The thyroid gland regulates growth and metabolism via production of thyroid hormone in follicles composed of thyrocytes. So far, thyrocytes have been assumed to be a homogenous population. To uncover ...heterogeneity in the thyrocyte population and molecularly characterize the non‐thyrocyte cells surrounding the follicle, we developed a single‐cell transcriptome atlas of the region containing the zebrafish thyroid gland. The 6249‐cell atlas includes profiles of thyrocytes, blood vessels, lymphatic vessels, immune cells, and fibroblasts. Further, the thyrocytes show expression heterogeneity, including bimodal expression of the transcription factor pax2a. To validate thyrocyte heterogeneity, we generated a CRISPR/Cas9‐based pax2a knock‐in line that monitors pax2a expression in the thyrocytes. A population of pax2a‐low mature thyrocytes interspersed in individual follicles can be distinguished. We corroborate heterogeneity within the thyrocyte population using RNA sequencing of pax2a‐high and pax2a‐low thyrocytes, which demonstrates 20% differential expression in transcriptome between the two subpopulations. Our results identify and validate transcriptional differences within the presumed homogenous thyrocyte population.
Synopsis
This study molecularly characterizes the zebrafish thyroid gland and surrounding structures at single‐cell resolution. It identifies the major cell‐types and potential cross‐talk and uncovers genetic heterogeneity in thyroid follicular cells.
A single‐cell transcriptome atlas of the zebrafish thyroid gland and the surrounding tissue identifies diverse cell‐types, including immune cells, fibroblasts and blood & lymphatic vessels, present in the organ.
An in silico connectome outlines the potential cross‐talk between the various cell‐types.
The thyroid follicular cells display heterogeneity in the expression of multiple genes, including the transcription factor pax2a.
A CRISPR/Cas9‐based knock‐in zebrafish line for pax2a corroborates the expression heterogeneity for pax2a in thyroid follicular cells in vivo.
This study molecularly characterizes the zebrafish thyroid gland and surrounding structures at single‐cell resolution. It identifies the major cell‐types and potential cross‐talk and uncovers genetic heterogeneity in thyroid follicular cells.
The thyroid gland captures iodide in order to synthesize hormones that act on almost all tissues and are essential for normal growth and metabolism. Low plasma levels of thyroid hormones lead to ...hypothyroidism, which is one of the most common disorder in humans and is not always satisfactorily treated by lifelong hormone replacement. Therefore, in addition to the lack of in vitro tractable models to study human thyroid development, differentiation and maturation, functional human thyroid organoids could pave the way to explore new therapeutic approaches. Here we report the generation of transplantable thyroid organoids derived from human embryonic stem cells capable of restoring plasma thyroid hormone in athyreotic mice as a proof of concept for future therapeutic development.
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.
Synopsis
In mouse embryo gastrulation, posterior epiblast cells rearrange in the shape of rosettes before the onset of epithelial‐mesenchymal transition at the primitive streak. Primitive streak cells display a higher frequency of mitosis; mitotic rounding is not limited to the apical side of the cell, and non‐apical mitosis favors asymmetric division giving rise to mesoderm cells, suggesting a role for cell cycle in cell delamination.
Epithelial rearrangement in the shape of rosettes is associated with primitive streak morphogenesis during mouse gastrulation.
Mitosis frequency is higher in the primitive streak region of the embryo.
Non‐apical mitosis is more frequent in the area were cells are undergoing epithelial‐mesenchymal transition.
Posterior non‐apical mitosis results in extrusion of one or two mesoderm daughter cells.
Live imaging of the mouse embryo epiblast reveals that delaminating cells of the posterior primitive streak undergo mitosis at a higher frequency and indicates that non‐apical mitosis might contribute to cell delamination.
Olfactory sensory neurons (OSNs) of the vertebrate olfactory epithelium (OE) undergo continuous turnover but also regenerate efficiently when the OE is acutely damaged by traumatic injury. Two ...distinct pools of neuronal stem/progenitor cells, the globose (GBCs), and horizontal basal cells (HBCs) have been shown to selectively contribute to intrinsic OSN turnover and damage‐induced OE regeneration, respectively. For both types of progenitors, their rate of cell divisions and OSN production must match the actual loss of cells to maintain or to re‐establish sensory function. However, signals that communicate between neurons or glia cells of the OE and resident neurogenic progenitors remain largely elusive. Here, we investigate the effect of purinergic signaling on cell proliferation and OSN neurogenesis in the zebrafish OE. Purine stimulation elicits transient Ca2+ signals in OSNs and distinct non‐neuronal cell populations, which are located exclusively in the basal OE and stain positive for the neuronal stem cell marker Sox2. The more apical population of Sox2‐positive cells comprises evenly distributed glia‐like sustentacular cells (SCs) and spatially restricted GBC‐like cells, whereas the more basal population expresses the HBC markers keratin 5 and tumor protein 63 and lines the entire sensory OE. Importantly, exogenous purine stimulation promotes P2 receptor‐dependent mitotic activity and OSN generation from sites where GBCs are located but not from HBCs. We hypothesize that purine compounds released from dying OSNs modulate GBC progenitor cell cycling in a dose‐dependent manner that is proportional to the number of dying OSNs and, thereby, ensures a constant pool of sensory neurons over time.
Olfactory sensory neurons (OSNs) undergo lifelong renewal from two distinct tissue‐resident progenitor pools. Globose basal cells (GBCs) predominantly contribute to olfactory neurogenesis in intact tissue, while horizontal basal cells (HBCs) become selectively activated upon injury. Here, Stefan Fuss and co‐authors investigate the effect of purinergic signalling on cell proliferation and olfactory neurogenesis using zebrafish. They show that purine compounds stimulate olfactory neurogenesis selectively from GBC but not HBC progenitors, suggesting a mechanistic link between the number of dying OSNs, ATP release, and the rate of GBC progenitor cell divisions.
A subpopulation of deeply quiescent, so-called dormant hematopoietic stem cells (dHSCs) resides at the top of the hematopoietic hierarchy and serves as a reserve pool for HSCs. The state of dormancy ...protects the HSC pool from exhaustion throughout life; however, excessive dormancy may prevent an efficient response to hematological stresses. Despite the significance of dHSCs, the mechanisms maintaining their dormancy remain elusive. Here, we identify CD38 as a novel and broadly applicable surface marker for the enrichment of murine dHSCs. We demonstrate that cyclic adenosine diphosphate ribose (cADPR), the product of CD38 cyclase activity, regulates the expression of the transcription factor c-Fos by increasing the release of Ca2+ from the endoplasmic reticulum (ER). Subsequently, we uncover that c-Fos induces the expression of the cell cycle inhibitor p57Kip2 to drive HSC dormancy. Moreover, we found that CD38 ecto-enzymatic activity at the neighboring CD38-positive cells can promote human HSC quiescence. Together, CD38/cADPR/Ca2+/c-Fos/p57Kip2 axis maintains HSC dormancy. Pharmacological manipulations of this pathway can provide new strategies to improve the success of stem cell transplantation and blood regeneration after injury or disease.
Mesoderm arises at gastrulation and contributes to both the mouse embryo proper and its extra‐embryonic membranes. Two‐photon live imaging of embryos bearing a keratin reporter allowed recording ...filament nucleation and elongation in the extra‐embryonic region. Upon separation of amniotic and exocoelomic cavities, keratin 8 formed apical cables co‐aligned across multiple cells in the amnion, allantois, and blood islands. An influence of substrate rigidity and composition on cell behavior and keratin content was observed in mesoderm explants. Embryos lacking all keratin filaments displayed a deflated extra‐embryonic cavity, a narrow thick amnion, and a short allantois. Single‐cell RNA sequencing of sorted mesoderm cells and micro‐dissected amnion, chorion, and allantois, provided an atlas of transcriptomes with germ layer and regional information. It defined the cytoskeleton and adhesion expression profile of mesoderm‐derived keratin 8‐enriched cells lining the exocoelomic cavity. Those findings indicate a novel role for keratin filaments in the expansion of extra‐embryonic structures and suggest mechanisms of mesoderm adaptation to the environment.
Synopsis
Keratin intermediate filaments form apical cables continuous across multiple cells in mesoderm‐derived extra‐embryonic tissues lining the exocoelomic cavity of the mouse embryo. Loss of keratin results in defective growth of extra‐embryonic membranes.
At late gastrulation, keratin filaments nucleate, elongate, and co‐align across multiple cells.
In explanted mesoderm cells, cell speed and keratin content vary according to the composition and rigidity of the substrate.
Embryos devoid of keratin filaments have a smaller cavity, amnion, and allantois.
A single cell transcriptomic atlas of extra‐embryonic membranes defines early differentiation of clusters in extra‐embryonic mesoderm, amnion, chorion, and allantois.
Cells lining the exocoelomic cavity in mouse embryos assemble keratin cables that are continuous intercellularly, and whose loss affects the growth of extra‐embryonic membranes.
The thyroid gland regulates metabolism and growth
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
holds great therapeutic promise. However, the utilization of
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
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
. Using pharmacological inhibition of TGF-beta pathway, we improve the level of thyroid maturation, in particular the induction of
expression. This in turn, leads to an enhancement of iodide organification
, 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.
An altered gut microbiota is associated with type 1 diabetes (T1D), affecting the production of short-chain fatty acids (SCFA) and glucose homeostasis. We previously demonstrated that enhancing serum ...acetate and butyrate using a dietary supplement (HAMSAB) improved glycemia in non-obese diabetic (NOD) mice and patients with established T1D. The effects of SCFA on immune-infiltrated islet cells remain to be clarified. Here, we performed single-cell RNA sequencing on islet cells from NOD mice fed an HAMSAB or control diet. HAMSAB induced a regulatory gene expression profile in pancreas-infiltrated immune cells. Moreover, HAMSAB maintained the expression of β-cell functional genes and decreased cellular stress. HAMSAB-fed mice showed preserved pancreatic endocrine cell identity, evaluated by decreased numbers of poly-hormonal cells. Finally, SCFA increased insulin levels in human β-like cells and improved transplantation outcome in NOD/SCID mice. Our findings support the use of metabolite-based diet as attractive approach to improve glucose control in T1D.
Display omitted
•HAMSAB induces a tolerogenic phenotype of infiltrated immune cells in NOD mice•HAMSAB reduces the stress response maintaining endocrine cell identity in NOD mice•SCFAs increase insulin levels and function of human beta-like cells•HAMSAB is an attractive strategy for clinical studies in early type 1 diabetes
Diabetology; Cell biology; Transcriptomics; Model organism
PDF
