Probes for INS

Spermatogonial stem cells (SSCs) fuel the production of male germ cells but the mechanisms behind SSC self-renewal, proliferation, and differentiation are still poorly understood. Using the Wnt target gene Axin2 and genetic lineage-tracing experiments, we found that undifferentiated spermatogonia, comprising SSCs and transit amplifying progenitor cells, respond to Wnt/β-catenin signals. Genetic elimination of β-catenin indicates that Wnt/β-catenin signaling promotes the proliferation of these cells. Signaling is likely initiated by Wnt6, which is uniquely expressed by neighboring Sertoli cells, the only somatic cells in the seminiferous tubule that support germ cells and act as a niche for SSCs. Therefore, unlike other stem cell systems where Wnt/β-catenin signaling is implicated in self-renewal, the Wnt pathway in the testis specifically contributes to the proliferation of SSCs and progenitor cells.

An increasing amount of evidence indicates that developmental programs are tightly regulated by the complex interplay between signalingpathways, as well as transcriptional and epigenetic processes. Here, we have uncovered coordination between transcriptional and morphogen cues to specify Merkel cells, poorly understood skin cells that mediate light touch sensations. In murine dorsal skin, Merkel cells are part of touch domes, which are skin structures consisting of specialized keratinocytes, Merkel cells, and afferent neurons, and are located exclusively around primary hair follicles. We show that the developing primary hair follicle functions as a niche required for Merkel cell specification. We find that intraepidermal Sonic hedgehog (Shh) signaling, initiated by the production of Shh ligand in the developing hair follicles, is required forMerkel cell specification. The importance of Shh for Merkel cell formation is further reinforced by the fact that Shh overexpression in embryonic epidermal progenitors leads to ectopic Merkel cells. Interestingly, Shh signaling is common to primary, secondary, and tertiary hair follicles, raising the possibility that there are restrictive mechanisms that regulate Merkel cell specification exclusively around primary hair follicles. Indeed, we find that loss of Polycomb repressive complex 2 (PRC2) in the epidermis results in the formation of ectopic Merkel cells that are associated with all hair types. We show that PRC2 loss expands the field of epidermal cells competent to differentiate into Merkel cells through the upregulation of key Merkel-differentiation genes, which are known PRC2 targets. Importantly, PRC2-mediated repression of the Merkel celldifferentiation program requires inductive Shh signaling to form mature Merkel cells. Our study exemplifies how the interplay between epigenetic and morphogen cues regulates the complex patterning and formation of the mammalian skin structures.

Abstract

PURPOSE:

Understanding tumour heterogeneity is an important challenge in current cancer research. Transcription and epigenetic profiling of cultured melanoma cells have defined at least two distinct cell phenotypes characterised by distinctive gene expression signatures associated with high or low/absent expression of Microphthalmia-associated transcription factor (MITF). Nevertheless, heterogeneity of cellpopulations and gene expression in primary human tumours is much less well characterised.

EXPERIMENTAL DESIGN:

We performed single cell gene expression analyses on 472 cells isolated from needle biopsies of 5 primary human melanomas, 4 superficial spreading and one acral melanoma. The expression of MITF-high and MITF-low signature genes was assessed and compared to investigate intra and inter-tumoural heterogeneity and correlated gene expression profiles.

RESULTS:

Single cell gene expression analyses revealed varying degrees of intra and inter-tumour heterogeneity conferred by the variable expression of distinct sets of genes in different tumours. Expression of MITF partially correlated with that of its known target genes while SOX10 expression correlated best with PAX3 and ZEB2. Nevertheless, cells simultaneously expressing MITF-high and MITF-low signature genes were observed both by single cell analyses and RNAscope.

CONCLUSIONS:

Single cell analyses can be performed on limiting numbers of cells from primary human melanomas revealing their heterogeneity. While tumours comprised variable proportions of cells with the MITF-high and MITF-low gene expression signatures characteristic of melanoma cultures, primary tumours also comprised cells expressing markers of both signatures defining a novel cell state in tumours in vivo.

Abstract

BACKGROUND:

Large animal models, such as the dog, are increasingly being used for studying diseases including gastrointestinal (GI) disorders. Dogs share similar environmental, genomic, anatomical, and intestinal physiologic features with humans. To bridge the gap between commonly used animal models, such as rodents, and humans, and expand the translational potential of the dog model, we developed a three-dimensional (3D) canine GI organoid (enteroid and colonoid) system. Organoids have recently gained interest in translational research as this model system better recapitulates the physiological and molecular features of the tissue environment in comparison with two-dimensional cultures.

RESULTS:

Organoids were derived from tissue of more than 40 healthy dogs and dogs with GI conditions, including inflammatory bowel disease (IBD) and intestinal carcinomas. Adult intestinal stem cells (ISC) were isolated from whole jejunal tissue as well as endoscopically obtained duodenal, ileal, and colonic biopsy samples using an optimized culture protocol. Intestinal organoids were comprehensively characterized using histology, immunohistochemistry, RNA in situ hybridization, and transmission electron microscopy, to determine the extent to which they recapitulated the in vivo tissue characteristics. Physiological relevance of the enteroid system was defined using functional assays such as optical metabolic imaging (OMI), the cystic fibrosis transmembrane conductance regulator (CFTR) function assay, and Exosome-Like Vesicles (EV) uptake assay, as a basis for wider applications of this technology in basic, preclinical and translational GI research. We have furthermore created a collection of cryopreserved organoids to facilitate future research.

CONCLUSIONS:

We establish the canine GI organoid systems as a model to study naturally occurring intestinal diseases in dogs and humans, and that can be used for toxicology studies, for analysis of host-pathogen interactions, and for other translational applications.

Chondrocytes and osteoblasts differentiate from a common mesenchymal precursor, the osteochondroprogenitor (OCP), and help build the vertebrate skeleton. The signaling pathways that control lineage commitment for OCPs are incompletely understood. We asked whether the ubiquitously expressed protein-tyrosine phosphatase SHP2 (encoded by Ptpn11) affects skeletal lineage commitment by conditionally deleting Ptpn11 in mouse limb and head mesenchyme using “Cre-loxP”-mediated gene excision. SHP2-deficient mice have increased cartilage mass and deficient ossification, suggesting that SHP2-deficient OCPs become chondrocytes and not osteoblasts. Consistent with these observations, the expression of the master chondrogenic transcription factor SOX9 and its target genes Acan, Col2a1, and Col10a1 were increased in SHP2-deficient chondrocytes, as revealed by gene expression arrays, qRT-PCR, in situ hybridization, and immunostaining. Mechanistic studies demonstrate that SHP2 regulates OCP fate determination via the phosphorylation and SUMOylation of SOX9, mediated at least in part via the PKA signaling pathway. Our data indicate that SHP2 is critical for skeletal cell lineage differentiation and could thus be a pharmacologic target for bone and cartilage regeneration.