Design principles and therapeutic applications of novel synthetic WNT signaling agonists.

Wingless-related integration site or Wingless and Int-1 or Wingless-Int (WNT) signaling is crucial for embryonic development, and adult tissue homeostasis and regeneration, through its essential roles in cell fate, patterning, and stem cell regulation. The biophysical characteristics of WNT ligands have hindered efforts to interrogate ligand activity in vivo and prevented their development as therapeutics. Recent breakthroughs have enabled the generation of synthetic WNT signaling molecules that possess characteristics of natural ligands and potently activate the pathway, while also providing distinct advantages for therapeutic development and manufacturing. This review provides a detailed discussion of the protein engineering of these molecular platforms for WNT signaling agonism. We discuss the importance of WNT signaling in several organs and share insights from the initial application of these new classes of molecules in vitro and in vivo. These molecules offer a unique opportunity to enhance our understanding of how WNT signaling agonism promotes tissue repair, enabling targeted development of tailored therapeutics.

A WNT mimetic with broad spectrum FZD-specificity decreases fibrosis and improves function in a pulmonary damage model.

BACKGROUND: Wnt/ß-catenin signaling is critical for lung development and AT2 stem cell maintenance in adults, but excessive pathway activation has been associated with pulmonary fibrosis, both in animal models and human diseases such as idiopathic pulmonary fibrosis (IPF). IPF is a detrimental interstitial lung disease, and although two approved drugs limit functional decline, transplantation is the only treatment that extends survival, highlighting the need for regenerative therapies. METHODS: Using our antibody-based platform of Wnt/ß-catenin modulators, we investigated the ability of a pathway antagonist and pathway activators to reduce pulmonary fibrosis in the acute bleomycin model, and we tested the ability of a WNT mimetic to affect alveolar organoid cultures. RESULTS: A WNT mimetic agonist with broad FZD-binding specificity (FZD1,2,5,7,8) potently expanded alveolar organoids. Upon therapeutic dosing, a broad FZD-binding specific Wnt mimetic decreased pulmonary inflammation and fibrosis and increased lung function in the bleomycin model, and it impacted multiple lung cell types in vivo. CONCLUSIONS: Our results highlight the unexpected capacity of a WNT mimetic to effect tissue repair after lung damage and support the continued development of Wnt/ß-catenin pathway modulation for the treatment of pulmonary fibrosis.

Robust Colonic Epithelial Regeneration and Amelioration of Colitis via FZD-Specific Activation of Wnt Signaling.

BACKGROUND AND AIMS: Current management of inflammatory bowel disease leaves a clear unmet need to treat the severe epithelial damage. Modulation of Wnt signaling might present an opportunity to achieve histological remission and mucosal healing when treating IBD. Exogenous R-spondin, which amplifies Wnt signals by maintaining cell surface expression of Frizzled (Fzd) and low-density lipoprotein receptor-related protein receptors, not only helps repair intestine epithelial damage, but also induces hyperplasia of normal epithelium. Wnt signaling may also be modulated with the recently developed Wnt mimetics, recombinant antibody-based molecules mimicking endogenous Wnts. METHODS: We first compared the epithelial healing effects of RSPO2 and a Wnt mimetic with broad Fzd specificity in an acute dextran sulfate sodium mouse colitis model. Guided by Fzd expression patterns in the colon epithelium, we also examined the effects of Wnt mimetics with subfamily Fzd specificities. RESULTS: In the DSS model, Wnt mimetics repaired damaged colon epithelium and reduced disease activity and inflammation and had no apparent effect on uninjured tissue. We further identified that the FZD5/8 and LRP6 receptor-specific Wnt mimetic, SZN-1326-p, was associated with the robust repair effect. Through a range of approaches including single-cell transcriptome analyses, we demonstrated that SZN-1326-p directly impacted epithelial cells, driving transient expansion of stem and progenitor cells, promoting differentiation of epithelial cells, histologically restoring the damaged epithelium, and secondarily to epithelial repair, reducing inflammation. CONCLUSIONS: It is feasible to design Wnt mimetics such as SZN-1326-p that impact damaged intestine epithelium specifically and restore its physiological functions, an approach that holds promise for treating epithelial damage in inflammatory bowel disease.

Non-neuronal expression of SARS-CoV-2 entry genes in the olfactory system suggests mechanisms underlying COVID-19-associated anosmia.

Altered olfactory function is a common symptom of COVID-19, but its etiology is unknown. A key question is whether SARS-CoV-2 (CoV-2) - the causal agent in COVID-19 - affects olfaction directly, by infecting olfactory sensory neurons or their targets in the olfactory bulb, or indirectly, through perturbation of supporting cells. Here we identify cell types in the olfactory epithelium and olfactory bulb that express SARS-CoV-2 cell entry molecules. Bulk sequencing demonstrated that mouse, non-human primate and human olfactory mucosa expresses two key genes involved in CoV-2 entry, ACE2 and TMPRSS2. However, single cell sequencing revealed that ACE2 is expressed in support cells, stem cells, and perivascular cells, rather than in neurons. Immunostaining confirmed these results and revealed pervasive expression of ACE2 protein in dorsally-located olfactory epithelial sustentacular cells and olfactory bulb pericytes in the mouse. These findings suggest that CoV-2 infection of non-neuronal cell types leads to anosmia and related disturbances in odor perception in COVID-19 patients.

Cellular mechanisms of epithelial stem cell self-renewal and differentiation during homeostasis and repair.

Epithelia in adult mammals exhibit remarkable regenerative capacities owing to the presence of adult stem cells, which self-renew and differentiate to replace cells lost to normal turnover or injury. The mechanisms supporting tissue homeostasis and injury-induced repair often differ from each other as well as from those used in embryonic development. Recent studies have also highlighted the phenomenon of cellular plasticity in adult tissues, in which differentiated cells can change fate and even give rise to new stem cell populations to complement the canonical stem cells in promoting repair following injury. Signaling pathways such as WNT, bone morphogenetic protein, and Sonic Hedgehog play critical roles in stem cell maintenance and cell fate decisions across diverse epithelia and conditions, suggesting that conserved mechanisms underlie the regenerative capacity of adult epithelial structures. This article is categorized under: Gene Expression and Transcriptional Hierarchies > Regulatory Mechanisms Adult Stem Cells, Tissue Renewal, and Regeneration > Tissue Stem Cells and Niches Adult Stem Cells, Tissue Renewal, and Regeneration > Stem Cell Differentiation and Reversion Adult Stem Cells, Tissue Renewal, and Regeneration > Regeneration.

clusterExperiment and RSEC: A Bioconductor package and framework for clustering of single-cell and other large gene expression datasets.

Clustering of genes and/or samples is a common task in gene expression analysis. The goals in clustering can vary, but an important scenario is that of finding biologically meaningful subtypes within the samples. This is an application that is particularly appropriate when there are large numbers of samples, as in many human disease studies. With the increasing popularity of single-cell transcriptome sequencing (RNA-Seq), many more controlled experiments on model organisms are similarly creating large gene expression datasets with the goal of detecting previously unknown heterogeneity within cells. It is common in the detection of novel subtypes to run many clustering algorithms, as well as rely on subsampling and ensemble methods to improve robustness. We introduce a Bioconductor R package, clusterExperiment, that implements a general and flexible strategy we entitle Resampling-based Sequential Ensemble Clustering (RSEC). RSEC enables the user to easily create multiple, competing clusterings of the data based on different techniques and associated tuning parameters, including easy integration of resampling and sequential clustering, and then provides methods for consolidating the multiple clusterings into a final consensus clustering. The package is modular and allows the user to separately apply the individual components of the RSEC procedure, i.e., apply multiple clustering algorithms, create a consensus clustering or choose tuning parameters, and merge clusters. Additionally, clusterExperiment provides a variety of visualization tools for the clustering process, as well as methods for the identification of possible cluster signatures or biomarkers. The R package clusterExperiment is publicly available through the Bioconductor Project, with a detailed manual (vignette) as well as well documented help pages for each function.

Creating Lineage Trajectory Maps Via Integration of Single-Cell RNA-Sequencing and Lineage Tracing: Integrating transgenic lineage tracing and single-cell RNA-sequencing is a robust approach for mapping developmental lineage trajectories and cell fate changes.

Mapping the paths that stem and progenitor cells take en route to differentiate and elucidating the underlying molecular controls are key goals in developmental and stem cell biology. However, with population level analyses it is difficult - if not impossible - to define the transition states and lineage trajectory branch points within complex developmental lineages. Single-cell RNA-sequencing analysis can discriminate heterogeneity in a population of cells and even identify rare or transient intermediates. In this review, we propose that using these data, one can infer the lineage trajectories of individual stem cells and identify putative branch points. Clonal lineage tracing of stem cells allows one to define the outcome of differentiation. Integrating these single cell-based approaches provides a robust strategy for establishing and testing models of how an individual stem cell changes through time to differentiate and self-renew.

Slingshot: cell lineage and pseudotime inference for single-cell transcriptomics.

BACKGROUND: Single-cell transcriptomics allows researchers to investigate complex communities of heterogeneous cells. It can be applied to stem cells and their descendants in order to chart the progression from multipotent progenitors to fully differentiated cells. While a variety of statistical and computational methods have been proposed for inferring cell lineages, the problem of accurately characterizing multiple branching lineages remains difficult to solve. RESULTS: We introduce Slingshot, a novel method for inferring cell lineages and pseudotimes from single-cell gene expression data. In previously published datasets, Slingshot correctly identifies the biological signal for one to three branching trajectories. Additionally, our simulation study shows that Slingshot infers more accurate pseudotimes than other leading methods. CONCLUSIONS: Slingshot is a uniquely robust and flexible tool which combines the highly stable techniques necessary for noisy single-cell data with the ability to identify multiple trajectories. Accurate lineage inference is a critical step in the identification of dynamic temporal gene expression.

Injury Activates Transient Olfactory Stem Cell States with Diverse Lineage Capacities.

Tissue homeostasis and regeneration are mediated by programs of adult stem cell renewal and differentiation. However, the mechanisms that regulate stem cell fates under such widely varying conditions are not fully understood. Using single-cell techniques, we assessed the transcriptional changes associated with stem cell self-renewal and differentiation and followed the maturation of stem cell-derived clones using sparse lineage tracing in the regenerating mouse olfactory epithelium. Following injury, quiescent olfactory stem cells rapidly shift to activated, transient states unique to regeneration and tailored to meet the demands of injury-induced repair, including barrier formation and proliferation. Multiple cell fates, including renewed stem cells and committed differentiating progenitors, are specified during this early window of activation. We further show that Sox2 is essential for cells to transition from the activated to neuronal progenitor states. Our study highlights strategies for stem cell-mediated regeneration that may be conserved in other adult stem cell niches.

Deconstructing Olfactory Stem Cell Trajectories at Single-Cell Resolution.

A detailed understanding of the paths that stem cells traverse to generate mature progeny is vital for elucidating the mechanisms governing cell fate decisions and tissue homeostasis. Adult stem cells maintain and regenerate multiple mature cell lineages in the olfactory epithelium. Here we integrate single-cell RNA sequencing and robust statistical analyses with in vivo lineage tracing to define a detailed map of the postnatal olfactory epithelium, revealing cell fate potentials and branchpoints in olfactory stem cell lineage trajectories. Olfactory stem cells produce support cells via direct fate conversion in the absence of cell division, and their multipotency at the population level reflects collective unipotent cell fate decisions by single stem cells. We further demonstrate that Wnt signaling regulates stem cell fate by promoting neuronal fate choices. This integrated approach reveals the mechanisms guiding olfactory lineage trajectories and provides a model for deconstructing similar hierarchies in other stem cell niches.

How Azobenzene Photoswitches Restore Visual Responses to the Blind Retina.

Azobenzene photoswitches confer light sensitivity onto retinal ganglion cells (RGCs) in blind mice, making these compounds promising candidates as vision-restoring drugs in humans with degenerative blindness. Remarkably, photosensitization manifests only in animals with photoreceptor degeneration and is absent from those with intact rods and cones. Here we show that P2X receptors mediate the entry of photoswitches into RGCs, where they associate with voltage-gated ion channels, enabling light to control action-potential firing. All charged photoswitch compounds require permeation through P2X receptors, whose gene expression is upregulated in the blind retina. Photoswitches and membrane-impermeant fluorescent dyes likewise penetrate through P2X receptors to label a subset of RGCs in the degenerated retina. Electrophysiological recordings and mapping of fluorescently labeled RGC dendritic projections together indicate that photosensitization is highly selective for OFF-RGCs. Hence, P2X receptors are a natural conduit allowing cell-type-selective and degeneration-specific delivery of photoswitches to restore visual function in blinding disease.

p63 regulates olfactory stem cell self-renewal and differentiation.

The olfactory epithelium is a sensory neuroepithelium that supports adult neurogenesis and tissue regeneration following injury, making it an excellent model for investigating neural stem cell regulation in vivo. Previous studies have identified the horizontal basal cell (HBC) as the neural stem cell of the postnatal olfactory epithelium. However, the molecules and pathways regulating HBC self-renewal and differentiation are unknown. In the present study, we demonstrate that the transcription factor p63, a member of the p53 tumor suppressor gene family known to regulate stem cell dynamics in other epithelia, is highly enriched in HBCs. We show that p63 is required cell autonomously for olfactory stem cell renewal and further demonstrate that p63 functions to repress HBC differentiation. These results provide critical insight into the genetic regulation of the olfactory stem cell in vivo and more generally provide an entrée toward understanding the coordination of stem cell self-renewal and differentiation.

Expression cloning in Xenopus identifies RNA-binding proteins as regulators of embryogenesis and Rbmx as necessary for neural and muscle development.

We have performed an expression cloning screen in Xenopus laevis with the aim of isolating novel gene activities from the neural plate. Of 8,064 clones screened, we isolated 61 clones that affected either neural plate patterning or tadpole morphology. Of these, 20 clones encoded RNA binding proteins, and the majority of these are heterogeneous nuclear ribonucleoproteins (hnRNPs) or SR-proteins, which are associated with alternative splicing. All of these genes are expressed in the nervous system, and in several cases specific to neural tissue. Injecting mRNA encoding these proteins results in neural plate mispatterning and abnormal muscle segmentation. To initiate characterization of these proteins, we selected Rbmx as a candidate for deeper analysis. Using morpholino mediated knockdown, we show that Rbmx is necessary for normal anterior neural plate patterning, neurogenesis, neural crest development, and muscle segmentation.

The role of FGF signaling in the establishment and maintenance of mesodermal gene expression in Xenopus.

FGF signaling is important for the formation of mesoderm in vertebrates, and when it is perturbed in Xenopus, most trunk and tail mesoderm fails to form. Here we have further dissected the activities of FGF in patterning the embryo by addressing its inductive and maintenance roles. We show that FGF signaling is necessary for the establishment of xbra expression in addition to its well-characterized role in maintaining xbra expression. The role of FGF signaling in organizer formation is not clear in Xenopus. We find that FGF signaling is essential for the initial specification of paraxial mesoderm but not for activation of several pan-mesodermal and most organizer genes; however, early FGF signaling is necessary for the maintenance of organizer gene expression into the neurula stage. Inhibition of FGF signaling prevents VegT activation of specific mesodermal transcripts. These findings illuminate how FGF signaling contributes to the establishment of distinct types of mesoderm.

FGF8 spliceforms mediate early mesoderm and posterior neural tissue formation in Xenopus.

The relative contributions of different FGF ligands and spliceforms to mesodermal and neural patterning in Xenopus have not been determined, and alternative splicing, though common, is a relatively unexplored area in development. We present evidence that FGF8 performs a dual role in X. laevis and X. tropicalis early development. There are two FGF8 spliceforms, FGF8a and FGF8b, which have very different activities. FGF8b is a potent mesoderm inducer, while FGF8a has little effect on the development of mesoderm. When mammalian FGF8 spliceforms are analyzed in X. laevis, the contrast in activity is conserved. Using a loss-of-function approach, we demonstrate that FGF8 is necessary for proper gastrulation and formation of mesoderm and that FGF8b is the predominant FGF8 spliceform involved in early mesoderm development in Xenopus. Furthermore, FGF8 signaling is necessary for proper posterior neural formation; loss of either FGF8a or a reduction in both FGF8a and FGF8b causes a reduction in the hindbrain and spinal cord domains.

Expression of Xenopus tropicalis noggin1 and noggin2 in early development: two noggin genes in a tetrapod.

We report the identification of two distinct noggin genes in the tetrapod Xenopus tropicalis. Noggin functions to antagonize BMP signaling in many developmental contexts, and much work has explored its role in early vertebrate development. We have identified two noggin genes in the tropical clawed frog, X. tropicalis, a diploid anuran which is being explored for its potential as a genetic model system for early vertebrate development. Here we report the cloning and characterization of the Xenopus tropicalis noggin1 and noggin2 genes, which have distinct expression domains in the early embryo with one overlapping domain in the anterior neural tissue. X. tropicalis noggin1 expression is very similar to that of noggin in Xenopus laevis, with expression beginning in the blastula organizer region and continuing through gastrulation and neurulation in the organizer and notochord. Later, it is also expressed in the anterior neural ridge and subsequent forebrain; noggin1 is also expressed in the pharyngeal arches after neural tube closure. At the tadpole stage expression is maintained in the dorsal neural tube and is present in the otic vesicle. However, the expression of noggin2 is much more similar to the expression of noggin2 in D. rerio with expression in the forebrain, hindbrain, and somites, but unlike D. rerio, X. tropicalis noggin2 is expressed in the heart by stage 28. This work presents the first example of a tetrapod with at least two noggin genes.

Neural crest induction by paraxial mesoderm in Xenopus embryos requires FGF signals.

At the border of the neural plate, the induction of the neural crest can be achieved by interactions with the epidermis, or with the underlying mesoderm. Wnt signals are required for the inducing activity of the epidermis in chick and amphibian embryos. Here, we analyze the molecular mechanisms of neural crest induction by the mesoderm in Xenopus embryos. Using a recombination assay, we show that prospective paraxial mesoderm induces a panel of neural crest markers (Slug, FoxD3, Zic5 and Sox9), whereas the future axial mesoderm only induces a subset of these genes. This induction is blocked by a dominant negative (dn) form of FGFR1. However, neither dnFGFR4a nor inhibition of Wnt signaling prevents neural crest induction in this system. Among the FGFs, FGF8 is strongly expressed by the paraxial mesoderm. FGF8 is sufficient to induce the neural crest markers FoxD3, Sox9 and Zic5 transiently in the animal cap assay. In vivo, FGF8 injections also expand the Slug expression domain. This suggests that FGF8 can initiate neural crest formation and cooperates with other DLMZ-derived factors to maintain and complete neural crest induction. In contrast to Wnts, eFGF or bFGF, FGF8 elicits neural crest induction in the absence of mesoderm induction and without a requirement for BMP antagonists. In vivo, it is difficult to dissociate the roles of FGF and WNT factors in mesoderm induction and neural patterning. We show that, in most cases, effects on neural crest formation were parallel to altered mesoderm or neural development. However, neural and neural crest patterning can be dissociated experimentally using different dominant-negative manipulations: while Nfz8 blocks both posterior neural plate formation and neural crest formation, dnFGFR4a blocks neural patterning without blocking neural crest formation. These results suggest that different signal transduction mechanisms may be used in neural crest induction, and anteroposterior neural patterning.