Probes for INS

Through their ability to regulate gene expression in most organs, glucocorticoid hormones influence numerous physiological processes and therefore are key regulators of organismal homeostasis. In bone, glucocorticoid hormones inhibit the expression of the hormone Osteocalcin for poorly understood reasons. Here we show that in a classical endocrine feedback loop, osteocalcin in return enhances the biosynthesis of glucocorticoid but also mineralocorticoid hormones (adrenal steroidogenesis) in rodents and primates. Conversely, inactivating osteocalcin signalling in adrenal glands significantly impairs adrenal growth and steroidogenesis in mice. Embryo-made osteocalcin is necessary for normal Sf1 expression in foetal adrenal cells and adrenal cell steroidogenic differentiation, it therefore determines the number of steroidogenic cells present in adrenal glands of adult animals. Embryonic not postnatal osteocalcin also governs adrenal growth, adrenal steroidogenesis, blood pressure, electrolyte equilibrium and the rise of circulating corticosterone during the acute stress response in adult offspring. This osteocalcin-dependent regulation of adrenal development and steroidogenesis occurs even in the absence of a functional of hypothalamus-pituitary-adrenal axis; this explains why osteocalcin administration during pregnancy promotes adrenal growth and steroidogenesis and improves survival of adrenocorticotropic hormone signalling-deficient animals. This study reveals that a bone-derived, embryonic hormone influences lifelong adrenal functions and organismal homeostasis in the mouse.

ADAMTS18 is an orphan member of the ADAMTS family of metalloproteinase. ADAMTS18 mutation has been linked to developmental eye disorders, such as retinal dystrophies and ectopia lentis. Here, we report a new function of ADAMTS18 in modulating the lacrimal gland (LG) branching morphogenesis, and an association with dry eye in mice. Adamts18 mRNA was found to be enriched in the epithelium of branching tips of embryonic (E) LG, but its expression was barely detectable after 2 weeks of birth. Histological analyses of E16.5-E17.5 LG showed that ADAMTS18 deficiency resulted in a significant reduction of epithelial branching in embryonic LG. In vitro culture of E15.5 LG explants showed that the numbers of epithelial buds and branches in Adamts18 knockout (Adamts18-/-) LGs were significantly decreased when compared to those of wild type (Adamts18+/+) LGs after 0 h, 24 h, and 48 h of culture. Increased fibronectin deposition was detected in LG mesenchyme of E16.5 Adamts18-/- mice. At 14 months of age, Adamts18-/- mice manifested multiple LG pathological changes, including acinar atrophy and irregular duct ectasis with periductal fibrosis. The tear volume was significantly decreased in Adamts18-/- mice at 4 months of age, which corresponds to early adulthood in humans.

Bone marrow (BM) fibrosis was thought to be induced exclusively by mesenchymal stromal cells (MSCs). However, we and others found that neoplastic fibrocytes induce BM fibrosis in myelofibrosis (MF). Because glioma-associated oncogene-1 (GLI1), an effector of the Hedgehog pathway, plays a role in the induction of BM fibrosis, we wondered whether GLI1 affects fibrocyte-induced BM fibrosis in MF. Multiplexed fluorescence immunohistochemistry analysis of MF patients' BM detected high levels of GLI1 in MF fibrocytes compared to MSCs or normal fibrocytes. Immunostaining, RNA in situ hybridization, gene expression analysis, and western immunoblotting detected high levels of GLI1 and GLI1-induced matrix metalloproteases (MMP) 2 and 9 in MF patients BM-derived cultured fibrocytes. Similarly, MF patients' BM-derived GLI1+ fibrocytes were found in BMs and spleens of MF xenograft mice. GLI1 silencing reduced the levels of MMP2/9, phosphorylated SMAD2/3, and procollagen-I, and knockdown or inhibition of GLI1 decreased fibrocyte formation and induced apoptosis of both fibrocytes and fibrocyte progenitors. Because Janus kinase (JAK)2-induced STAT3 is constitutively activated in MF and because STAT3 induces GLI1 expression, we sought to determine whether STAT3 activates GLI1 in MF fibrocytes. Imaging analysis detected phosphotyrosine STAT3 in MF patients' BM fibrocytes, and transfection of fibrocytes with STAT3-siRNA or treatment with a JAK1/2 inhibitor ruxolitinib reduced GLI1 and MMP2/9 levels. Chromatin immunoprecipitation and a luciferase assay revealed that STAT3 induced the expression of the GLI1 gene in both MF BM fibrocytes and fibrocyte progenitors. Together, our data suggest that STAT3-activated GLI1 contributes to the induction of BM fibrosis in MF.

The ADAMTS (A Disintegrin and Metalloproteinase with Thrombospondin Motifs) enzymes are secreted metalloproteinases with major roles in development, morphogenesis, and tissue repair via the assembly and degradation of extracellular matrix (ECM). In this study, we investigated the role of ADAMTS18 in the development of the reproductive tract in female mice by phenotyping Adamts18 knockout (Adamts18-/-) mice. The results showed that Adamst18 mRNAs were abundantly expressed in vaginal epithelial cells and muscularis cells of the developing vagina. At the time of vaginal opening (5 weeks of age), about 41 % of Adamts18-/- females showed enlarged protrusions in the upper and middle parts of the vagina, reduced vaginal length, and simultaneously exhibited vaginal atresia. 6% Adamts18-/- females exhibited vaginal septum. Histological analyses revealed that the paired Mullerian ducts in ∼33 % female Adamts18-/- embryos failed to fuse at embryonic day 15.5 (E15.5) resulting in the formation of two vaginal cavities. Results of TUNEL assay and immunohistochemistry for caspase-3 showed that the number of apoptotic cells in the terminal portion of the vagina of 5-week-old Adamts18-/- females with vaginal atresia was significantly decreased. Adamts18-/- females also showed a significant decrease in serum estradiol E2 compared to age-matched Adamts18+/+ females. Results of qRT-PCR showed that the expression level of the anti-apoptosis gene Bcl-2 was significantly increased and that of the apoptosis-related gene Epha1 was decreased in the vagina of 5-week-old Adamts18-/- females. These results suggest that ADAMTS18 regulates vaginal opening through influencing the fusion of Mullerian ducts and apoptosis of vaginal cells in mice.

Birth defects of the external genitalia are among the most common in the world. Proper formation of the external genitalia requires a highly orchestrated process that involves special cell populations and sexually dimorphic hormone signaling. It is clear what the end result of the sexually dimorphic development is (a penis in the male versus clitoris in the female); however, the cell populations involved in the process remain poorly defined. Here, we used single-cell messenger RNA sequencing in mouse embryos to uncover the dynamic changes in cell populations in the external genitalia during the critical morphogenetic window. We found that overall, male and female external genitalia are largely composed of the same core cellular components. At the bipotential stage of development (embryonic day or E14.5), few differences in cell populational composition exist between male and female. Although similar in cell population composition, genetic differences in key sexual differentiation developmental pathways arise between males and females by the early (E16.5) and late (E18.5) differentiation stages. These differences include discrete cell populations with distinct responsiveness to androgen and estrogen. By late sexual differentiation (E18.5), unique cell populations in both male and female genitalia become apparent and are enriched with androgen- and estrogen-responsive genes, respectively. These data provide insights into the morphogenesis of the external genitalia that could be used to understand diseases associated with defects in the external genitalia.

The vestibular lamina (VL) forms the oral vestibule, creating a gap between the teeth, lips and cheeks. In a number of ciliopathies, formation of the vestibule is defective, leading to the creation of multiple frenula. In contrast to the neighbouring dental lamina, which forms the teeth, little is known about the genes that pattern the VL. Here, we establish a molecular signature for the usually non-odontogenic VL in mice and highlight several genes and signalling pathways that may play a role in its development. For one of these, the Sonic hedgehog (Shh) pathway, we show that co-receptors Gas1, Cdon and Boc are highly expressed in the VL and act to enhance the Shh signal from the forming incisor region. In Gas1 mutant mice, expression of Gli1 was disrupted and the VL epithelium failed to extend due to a loss of proliferation. This defect was exacerbated in Boc/Gas1 double mutants and could be phenocopied using cyclopamine in culture. Signals from the forming teeth, therefore, control development of the VL, coordinating the development of the dentition and the oral cavity.

ADAMTS18 was identified in 2002 as a member of the ADAMTS family of 19 secreted Zinc-dependent metalloproteinases. Prior to 2016, ADAMTS18 was known as a candidate gene associated with a wide range of pathologies, particularly various malignancies and eye disorders. However, functions and substrates of ADAMTS18 in normal conditions were unknown. Since 2016, with the development of Adamts18 knockout models, many studies had been conducted on the Adamts18 gene in vivo. These studies revealed that ADAMTS18 is essential for the morphology and organogenesis of several epithelial organs (e.g., lung, kidney, breast, salivary glands, and lacrimal glands), vascular and neuronal systems, adipose tissue, and reproductive tracts. In this review, we describe the current understanding of ADAMTS18 and its substrates and regulators. Limitations in translating new findings on ADAMTS18 to clinical practice are also discussed.

Members of the ADAMTS family have been implicated in various vascular diseases. However, their functional roles in early embryonic vascular development are unknown. In this study, we showed that Adamts18 is highly expressed at E11.5-E14.5 in cells surrounding the embryonic aortic arch (AOAR) and the common carotid artery (CCA) during branchial arch artery development in mice. Adamts18 deficiency was found to cause abnormal development of AOAR, CCA, and the 3rd and 4th branchial arch appendages, leading to hypoplastic carotid body, thymus, and variation of middle cerebral artery. Adamts18 was shown to affect the accumulation of extracellular matrix (ECM) components, in particular fibronectin (Fn), around AOAR and CCA. As a result of increased Fn accumulation, the Notch3 signaling pathway was activated to promote the differentiation of cranial neural crest cells (CNCCs) to vascular smooth muscle cells. These data indicate that Adamts18-mediated ECM homeostasis is crucial for the differentiation of CNCCs.

In comparison to our understanding of endochondral ossification, much less is known about the coordinated arrest of growth defined by the narrowing and fusion of the cartilaginous growth plate. Throughout the musculoskeletal system, appropriate cell and tissue responses to mechanical force delineate morphogenesis and ensure lifelong health. It remains unclear how mechanical cues are integrated into many biological programmes including those coordinating the ossification of the adolescent growth plate at the cessation of growth. Primary cilia are microtubule-based organelles tuning a range of cell activities, including signalling cascades activated or modulated by extracellular biophysical cues. Cilia have been proposed to directly facilitate cell mechanotransduction. To explore the influence of primary cilia in the mouse adolescent limb, we conditionally targeted the ciliary gene Intraflagellar transport protein 88 (Ift88fl/fl ) in the juvenile and adolescent skeleton using a cartilage-specific, inducible, Cre (AggrecanCreERT2 Ift88fl/fl ). Deletion of IFT88 in cartilage, which reduced ciliation in the growth plate, disrupted chondrocyte differentiation, cartilage resorption and mineralisation. These effects were largely restricted to peripheral tibial regions beneath the load-bearing compartments of the knee. These regions were typified by an enlarged population of hypertrophic chondrocytes. While normal patterns of hedgehog signalling were maintained, targeting IFT88 inhibited hypertrophic chondrocyte VEGF expression and downstream vascular recruitment, osteoclastic activity and the replacement of cartilage with bone. In control mice, increases to physiological loading also impair ossification in the peripheral growth plate, mimicking the effects of IFT88 deletion. Limb immobilisation inhibited changes to VEGF expression and epiphyseal morphology in Ift88cKO mice, indicating the effects of depletion of IFT88 in the adolescent growth plate are mechano-dependent. We propose that during this pivotal phase in adolescent skeletal maturation, ciliary IFT88 protects uniform, coordinated ossification of the growth plate from an otherwise disruptive heterogeneity of physiological mechanical forces. This article is protected by

There are major differences in duration and scale at which limb development and regeneration proceed, raising the question to what extent regeneration is a recapitulation of development. We address this by analyzing skeletal elements using a combination of micro-CT imaging, molecular profiling and clonal cell tracing. We find that, in contrast to development, regenerative skeletal growth is accomplished based entirely on cartilage expansion prior to ossification, not limiting the transversal cartilage expansion and resulting in bulkier skeletal parts. The oriented extension of salamander cartilage and bone appear similar to the development of basicranial synchondroses in mammals, as we found no evidence for cartilage stem cell niches or growth plate-like structures during neither development nor regeneration. Both regenerative and developmental ossification in salamanders start from the cortical bone and proceeds inwards, showing the diversity of schemes for the synchrony of cortical and endochondral ossification among vertebrates.

Sonic hedgehog (Shh) signaling plays a critical role in regulating cerebellum development by maintaining the physiological proliferation of granule neuron precursors (GNPs), and its dysregulation leads to the oncogenesis of medulloblastoma. O-GlcNAcylation (O-GlcNAc) of proteins is an emerging regulator of brain function that maintains normal development and neuronal circuitry. Here, we demonstrate that O-GlcNAc transferase (OGT) in GNPs mediate the cerebellum development, and the progression of the Shh subgroup of medulloblastoma. Specifically, OGT regulates the neurogenesis of GNPs by activating the Shh signaling pathway via O-GlcNAcylation at S355 of GLI family zinc finger 2 (Gli2), which in turn promotes its deacetylation and transcriptional activity via dissociation from p300, a histone acetyltransferases. Inhibition of OGT via genetic ablation or chemical inhibition improves survival in a medulloblastoma mouse model. These data uncover a critical role for O-GlcNAc signaling in cerebellar development, and pinpoint a potential therapeutic target for Shh-associated medulloblastoma.

Chromatin remodelers often show broad expression patterns in multiple cell types yet can elicit cell-specific effects in development and diseases. Arid1a binds DNA and regulates gene expression during tissue development and homeostasis. However, it is unclear how Arid1a achieves its functional specificity in regulating progenitor cells. Using the tooth root as a model, we show that loss of Arid1a impairs the differentiation-associated cell cycle arrest of tooth root progenitors through Hedgehog (Hh) signaling regulation, leading to shortened roots. Our data suggest that Plagl1, as a co-factor, endows Arid1a with its cell-type/spatial functional specificity. Furthermore, we show that loss of Arid1a leads to increased expression of Arid1b, which is also indispensable for odontoblast differentiation but is not involved in regulation of Hh signaling. This study expands our knowledge of the intricate interactions among chromatin remodelers, transcription factors, and signaling molecules during progenitor cell fate determination and lineage commitment.