Development of a functional musculoskeletal system requires coordinated generation of muscles, bones, and tendons. However, how axial tendon cells (tenocytes) are generated during embryo development is still poorly understood. Here, we show that axial tenocytes arise from the sclerotome in zebrafish. In contrast to mouse and chick, the zebrafish sclerotome consists of two separate domains: a ventral domain and a previously undescribed dorsal domain. While dispensable for sclerotome induction, Hedgehog (Hh) signaling is required for the migration and maintenance of sclerotome derived cells. Axial tenocytes are located along the myotendinous junction (MTJ), extending long cellular processes into the intersomitic space. Using time-lapse imaging, we show that both sclerotome domains contribute to tenocytes in a dynamic and stereotypic manner. Tenocytes along a given MTJ always arise from the sclerotome of the adjacent anterior somite. Inhibition of Hh signaling results in loss of tenocytes and enhanced sensitivity to muscle detachment. Together, our work shows that axial tenocytes in zebrafish originate from the sclerotome and are essential for maintaining muscle integrity.

Blood vessels are vital to sustain life in all vertebrates. While it is known that mural cells (pericytes and smooth muscle cells) regulate vascular integrity, the contribution of other cell types to vascular stabilization has been largely unexplored. Using zebrafish, we identified sclerotome-derived perivascular fibroblasts as a novel population of blood vessel associated cells. In contrast to pericytes, perivascular fibroblasts emerge early during development, express the extracellular matrix (ECM) genes col1a2 and col5a1, and display distinct morphology and distribution. Time-lapse imaging reveals that perivascular fibroblasts serve as pericyte precursors. Genetic ablation of perivascular fibroblasts markedly reduces collagen deposition around endothelial cells, resulting in dysmorphic blood vessels with variable diameters. Strikingly, col5a1 mutants show spontaneous hemorrhage, and the penetrance of the phenotype is strongly enhanced by the additional loss of col1a2. Together, our work reveals dual roles of perivascular fibroblasts in vascular stabilization where they establish the ECM around nascent vessels and function as pericyte progenitors.

Lindane (gamma-hexachlorocyclohexane) is a lipid-soluble pesticide that exerts carcinogenic and reprotoxic properties. The mechanisms by which lindane alters testicular function are unclear. Sertoli cells control germ cell proliferation and differentiation through cell-cell communication, including gap junction intercellular communication. Using the 42GPA9 Sertoli cell line, we show that lindane, at a non-cytotoxic dose (50 microM), abolished gap junction intercellular communication (GJIC) between adjacent cells. This change was associated with a time-related diminution and redistribution of Cx43 from the membrane to the cytoplasmic perinuclear region. A similar alteration was observed for ZO-1, a tight junction component associated with Cx43, but not for occludin, an integral tight junction protein. After a 24 h lindane exposure, Cx43 and ZO-1 colocalized within the cytoplasm and no modification of non-phosphorylated and phosphorylated isoforms of Cx43 was observed. By double immunofluorescent labelling we demonstrate that the cytoplasmic Cx43 signal was not present in either the endoplasmic reticulum/Golgi apparatus or lysosomes. These results suggest that lindane inhibits GJIC between Sertoli cells and that aberrant Cx43/ZO-1 localization may be responsible for this effect. The alterations in gap junctions induced by lindane in 42GPA9 Sertoli cells are similar to those observed in tumour cells and may be involved in the pathogenesis of neoplastic seminomal proliferation.

Clinical and experimental studies have suggested that estrogens, the archetype of female hormones, participate in the control of male germ cell proliferation and that fetal exposure to environmental estrogens may contribute to hypofertility and/or to testicular germ cell cancer. However, the underlying mechanisms remain to be elucidated. 17beta-Estradiol (E2) conjugated to BSA was able to stimulate human testicular seminoma cell proliferation by triggering a rapid, nongenomic, membrane-mediated activation of ERK1/2 and cAMP-dependent protein kinase A (PKA). Both ERK1/2 and PKA participated in this promoting effect. This activation was associated with phosphorylation of the transcription factor cAMP response element-binding protein and the nuclear factor retinoblastoma protein. Enhanced proliferation together with ERK activation could be reversed by pertussis toxin, a G protein inhibitor. Estrogen receptors (ERs) in JKT-1 were characterized by immunofluorescence, subcellular fractioning, and Western blot. JKT-1 cells did not express ERalpha but ERbeta, which localized to the mitochondria and the nucleus but not to the membrane. Moreover, neither ICI-182,780, a classical ER antagonist, nor tamoxifen, a selective ER modulator, could reverse the 17beta-estradiol-BSA-induced promoting effect. Estrogens contribute to human testicular germ cell cancer proliferation by rapid activation of ERK1/2 and PKA through a membrane nonclassical ER. This nongenomic effect represents a new basis for understanding the estrogenic control of spermatogenesis and evaluating the role of fetal exposure to xenoestrogens during malignant transformation of testicular germ stem cells.

Connexins, the constitutive proteins of gap junctions, are considered to be tumour suppressive agents and are often impaired in the tumourigenic processes. In the present study, the expression of connexin 43 (Cx43), which is involved in the control of spermatogenesis through Sertoli/germ cell coupling, has been investigated in human testicular seminoma cells (tumours and the JKT-1 cell line). Cx43 was immunolocalized in the Golgi apparatus without membrane expression and was detected by immunoblotting in JKT-1 as exclusive 70 kD bands. No mutation could be found by sequencing the transcript obtained by RT-PCR. Transfection with a Cx43-V5 vector reproduced the same gel shift, identifying these 70 kD bands as Cx43. The Cx43-70 kD bands were also expressed in normal testicular tissue, associated with the classical 43 kD isoforms. Stable transfection of JKT-1 with a Cx43-GFP vector allowed restoration of Cx43 membrane expression, functional cell coupling, and inhibition of the cell proliferation rate. Storage of Cx43 in the Golgi apparatus may correspond during spermatogenesis to an intermittent physiological process that becomes permanent in malignant seminoma cells as a result of the tumourigenic process. By preventing Cx43 membrane expression, this disrupted traffic may itself participate in tumour promotion.

It is now well established that estrogens participate in the control of normal spermatogenesis and endogenous or environmental estrogens are involved in pathological germ cell proliferation including testicular germ cell tumors. Studying a human testicular seminoma cell line, JKT-1, we show here that 17 -estradiol (10 −12 to 10 −6 M) induced in vitro a significant dose-dependent decrease of cell growth. This antiproliferative effect was maximum after 4 days of exposure at a physiologically intratesticular concentration of 10 −9 M, close to the K d of ER, and reversed by ICI 182780, an ER antagonist, suggesting an ER-mediated pathway. By RT-PCR and Western blot we were able to confirm that JKT-1, like tumoral seminoma cells and normal human testicular basal germ cells, expresses estrogen receptor (ER ), including ER 1 and ER 2, a dominant negative variant, but not ER . Using immunofluorescence and confocal microscopy, ER was observed as perinuclear intracytoplasmic spots in JKT-1 and tumoral seminoma cells without significant translocation of ER into the nucleus, under 17 -estradiol exposure. Double staining observed by confocal microscopy revealed that ER colocalized in JKT-1 cells with cytochrome C, a mitochondrial marker. We report for the first time the expression of a functional aromatase complex in seminoma cells as assessed by RT-PCR, Western blot and enzymatic assay. Seminoma cells are able to respond to estrogens through a possible autocrine or paracrine loop. These preliminary results support estrogen-dependency of human testicular seminoma, the most frequent tumor of young men, and suggest potential pharmacological use. Whether this estrogen control, however, involves an ER -mediated stimulation of cell apoptosis and/or an ER -mediated inhibition of cell proliferation, remains to be further determined.

Background Occipital nerve stimulation (ONS) has been proposed to treat chronic medically-intractable cluster headache (iCCH) in small series of cases without evaluation of its functional and emotional impacts. Methods We report the multidimensional outcome of a large observational study of iCCH patients, treated by ONS within a nationwide multidisciplinary network ( https://clinicaltrials.gov NCT01842763), with a one-year follow-up. Prospective evaluation was performed before surgery, then three and 12 months after. Results One year after ONS, the attack frequency per week was decreased >30% in 64% and >50% in 59% of the 44 patients. Mean (Standard Deviation) weekly attack frequency decreased from 21.5 (16.3) to 10.7 (13.8) ( p = 0.0002). About 70% of the patients responded to ONS, 47.8% being excellent responders. Prophylactic treatments could be decreased in 40% of patients. Functional (HIT-6 and MIDAS scales) and emotional (HAD scale) impacts were significantly improved, as well as the health-related quality of life (EQ-5D). The mean (SD) EQ-5D visual analogic scale score increased from 35.2 (23.6) to 51.9 (25.7) ( p = 0.0037). Surgical minor complications were observed in 33% of the patients. Conclusion ONS significantly reduced the attack frequency per week, as well as the functional and emotional headache impacts in iCCH patients, and dramatically improved the health-related quality of life of responders.

24Blood vessels are vital to sustain life in all vertebrates. While it is known that mural cells (pericytes 25 and smooth muscle cells) regulate vascular integrity, the contribution of other cell types to vascular 26 stabilization has been largely unexplored. Using zebrafish, we identified sclerotome-derived 27 perivascular fibroblasts as a novel population of blood vessel associated cells. In contrast to pericytes, 28 perivascular fibroblasts emerge early during development, express the extracellular matrix (ECM) 29 genes col1a2 and col5a1, and display distinct morphology and distribution. Time-lapse imaging 30 reveals that perivascular fibroblasts serve as pericyte precursors. Genetic ablation of perivascular 31 fibroblasts results in dysmorphic blood vessels with variable diameters. Strikingly, col5a1 mutants 32show spontaneous hemorrhage, and the penetrance of the phenotype is strongly enhanced by the 33 additional loss of col1a2. Together, our work reveals dual roles of perivascular fibroblasts in vascular 34 stabilization where they establish the ECM around nascent vessels and function as pericyte 35 progenitors. 36 3 AUTHOR SUMMARY 37 38 Blood vessels are essential to sustain life in humans. Defects in blood vessels can lead to serious 39 diseases, such as hemorrhage, tissue ischemia, and stroke. However, how blood vessel stability is 40 maintained by surrounding support cells is still poorly understood. Using the zebrafish model, we 41 identify a new population of blood vessel associated cells termed perivascular fibroblasts, which 42 originate from the sclerotome, an embryonic structure that is previously known to generate the 43 skeleton of the animal. Perivascular fibroblasts are distinct from pericytes, a known population of 44 blood vessel support cells. They become associated with blood vessels much earlier than pericytes 45 and express several collagen genes, encoding main components of the extracellular matrix. Loss of 46 perivascular fibroblasts or mutations in collagen genes result in fragile blood vessels prone to 47 53 54The vascular system is crucial to the survival of vertebrates. Blood vessels must rapidly expand 55 and contract in response to systemic cues, but also maintain the stability to withstand the stress of 56 blood flow. To maintain their integrity, blood vessels are supported by a highly specialized 57 perivascular architecture comprised of blood vessel associated cells and the surrounding extracellular 58 matrix (ECM) (1,2). Compromised vascular integrity can result in devastating human diseases, such 59 as aneurysms, vascular malformations, and hemorrhagic strokes (3-6). However, how blood vessels 60 are stabilized by different perivascular components is still poorly understood. 61The prevailing model is that vascular stability is maintained at three different levels: endothelial 62 cells, mural cells and the surrounding ECM (2). First, blood vessels are lined by endothelial cells. 63Adherens and tight junctions between endothelial cells provide the primary barrier to passage o...