Wnt/β-catenin signaling affects two modes of neurogenesis in the zebrafish olfactory epithelium

Olfactory sensory neurons undergo continuous turnover and the olfactory epithelium (OE) iscapable of efficient regeneration following traumatic injury. We have identified distinct progenitorcell populations in the zebrafish OE that selectively contribute to maintenance and repairneurogenesis. Gene expression analysis during OE regeneration suggests that canonicalWnt/β-catenin signaling contributes to the regulation of OE neurogenesis. To test thecontribution of Wnt signaling functionally, we pharmacologically manipulated the pathway in theintact OE and an experimental model of OE regeneration. Stimulation of the pathway resulted inan enhanced cell proliferation response that shares similarity with the pattern of neurogenesisthat can be observed in the damaged OE. Inhibitors of Wnt signaling suppressed both, the baserate of maintenance neurogenesis and damage-induced proliferation. Our results suggest thatWnt/β-catenin signaling is both necessary and sufficient to induce repair and maintenanceneurogenesis.Abstract selected by the committee for an oral presentationinfo:eu-repo/semantics/nonPublishe

Rapid de- and Regeneration of The Zebrafish Olfactory System

The peripheral olfactory epithelium (OE) has an exceptionallyhigh regenerative capacity. We use an experimental injury model inzebrafish to study the cellular responses underlying OE regeneration.Nasal irrigation with the detergent Triton X-100 results in the loss ofup to 80% of the olfactory sensory neuron population within 24 h andnear complete recovery within 7 d. We use BrdU incorporation assaysand expression analysis of cell type-specific markers to describethe dynamics of the regeneration response and to identify relevantprogenitor populations contributing to OE regeneration. We identified abasally located cell population, which expresses the stem cell markerssox2, krt5, and tp63, which shows increased neurogenic activityupon injury and is distinct from cells that contribute to maintenanceneurogenesis. Using transcriptome analysis over the time course of de- and regeneration, we have identified candidate molecular pathwaysthat may be critical for the selective activation of repair neurogenesis.Supported by TUBİTAK 113T038.Award for the second Best Poster Presentationinfo:eu-repo/semantics/publishe

Nuclei isolation from whole tissue using a detergent and enzyme-free method

High-throughput transcriptome and epigenome profiling requires preparation of a single cell or single nuclei suspension. Preparation of the suspension with intact cell or nuclei involves dissociation and permeabilization, steps that can introduce unwanted noise and undesirable damage. Particularly, certain cell-types such as neurons are challenging to dissociate into individual cells. Additionally, permeabilization of the cellular membrane to release nuclei requires optimization by trial-and-error, which can be time consuming, labor intensive and financially nonviable. To enhance the robustness and reproducibility of sample preparation for high-throughput sequencing, we describe a rapid enzyme and detergent-free column-based nuclei isolation method. The protocol enables efficient isolation of nuclei from the entire zebrafish brain within 20 minutes. The isolated nuclei display intact nuclear morphology and low propensity to aggregate. Further, flow cytometry allows nuclei enrichment and clearance of cellular debris for downstream application. The protocol, which should work on soft tissues and cultured cells, provides a simple and accessible method for sample preparation that can be utilized for high-throughput profiling, simplifying the steps required for successful single-nuclei RNA-seq and ATAC-seq experiments.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Wnt/beta-catenin Signaling Affects Two Modes of Neurogenesis in the Zebrafish Olfactory Epithelium

Olfactory sensory neurons (OSNs) have a limited life span and need to be generated constantly by maintenance neurogenesis to prevent olfactory loss at old age. In addition, the olfactory epithelium (OE) is capable of mounting efficient regenerative responses to acute tissue damage. In zebrafish, non-identical progenitor pools with distinct tissue distribution contribute to OSN maintenance and OE repair, however, the signals that regulate these two modes of OSN neurogenesis are not well characterized. We have used gene expression profiling by RNA sequencing to identify molecular signaling pathways that are significantly upregulated in an experimental model of OE regeneration. We find that components of the canonical Wnt/β-catenin signaling pathway are strongly activated early after damage to the OE. In the intact OE, β-catenin-positive cells are restricted to regions of maintenance neurogenesis at the central and peripheral edge of the sensory tissue, while repair neurogenesis is induced in the sensory OE upon damage. To test the contribution to Wnt/β-catenin signaling to these two modes of OSN neurogenesis functionally, we manipulated Wnt activity pharmacologically in the intact and lesioned OE. Activation of the Wnt pathway promoted strong cell proliferation responses, including responses in the sensory OE that resembled the pattern of neurogenesis under damage conditions. Inhibitors of the pathway, on the other hand, suppressed, but did not abolish, maintenance neurogenesis and had a suppressive effect on damage-induced proliferation in the sensory OE. Our results suggest that Wnt/β-catenin signaling is necessary and sufficient to induce cell proliferation from two types of neuronal progenitor populations in the OE that selectively contribute to maintenance and repair neurogenesis.info:eu-repo/semantics/publishe

A non-invasive partial ablation study in zebrafish liver

INTRODUCTIONAward for the Best Poster Presentationinfo:eu-repo/semantics/publishe

Disparate progenitor cell populations contribute to maintenance and repair neurogenesis in the zebrafish olfactory epithelium

Olfactory sensory neurons (OSNs) undergo constant turnover under physiological conditions but also regenerate efficiently following tissue injury. Maintenance and repair neurogenesis in the olfactory epithelium (OE) have been attributed to the selective activity of globose (GBCs) and horizontal basal cells (HBCs), respectively. In zebrafish, cells with GBC-like properties are localized to the peripheral margins of the sensory OE and contribute to OSN neurogenesis in the intact OE, while cells that resemble HBCs at the morphological and molecular level are more uniformly distributed. However, the contribution of these cells to the restoration of the injured OE has not been demonstrated. Here, we provide a detailed cellular and molecular analysis of the tissue response to injury and show that a dual progenitor cell system also exists in zebrafish. Zebrafish HBCs respond to the structural damage of the OE and generate a transient population of proliferative neurogenic progenitors that restores OSNs. In contrast, selective ablation of OSNs by axotomy triggers neurogenic GBC proliferation, suggesting that distinct signaling events activate GBC and HBC responses. Molecular analysis of differentially expressed genes in lesioned and regenerating OEs points toward an involvement of the canonical Wnt/β-catenin pathway. Activation of Wnt signaling appears to be sufficient to stimulate mitotic activity, while inhibition significantly reduces, but does not fully eliminate, HBC responses. Zebrafish HBCs are surprisingly active even under physiological conditions with a strong bias toward the zones of constitutive OSN neurogenesis, suggestive of a direct lineage relationship between progenitor cell subtypes.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Single-Cell Trajectory Inference Guided Enhancement of Thyroid Maturation In Vitro Using TGF-Beta Inhibition

The thyroid gland regulates metabolism and growth via secretion of thyroid hormones by thyroid follicular cells (TFCs). Loss of TFCs, by cellular dysfunction, autoimmune destruction or surgical resection, underlies hypothyroidism. Recovery of thyroid hormone levels by transplantation of mature TFCs derived from stem cells in vitro holds great therapeutic promise. However, the utilization of in vitro derived tissue for regenerative medicine is restricted by the efficiency of differentiation protocols to generate mature organoids. Here, to improve the differentiation efficiency for thyroid organoids, we utilized single-cell RNA-Seq to chart the molecular steps undertaken by individual cells during the in vitro transformation of mouse embryonic stem cells to TFCs. Our single-cell atlas of mouse organoid systematically and comprehensively identifies, for the first time, the cell types generated during production of thyroid organoids. Using pseudotime analysis, we identify TGF-beta as a negative regulator of thyroid maturation in vitro .Using pharmacological inhibition of TGF-beta pathway, we improve the level of thyroid maturation, in particular the induction of Nis expression. This in turn, leads to an enhancement of iodide organification in vitro ,suggesting functional improvement of the thyroid organoid. Our study highlights the potential of single-cell molecular characterization in understanding and improving thyroid maturation and paves the way for identification of therapeutic targets against thyroid disorders.info:eu-repo/semantics/publishe

RNA velocity unveils the dynamics of differentiation and maturation of mouse thyroid cells

More than 10 % of the human population suffers from thyroid-related disorders, against which stem-cell derived organoid models provide therapeutic promise. Previous work from our group reported generation of functional thyroid tissue from mouse embryonic stem cells in vitro. However, the efficiency of obtaining mature thyroid cells in vitro remains limited. To enhance the efficiency of in vitro differentiation protocol, we developed a molecular map of the thyroid gland lineage at single-cell resolution. For this, we performed unbiased droplet-based single-cell RNA sequencing of cells from the organoid differentiation protocol. Cluster analysis identified the intermediate stages between stem-cell and mature thyroid. By taking advantage of RNA velocity and pseudo-time trajectory analysis, we identified the key signaling pathways driving thyroid cell differentiation. Specifically, our analysis demonstrated the involvement of TGF-β along with non-canonical Wnt/PCP pathway in regulating thyroid gland lineage. In future, we will pharmacologically manipulate the identified pathways to generate mature and functional thyroid follicular cells in a dish. Overall, our study portrays the power of single-cell gene expression profiling towards improving in vitro organoid models for therapeutic applications.Award for the Best Poster Presentationinfo:eu-repo/semantics/publishe

Asymmetry in the frequency and position of mitosis in the mouse embryo epiblast at gastrulation.

At gastrulation, a subpopulation of epiblast cells constitutes a transient posteriorly located structure called the primitive streak, where cells that undergo epithelial-mesenchymal transition make up the mesoderm and endoderm lineages. Mouse embryo epiblast cells were labelled ubiquitously or in a mosaic fashion. Cell shape, packing, organization and division were recorded through live imaging during primitive streak formation. Posterior epiblast displays a higher frequency of rosettes, some of which associate with a central cell undergoing mitosis. Cells at the primitive streak, in particular delaminating cells, undergo mitosis more frequently than other epiblast cells. In pseudostratified epithelia, mitosis takes place at the apical side of the epithelium. However, mitosis is not restricted to the apical side of the epiblast, particularly on its posterior side. Non-apical mitosis occurs specifically in the streak even when ectopically located. Posterior non-apical mitosis results in one or two daughter cells leaving the epiblast layer. Cell rearrangement associated with mitotic cell rounding in posterior epiblast, in particular when non-apical, might thus facilitate cell ingression and transition to a mesenchymal phenotype.info:eu-repo/semantics/publishe

An asymmetry in the frequency and position of mitosis in the epiblast precedes gastrulation and suggests a role for mitotic rounding in cell delamination during primitive streak epithelial-mesenchymal transition

info:eu-repo/semantics/publishedin revision a EMBO Report