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Stem cells in the anterior pituitary gland can give rise to all resident endocrine cells and are integral components for the appropriate development and subsequent maintenance of the organ. Located in discreet niches within the gland, stem cells are involved in bi-directional signalling with their surrounding neighbours, interactions which underpin pituitary gland homeostasis and response to organ challenge or physiological demand. In this review we highlight core signalling pathways that steer pituitary progenitors towards specific endocrine fate decisions throughout development. We further elaborate on those which are conserved in the stem cell niche postnatally, including WNT, YAP/TAZ and Notch signalling. Furthermore, we have collated a directory of single cell RNA sequencing studies carried out on pituitaries across multiple organisms, which have the potential to provide a vast database to study stem cell niche components in an unbiased manner. Reviewing published data, we highlight that stem cells are one of the main signalling hubs within the anterior pituitary. In future, coupling single cell sequencing approaches with genetic manipulation tools in vivo, will enable elucidation of how previously understudied signalling pathways function within the anterior pituitary stem cell niche.

The inhibins control reproduction by suppressing follicle-stimulating hormone synthesis in pituitary gonadotrope cells. The newly discovered inhibin B coreceptor, TGFBR3L, is selectively and highly expressed in gonadotropes in both mice and humans. Here, we describe our initial characterization of mechanisms controlling cell-specific Tgfbr3l/TGFBR3L transcription. We identified two steroidogenic factor 1 (SF-1 or NR5A1) cis-elements in the proximal Tgfbr3l promoter in mice. SF-1 induction of murine Tgfbr3l promoter–reporter activity was inhibited by mutations in one or both sites in heterologous cells. In homologous cells, mutation of these cis-elements or depletion of endogenous SF-1 similarly decreased reporter activity. We observed nearly identical results when using a human TGFBR3L promoter–reporter. The Tgfbr3l gene was tightly compacted and Tgfbr3l mRNA expression was essentially absent in gonadotropes of SF-1 (Nr5a1) conditional knockout mice. During murine embryonic development, Tgfbr3l precedes Nr5a1 expression, though the two transcripts are fully colocalized by embryonic day 18.5 and thereafter. Collectively, these data indicate that SF-1 directly regulates Tgfbr3l/TGFBR3L transcription and is required for postnatal expression of the gene in gonadotropes.

Introduction Hormone-producing pituitary cell lineages are derived from a population of embryonic precursors expressing SOX2. These cells maintain multipotency into early postnatal life, acting as the resident population of pituitary stem cells (PSCs) and contributing extensively to all the endocrine cell lineages. In addition to this direct contribution to pituitary turnover, paracrine signalling from PSCs has been shown to be important for cell proliferation of neighbouring progenitors (PMC7803373). It is not known if SOX2+ PSCs are involved in the regulation of additional cell attributes during normal physiology and if there is functional heterogeneity among the SOX2+ PSC population. Experimental Methods We have carried out single-cell RNA-Sequencing of SOX2+ PSCs from Sox2Egfp/+ mouse pituitaries at three postnatal stages from P3 to P56 and used computational approaches to analyse their molecular signatures. A novel conditional mouse model expressing a constitutively active mutant form of the RNA binding factor BRF1 (R26stop-mBRF1) has been used to attenuate the expression of several cytokines and chemokines in SOX2+ cells embryonically and postnatally (PMC4589897). Results We show that the SOX2+ PSC population consists of three subgroups (SC1, SC2 and SC3). We reveal that SC1-SC2 express abundant cytokines and secreted factors, suggesting paracrine function. In contrast, SC3 is characterised by robust expression of Lef1, is identified as a committing PSC cluster, and its presence diminishes with age. Key markers of PSC clusters SC1-SC2 include the RNA binding factor BRF1. We show that BRF1 is highly expressed in PSCs and validate its expression by immunohistochemistry in both mouse and human pituitaries. Secondly, we show that the dysregulation of BRF1 in embryonic SOX2+ cells using the Hesx1-Cre driver (PMC3461924) results in pituitary hypoplasia and severe hypopituitarism due to a failure of the PIT1 and SF1 cell-lineage committed progenitors to terminally differentiate into hormone-producing cells. Additionally, there is a significant reduction of the stem cell compartment, manifested by lower numbers of SOX2/SOX9+ stem cells. This phenotype is recapitulated when using a Sox2-CreERT2 driver (PMID24094324). The differentiation failure can be rescued in vitro through co-culture of mutant cells with wild-type stem cells, as well as in vivo, in mutant pituitaries where activation of constitutively active BRF1 is restricted to few SOX2+ PSCs in a mosaic manner. Finally, we identify key ligands underlying this differentiation phenotype and demonstrate a partial restoration of terminal differentiation in the mutant, when cultured in the presence of these ligands. Conclusion We provide evidence indicating the presence of functionally distinct groups of SOX2+ pituitary stem cells and reveal a critical role for a PSC subset in the development of the stem cell compartment and in driving terminal differentiation of committed progenitors. Presentation: Sunday, June 12, 2022 1:00 p.m. - 1:05 p.m., Monday, June 13, 2022 12:30 p.m. - 2:30 p.m.

Stem cells in the anterior pituitary gland can give rise to all resident endocrine cells and are integral components for the appropriate development and subsequent maintenance of the organ. Located in discreet niches within the gland, stem cells are involved in bi-directional signalling with their surrounding neighbours, interactions which underpin pituitary gland homeostasis and response to organ challenge or physiological demand. In this review we highlight core signalling pathways that steer pituitary progenitors towards specific endocrine fate decisions throughout development. We further elaborate on those which are conserved in the stem cell niche postnatally, including WNT, YAP/TAZ and Notch signalling. Furthermore, we have collated a directory of single cell RNA sequencing studies carried out on pituitaries across multiple organisms, which have the potential to provide a vast database to study stem cell niche components in an unbiased manner. Reviewing published data, we highlight that stem cells are one of the main signalling hubs within the anterior pituitary. In future, coupling single cell sequencing approaches with genetic manipulation tools in vivo, will enable elucidation of how previously understudied signalling pathways function within the anterior pituitary stem cell niche.

Despite their importance in tissue homeostasis and renewal, human pituitary stem cells (PSCs) are incompletely characterized. We describe a human single nucleus (sn) RNAseq and ATACseq resource from pediatric, adult, and aged pituitaries (snpituitaryatlas.princeton.edu) and characterize cell type-specific gene expression and chromatin accessibility programs for all major pituitary cell lineages. We identify uncommitted PSCs, committing progenitor cells, and sex differences. Pseudotime trajectory analysis indicates that early life PSCs are distinct from the other age groups. Linear modeling of same-cell multiome data identifies regulatory domain accessibility sites and transcription factors (TFs) that are significantly associated with gene expression in PSCs compared to other cell types and within PSCs. Modeling the heterogeneous expression of two markers for committing cell lineages among PSCs shows significant correlation with regulatory domain accessibility forGATA3, but with TF expression forPOMC. These findings characterize human stem cell lineages and reveal diverse mechanisms regulating key PSC genes.

Cellular quiescence is a dormant but reversible cellular state in which cell-cycle entry and proliferation are prevented. Recent studies both in vivo and in vitro demonstrate that quiescence is actively maintained through synergistic interactions between intrinsic and extrinsic signals. Subtypes of adult mammalian stem cells can be maintained in this poised, quiescent state, and subsequently reactivated upon tissue injury to restore homeostasis. However, quiescence can become deregulated in pathological settings. In this review, we discuss the recent advances uncovering intracellular signaling pathways, transcriptional changes, and extracellular cues within the stem cell niche that control induction and exit from quiescence in tissue stem cells. We discuss the implications of quiescence as well as the pharmacological and genetic approaches that are being explored to either induce or prevent quiescence as a therapeutic strategy. : Stem cell quiescence is a dormant but reversible cellular state in which cell-cycle entry and proliferation are prevented. Intrinsic regulators include the cell-cycle machinery, autophagy, chromatin modifiers, and microRNAs. Extrinsic regulators include cell-adhesion molecules and secreted factors. Therapeutic modulation of stem cell quiescence could restore tissue homeostasis. Keywords: stem cells, cancer, quiescence, cell cycle, autophagy, niche, cell therapy

In mammals, imprinted genes regulate many critical endocrine processes such as growth, the onset of puberty and maternal reproductive behaviour. Human imprinting disorders (IDs) are caused by genetic and epigenetic mechanisms that alter the expression dosage of imprinted genes. Due to improvements in diagnosis, increasing numbers of patients with IDs are now identified and monitored across their lifetimes. Seminal work has revealed that IDs have a strong endocrine component, yet the contribution of imprinted gene products in the development and function of the hypothalamo-pituitary axis are not well defined. Postnatal endocrine processes are dependent upon the production of hormones from the pituitary gland. While the actions of a few imprinted genes in pituitary development and function have been described, to date there has been no attempt to link the expression of these genes as a class to the formation and function of this essential organ. This is important because IDs show considerable overlap, and imprinted genes are known to define a transcriptional network related to organ growth. This knowledge deficit is partly due to technical difficulties in obtaining useful transcriptomic data from the pituitary gland, namely, its small size during development and cellular complexity in maturity. Here we utilise high-sensitivity RNA sequencing at the embryonic stages, and single-cell RNA sequencing data to describe the imprinted transcriptome of the pituitary gland. In concert, we provide a comprehensive literature review of the current knowledge of the role of imprinted genes in pituitary hormonal pathways and how these relate to IDs. We present new data that implicate imprinted gene networks in the development of the gland and in the stem cell compartment. Furthermore, we suggest novel roles for individual imprinted genes in the aetiology of IDs. Finally, we describe the dynamic regulation of imprinted genes in the pituitary gland of the pregnant mother, with implications for the regulation of maternal metabolic adaptations to pregnancy.

The pituitary gland regulates key physiological functions, including growth, sexual maturation, reproduction, and lactation. Here, we present a paired single-nuclei (sn) transcriptome and chromatin accessibility characterization of six post-mortem human pituitaries. These samples were from juvenile, adult, and elderly male and female subjects. Well-correlated snRNAseq and snATACseq datasets facilitated robust identification of the major pituitary cell types in each sample. Using latent variable pathway analysis, we uncovered previously unreported coordinated gene expression modules and chromatin accessibility programs for each major cell type as well as an age-specific program across all the endocrine cell types. These largely appear to be congruent between human and mouse datasets. Given the importance of murine models in the study of human pituitary disorders and pituitary physiology, we next sought to compare expression profiles of pituitary cell types in mouse vs. human. Murine and human cell types were well correlated, exemplified by coordinated gene expression programs, especially for undifferentiated stem cells (SCs). In both species, we identified clusters corresponding to naive and committing SCs. All human SC clusters expressed the established SC markers SOX2 and SOX9, as well as genes involved in SC regulatory pathways (WWTR1, YAP1 andPITX2). Additional markers previously reported in murine pituitary SCs were also found in human SC, including WIF1, LGR5, FOS, CDH1, EGFR, LGR4, and WLS. Remarkably, in human, the main naive SC cluster was roughly divided into a high-JUN and a low-JUN expressing subgroup, whereas Jun expression was less pronounced in the murine SC cluster. In both species, committing SC clusters expressed the endocrine markers for POU1F1, TSHB, or POMC, while SCs committing to an intermediate lobe/melanotrope cell identity were distinguishable based on PAX7 expression. In addition, in the human datasets we identify a population of cells as originating from the pars tuberalis. We offer a range of markers that can be utilized for in vivo validation of these cells. Overall, the characterization of the murine and human pituitary SCs strongly suggests the co-existence of subpopulations with different lineage commitments in addition to a single uncommitted SC population. This sn atlas of the human pituitary is a valuable resource that will be made web-accessible.

Autophagy comprises the processes of autophagosome synthesis and lysosomal degradation. In certain stress conditions, increased autophagosome synthesis may be associated with decreased lysosomal activity, which may result in reduced processing of the excessive autophagosomes by the rate-limiting lysosomal activity. Thus, the excessive autophagosomes in such situations may be largely unfused to lysosomes, and their formation/accumulation under these conditions is assumed to be futile for autophagy. The role of cytotoxicity in accumulating autophagosomes (representing synthesis of autophagosomes subsequently unfused to lysosomes) has not been investigated previously. Here, we found that accumulation of autophagosomes compromised cell viability, and this effect was alleviated by depletion of autophagosome machinery proteins. We tested whether reduction in autophagosome synthesis could affect cell viability in cell models expressing mutant huntingtin and α-synuclein, given that both of these proteins cause increased autophagosome biogenesis and compromised lysosomal activity. Importantly, partial depletion of autophagosome machinery proteins Atg16L1 and Beclin 1 significantly ameliorated cell death in these conditions. Our data suggest that production/accumulation of autophagosomes subsequently unfused to lysosomes (or accumulation of autophagosomes) directly induces cellular toxicity, and this process may be implicated in the pathogenesis of neurodegenerative diseases. Therefore, lowering the accumulation of autophagosomes may represent a therapeutic strategy for tackling such diseases.

Autophagy cargo recognition and clearance are essential for intracellular protein quality control. SQSTM1/p62 sequesters intracellular aberrant proteins and mediates cargo delivery for their selective autophagic degradation. The formation of p62 non-membrane-bound liquid compartments is critical for its function as a cargo receptor. The regulation of p62 phase separation/condensation has yet been poorly characterised. Using an unbiased yeast two-hybrid screening and complementary approaches, we found that DAXX physically interacts with p62. Cytoplasmic DAXX promotes p62 puncta formation. We further elucidate that DAXX drives p62 liquid phase condensation by inducing p62 oligomerisation. This effect promotes p62 recruitment of Keap1 and subsequent Nrf2-mediated stress response. The present study suggests a mechanism of p62 phase condensation by a protein interaction, and indicates that DAXX regulates redox homoeostasis, providing a mechanistic insight into the prosurvival function of DAXX., The autophagy protein p62 undergoes liquid-liquid phase separation but how this is regulated is unclear. Here, the authors report that the histone chaperone DAXX interacts with p62 in the cytoplasm to drive its phase separation.