Collectively, these findings provide experimental evidence for how the microenvironmental architecture of the SAN is patterned and demonstrate that proper cellular arrangement is critical for cardiac pacemaker biorhythmicity.

Hemodynamic forces regulate developmental patterning of atrial conduction.

Anomalous action potential conduction through the atrial chambers of the heart can lead to severe cardiac arrhythmia. To date, however, little is known regarding the mechanisms that pattern proper atrial conduction during development. Here we demonstrate that atrial muscle functionally diversifies into at least two heterogeneous subtypes, thin-walled myocardium and rapidly conducting muscle bundles, during a developmental window just following cardiac looping. During this process, atrial muscle bundles become enriched for the fast conduction markers Cx40 and Nav1.5, similar to the precursors of the fast conduction Purkinje fiber network located within the trabeculae of the ventricles. In contrast to the ventricular trabeculae, however, atrial muscle bundles display an increased proliferation rate when compared to the surrounding myocardium. Interestingly, mechanical loading of the embryonic atrial muscle resulted in an induction of Cx40, Nav1.5 and the cell cycle marker Cyclin D1, while decreasing atrial pressure via in vivo ligation of the vitelline blood vessels results in decreased atrial conduction velocity. Taken together, these data establish a novel model for atrial conduction patterning, whereby hemodynamic stretch coordinately induces proliferation and fast conduction marker expression, which in turn promotes the formation of large diameter muscle bundles to serve as preferential routes of conduction.

Reciprocal myocardial-endocardial interactions pattern the delay in atrioventricular junction conduction.

Efficient blood flow depends on two developmental processes that occur within the atrioventricular junction (AVJ) of the heart: conduction delay, which entrains sequential chamber contraction; and valve formation, which prevents retrograde fluid movement. Defects in either result in severe congenital heart disease; however, little is known about the interplay between these two crucial developmental processes. Here, we show that AVJ conduction delay is locally assigned by the morphogenetic events that initiate valve formation. Our data demonstrate that physical separation from endocardial-derived factors prevents AVJ myocardium from becoming fast conducting. Mechanistically, this physical separation is induced by myocardial-derived factors that support cardiac jelly deposition at the onset of valve formation. These data offer a novel paradigm for conduction patterning, whereby reciprocal myocardial-endocardial interactions coordinate the processes of valve formation with establishment of conduction delay. This, in turn, synchronizes the electrophysiological and structural events necessary for the optimization of blood flow through the developing heart.

Distinct patterns of human medulloblastoma dissemination in the developing chick embryo nervous system.

Medulloblastoma (MB) is the most common malignant primary brain tumor in children. Aggressive tumors that disseminate along the leptomeninges carry extremely poor prognoses. Mechanisms that predict dissemination are poorly understood. Our objective was to develop a reliable and reproducible model to study MB dissemination. We have created a chicken-human xenograft to study features of MB with leptomeningeal dissemination. Human MB cell lines (D283, Daoy), primary human MB cells (SF8113), and primary genetic mouse model (Math1cre:SmoM2 flox/flox) MB cells were either transfected to express green fluorescent protein (GFP) or were labeled with a membrane permeable green fluorescent probe. Cells were then injected as aggregates or implanted as pellets into the developing chicken brain immediately after neural tube closure at embryonic day 2 (E2). Most embryos were harvested three days after implantation (E5) though some were harvested up to E15. The developing brain was analyzed via whole mount fluorescent imaging and tissue section immunohistochemistry. Human and mouse MBs survived in the developing chicken central nervous system (CNS). They exhibited distinct patterns of incorporation and dissemination into the CNS that were consistent with observed phenotypes of the corresponding human patient or mouse host. Specifically, metastatic D283 cells disseminated along the leptomeninges whereas Daoy, primary mouse MB, and primary human MB cells did not. This work supports an avian-human xenograft as a successful model to study patterns of MB dissemination. Our model provides a basis for manipulating cell signaling mechanisms to understand critical targets involved in MB dissemination.