Development (Cambridge, England)
Marczenke, M;Sunaga-Franze, DY;Popp, O;Althaus, IW;Sauer, S;Mertins, P;Christ, A;Allen, BL;Willnow, TE;
PMID: 34698766 | DOI: 10.1242/dev.200080
Growth arrest-specific 1 (GAS1) acts as a co-receptor to Patched 1 promoting sonic hedgehog (SHH) signaling in the developing nervous system. GAS1 mutations in humans and animal models result in forebrain and craniofacial malformations, defects ascribed to a function for GAS1 in SHH signaling during early neurulation. Here, we confirm loss of SHH activity in the forebrain neuroepithelium in GAS1-deficient mice and in iPSC-derived cell models of human neuroepithelial differentiation. However, our studies document that this defect can be attributed, at least in part, to a novel role for GAS1 in facilitating Notch signaling, essential to sustain a persistent SHH activity domain in the forebrain neuroepithelium. GAS1 directly binds NOTCH1, enhancing ligand-induced processing of the NOTCH1 intracellular domain, which drives Notch pathway activity in the developing forebrain. Our findings identify a unique role for GAS1 in integrating Notch and SHH signal reception in neuroepithelial cells, and they suggest that loss of GAS1-dependent NOTCH1 activation contributes to forebrain malformations in individuals carrying GAS1 mutations.
Investigative ophthalmology & visual science
Ramberg, I;Vieira, FG;Toft, PB;von Buchwald, C;Funding, M;Nielsen, FC;Heegaard, S;
PMID: 34779821 | DOI: 10.1167/iovs.62.14.11
The genomic alterations contributing to the pathogenesis of conjunctival squamous cell carcinomas (SCCs) and their precursor lesions are poorly understood and hamper our ability to develop molecular therapies to reduce the recurrence rates and treatment-related morbidities of this disease. We aimed to characterize the somatic DNA alterations in human papillomavirus (HPV)-positive and HPV-negative conjunctival SCC.Patients diagnosed with conjunctival SCC in situ or SCC treated in ocular oncology referral centers in Denmark were included. HPV detection (HPV DNA PCR, p16 immunohistochemistry, and mRNA in situ hybridization) and targeted capture-based next-generation sequencing of 523 genes frequently involved in cancer were performed to describe the mutational profile based on HPV status.Tumor tissue was available in 33 cases (n = 8 conjunctival SCCs in situ, n = 25 conjunctival SCCs), constituting 25 male and 8 female patients. Nine cases were HPV positive. The HPV-positive SCCs in situ and SCCs were characterized by transcriptionally active high-risk HPV (types 16 and 39) within the tumor cells, frequent mutations in PIK3CA (n = 5/9), and wild-type TP53, CDKN2A, and RB1, while the HPV-negative counterparts harbored frequent mutations in TP53 (n = 21/24), CDKN2A (n = 7/24), and RB1 (n = 6/24).Our findings have delineated two potentially distinct distributions of somatic mutations in conjunctival SCC based on HPV status-pointing to different biological mechanisms of carcinogenesis. The present findings support a causal role of HPV in a subset of conjunctival SCC.
Virchows Archiv (2015): 1-9.
Hauck F, Oliveira-Silva M, Dreyer JH, Ferreira Perrusi VJ, Arcuri RA, Hassan R, Bonvicino CR, Barros MHM, Niedobitek G.
PMID: 25820374 | DOI: 10.1007/s00428-015-1761-4
Rising prevalence rates of high-risk human papillomaviruses (hrHPV) infection in oropharyngeal carcinoma (up to 80 %) have been reported in North America and Scandinavia. We have analysed 424 German and 163 Brazilian head and neck squamous cell carcinomas (HNSCC) from the oral cavity (OSCC), oropharynx (OPSCC) and hypopharynx (HPSCC) using p16 immunohistochemistry, HPV DNA PCR and sequencing, hrHPV DNA in situ hybridisation (ISH) and hrHPV E6/E7 RNA ISH. In the German series, 52/424 cases (12.3 %) were p16-positive/hrHPV-positive (OSCC 3.8 % [10/265], OPSCC 34.4 % [42/122], HPSCC 0 % [0/37]). In addition, there were 9 cases that were p16-positive/hrHPV-negative (5 OPSCC and 4 OSCC). In the Brazilian series, the overall hrHPV DNA prevalence by PCR was 11.0 % ([18/163]; OSCC 6 % [5/83], OPSCC 15.5 % [11/71], HPSCC 22.2 % [2/9]). Ten of these cases were hrHPV-positive/p16-positive. The remaining 8 hrHPV-positive/p16-negative cases were also negative in both ISH assays. Furthermore, 5 p16-positive/hrHPV-negative cases (2 OPSCC and 3 OSCC) were identified. In both series, HPV16 was by far the most common HPV type detected. We confirm that regardless of geographical origin, the highest hrHPV prevalence in HNSCC is observed in oropharyngeal carcinomas. The proportion of HPV-associated OPSCC was substantially higher in the German cohort than in the Brazilian series (34.4 vs. 15.5 %), and in both groups, the prevalence of hrHPV in OPSCC was much lower than in recent reports from North America and Scandinavia. We suggest, therefore, that it may be possible to define areas with high (e.g. USA, Canada, Scandinavia), intermediate (e.g. Germany) and low (e.g. Brazil) prevalences of HPV infection in OPSCC.
The Journal of clinical investigation
Yadav, VK;Berger, JM;Singh, P;Nagarajan, P;Karsenty, G;
PMID: 34905510 | DOI: 10.1172/JCI153752
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.
Teng CS, Ting MC, Farmer DT, Brockop M, Maxson RE, Crump JG.
PMID: 30375332 | DOI: 10.7554/eLife.37024
Cranial sutures separate the skull bones and house stem cells for bone growth and repair. In Saethre-Chotzen syndrome, mutations in TCF12 or TWIST1 ablate a specific suture, the coronal. This suture forms at a neural-crest/mesoderm interface in mammals and a mesoderm/mesoderm interface in zebrafish. Despite this difference, we show that combinatorial loss of TCF12 and TWIST1 homologs in zebrafish also results in specific loss of the coronal suture. Sequential bone staining reveals an initial, directional acceleration of bone production in the mutant skull, with subsequent localized stalling of bone growth prefiguring coronal suture loss. Mouse genetics further reveal requirements for Twist1 and Tcf12 in both the frontal and parietal bones for suture patency, and to maintain putative progenitors in the coronal region. These findings reveal conservation of coronal suture formation despite evolutionary shifts in embryonic origins, and suggest that the coronal suture might be especially susceptible to imbalances in progenitor maintenance and osteoblast differentiation.
Li J, Yuan Y, He J, Feng J, Han X, Jing J, Ho TV, Xu J, Chai Y.
PMID: 29981310 | DOI: 10.1016/j.ydbio.2018.07.003
Cleft palate is one of the most common craniofacial congenital defects in humans. It is associated with multiple genetic and environmental risk factors, including mutations in the genes encoding signaling molecules in the sonic hedgehog (Shh) pathway, which are risk factors for cleft palate in both humans and mice. However, the function of Shh signaling in the palatal epithelium during palatal fusion remains largely unknown. Although components of the Shh pathway are localized in the palatal epithelium, specific inhibition of Shh signaling in palatal epithelium does not affect palatogenesis. We therefore utilized a hedgehog (Hh) signaling gain-of-function mouse model, K14-Cre;R26SmoM2, to uncover the role of Shh signaling in the palatal epithelium during palatal fusion. In this study, we discovered that constitutive activation of Hh signaling in the palatal epithelium results in submucous cleft palate and persistence of the medial edge epithelium (MEE). Further investigation revealed that precise downregulation of Shh signaling is required at a specific time point in the MEE during palatal fusion. Upregulation of Hh signaling in the palatal epithelium maintains the proliferation of MEE cells. This may be due to a dysfunctional p63/Irf6 regulatory loop. The resistance of MEE cells to apoptosis is likely conferred by enhancement of a cell adhesion network through the maintenance of p63 expression. Collectively, our data illustrate that persistent Hh signaling in the palatal epithelium contributes to the etiology and pathogenesis of submucous cleft palate through its interaction with a p63/Irf6-dependent biological regulatory loop and through a p63-induced cell adhesion network.
Takizawa N, Tanaka S, Oe S, Koike T, Yoshida T, Hirahara Y, Matsuda T, Yamada H.
PMID: 30266964 | DOI: 10.1038/s41598-018-32870-9
Bilateral adrenalectomy forces the patient to undergo glucocorticoid replacement therapy and bear a lifetime risk of adrenal crisis. Adrenal autotransplantation is considered useful to avoid adrenal crisis and glucocorticoid replacement therapy. However, the basic process of regeneration in adrenal autografts is poorly understood. Here, we investigated the essential regeneration factors in rat adrenocortical autografts, with a focus on the factors involved in adrenal development and steroidogenesis, such as Hh signalling. A remarkable renewal in cell proliferation and increase in Cyp11b1, which encodes 11-beta-hydroxylase, occurred in adrenocortical autografts from 2-3 weeks after autotransplantation. Serum corticosterone and adrenocorticotropic hormone levels were almost recovered to sham level at 4 weeks after autotransplantation. The adrenocortical autografts showed increased Dhh expression at 3 weeks after autotransplantation, but not Shh, which is the only Hh family member to have been reported to be expressed in the adrenal gland. Increased Gli1 expression was also found in the regenerated capsule at 3 weeks after autotransplantation. Dhh and Gli1 might function in concert to regenerate adrenocortical autografts. This is the first report to clearly show Dhh expression and its elevation in the adrenal gland.
Razumilava N, Shiota J, Mohamad Zaki NH, Ocadiz-Ruiz R, Cieslak CM, Zakharia K, Allen BL, Gores GJ, Samuelson LC, Merchant JL.
| DOI: 10.1002/hep4.1295
Hedgehog (HH) signaling participates in hepatobiliary repair after injury and is activated in patients with cholangiopathies. Cholangiopathies are associated with bile duct (BD) hyperplasia, including expansion of peribiliary glands, the niche for biliary progenitor cells. The inflammation‐associated cytokine interleukin (IL)‐33 is also up‐regulated in cholangiopathies, including cholangiocarcinoma. We hypothesized that HH signaling synergizes with IL‐33 in acute inflammation‐induced BD hyperplasia. We measured extrahepatic BD (EHBD) thickness and cell proliferation with and without an IL‐33 challenge in wild‐type mice, mice overexpressing Sonic HH (pCMV‐Shh), and mice with loss of the HH pathway effector glioma‐associated oncogene 1 (Gli1lacZ/lacZ). LacZ reporter mice were used to map the expression of HH effector genes in mouse EHBDs. An EHBD organoid (BDO) system was developed to study biliary progenitor cells in vitro. EHBDs from the HH overexpressing pCMV‐Shh mice showed increased epithelial cell proliferation and hyperplasia when challenged with IL‐33. In Gli1lacZ/lacZ mice, we observed a decreased proliferative response to IL‐33 and decreased expression of Il6. The HH ligands Shh and Indian HH (Ihh) were expressed in epithelial cells, whereas the transcriptional effectors Gli1, Gli2, and Gli3 and the HH receptor Patched1 (Ptch1) were expressed in stromal cells, as assessed by in situ hybridization and lacZ reporter mice. Although BDO cells lacked canonical HH signaling, they expressed the IL‐33 receptor suppression of tumorigenicity 2. Accordingly, IL‐33 treatment directly induced BDO cell proliferation in a nuclear factor κB‐dependent manner. Conclusion: HH ligand overexpression enhances EHBD epithelial cell proliferation induced by IL‐33. This proproliferative synergism of HH and IL‐33 involves crosstalk between HH ligand‐producing epithelial cells and HH‐responding stromal cells.
Manshouri, T;Veletic, I;Li, P;Yin, CC;Post, SM;Verstovsek, S;Estrov, Z;
PMID: 35595725 | DOI: 10.1038/s41419-022-04932-4
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.
Nguyen V, Sabeur K, Maltepe E, Ameri K, Bayraktar O, Rowitch DH.
PMID: 29134361 | DOI: 10.1007/s12311-017-0895-0
The cerebellum undergoes rapid growth during the third trimester and is vulnerable to injury and deficient growth in infants born prematurely. Factors associated with preterm cerebellar hypoplasia include chronic lung disease and postnatal glucocorticoid administration. We modeled chronic hypoxemia and glucocorticoid administration in neonatal mice to study whole cerebellar and cell type-specific effects of dual exposure. Chronic neonatal hypoxia resulted in permanent cerebellar hypoplasia. This was compounded by administration of prednisolone as shown by greater volume loss and Purkinje cell death. In the setting of hypoxia and prednisolone, administration of a small molecule Smoothened-Hedgehog agonist (SAG) preserved cerebellar volume and protected against Purkinje cell death. Such protective effects were observed even when SAG was given as a one-time dose after dual insult. To model complex injury and determine cell type-specific roles for the hypoxia inducible factor (HIF) pathway, we performed conditional knockout of von Hippel Lindau (VHL) to hyperactivate HIF1α in cerebellar granule neuron precursors (CGNP) or Purkinje cells. Surprisingly, HIF activation in either cell type resulted in no cerebellar deficit. However, in mice administered prednisolone, HIF overactivation in CGNPs resulted in significant cerebellar hypoplasia, whereas HIF overactivation in Purkinje cells caused cell death. Together, these findings indicate that HIF primes both cell types for injury via glucocorticoids, and that hypoxia/HIF + postnatal glucocorticoid administration act on distinct cellular pathways to cause cerebellar injury. They further suggest that SAG is neuroprotective in the setting of complex neonatal cerebellar injury.
Developmental and sexual dimorphic atlas of the prenatal mouse external genitalia at the single-cell level
Proceedings of the National Academy of Sciences of the United States of America
Amato, CM;Yao, HH;
PMID: 34155146 | DOI: 10.1073/pnas.2103856118
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.
SLITRK5 is a negative regulator of hedgehog signaling in osteoblasts
Sun, J;Shin, DY;Eiseman, M;Yallowitz, AR;Li, N;Lalani, S;Li, Z;Cung, M;Bok, S;Debnath, S;Marquez, SJ;White, TE;Khan, AG;Lorenz, IC;Shim, JH;Lee, FS;Xu, R;Greenblatt, MB;
PMID: 34326333 | DOI: 10.1038/s41467-021-24819-w
Hedgehog signaling is essential for bone formation, including functioning as a means for the growth plate to drive skeletal mineralization. However, the mechanisms regulating hedgehog signaling specifically in bone-forming osteoblasts are largely unknown. Here, we identified SLIT and NTRK-like protein-5(Slitrk5), a transmembrane protein with few identified functions, as a negative regulator of hedgehog signaling in osteoblasts. Slitrk5 is selectively expressed in osteoblasts and loss of Slitrk5 enhanced osteoblast differentiation in vitro and in vivo. Loss of SLITRK5 in vitro leads to increased hedgehog signaling and overexpression of SLITRK5 in osteoblasts inhibits the induction of targets downstream of hedgehog signaling. Mechanistically, SLITRK5 binds to hedgehog ligands via its extracellular domain and interacts with PTCH1 via its intracellular domain. SLITRK5 is present in the primary cilium, and loss of SLITRK5 enhances SMO ciliary enrichment upon SHH stimulation. Thus, SLITRK5 is a negative regulator of hedgehog signaling in osteoblasts that may be attractive as a therapeutic target to enhance bone formation.
