Hindbrain boundaries as niches of neural progenitor and stem cells regulated by the extracellular matrix proteoglycan chondroitin sulphate.

The interplay between neural progenitors and stem cells (NPSCs), and their extracellular matrix (ECM) is a crucial regulatory mechanism that determines their behavior. Nonetheless, how the ECM dictates the state of NPSCs remains elusive. The hindbrain is valuable to examine this relationship, as cells in the ventricular surface of hindbrain boundaries (HBs), which arise between any two neighboring rhombomeres, express the NPSC marker Sox2, while being surrounded with the membrane-bound ECM molecule chondroitin sulphate proteoglycan (CSPG), in chick and mouse embryos. CSPG expression was used to isolate HB Sox2+ cells for RNA-sequencing, revealing their distinguished molecular properties as typical NPSCs, which express known and newly identified genes relating to stem cells, cancer, the matrisome and cell cycle. In contrast, the CSPG- non-HB cells, displayed clear neural-differentiation transcriptome. To address whether CSPG is significant for hindbrain development, its expression was manipulated in vivo and in vitro. CSPG manipulations shifted the stem versus differentiation state of HB cells, evident by their behavior and altered gene expression. These results provide further understanding of the uniqueness of hindbrain boundaries as repetitive pools of NPSCs in-between the rapidly growing rhombomeres, which rely on their microenvironment to maintain their undifferentiated state during development.

Differential Effects of Halofuginone Enantiomers on Muscle Fibrosis and Histopathology in Duchenne Muscular Dystrophy.

Progressive loss of muscle and muscle function is associated with significant fibrosis in Duchenne muscular dystrophy (DMD) patients. Halofuginone, an analog of febrifugine, prevents fibrosis in various animal models, including those of muscular dystrophies. Effects of (+)/(-)-halofuginone enantiomers on motor coordination and diaphragm histopathology in mdx mice, the mouse model for DMD, were examined. Four-week-old male mice were treated with racemic halofuginone, or its separate enantiomers, for 10 weeks. Controls were treated with saline. Racemic halofuginone-treated mice demonstrated better motor coordination and balance than controls. However, (+)-halofuginone surpassed the racemic form's effect. No effect was observed for (-)-halofuginone, which behaved like the control. A significant reduction in collagen content and degenerative areas, and an increase in utrophin levels were observed in diaphragms of mice treated with racemic halofuginone. Again, (+)-halofuginone was more effective than the racemic form, whereas (-)-halofuginone had no effect. Both racemic and (+)-halofuginone increased diaphragm myofiber diameters, with no effect for (-)-halofuginone. No effects were observed for any of the compounds tested in an in-vitro cell viability assay. These results, demonstrating a differential effect of the halofuginone enantiomers and superiority of (+)-halofuginone, are of great importance for future use of (+)-halofuginone as a DMD antifibrotic therapy.

Elevated Expression of Moesin in Muscular Dystrophies.

Fibrosis is the main complication of muscular dystrophies. We identified moesin, a member of the ezrin-radixin-moesin family, in dystrophic muscles of mice representing Duchenne and congenital muscular dystrophies (DMD and CMD, respectively) and dysferlinopathy, but not in the wild type. High levels of moesin were also observed in muscle biopsy specimens from DMD, Ullrich CMD, and merosin-deficient CMD patients, all of which present high levels of fibrosis. The myofibroblasts, responsible for extracellular matrix protein synthesis, and the macrophages infiltrating the dystrophic muscles were the source of moesin. Moesin-positive cells were embedded within the fibrotic areas between the myofibers adjacent to the collagen type I fibers. Radixin was also synthesized by the myofibroblasts, whereas ezrin colocalized with the myofiber membranes. In animal models and patients' muscles, part of the moesin was in its active phosphorylated form. Inhibition of fibrosis by halofuginone, an antifibrotic agent, resulted in a major decrease in moesin levels in the muscles of DMD and CMD mice. In summary, the results of this study may pave the way for exploiting moesin as a novel target for intervention in MDs, and as part of a battery of biomarkers to evaluate treatment success in preclinical studies and clinical trials.

The role of iNOS in cholesterol-induced liver fibrosis.

Accumulation of cholesterol in the liver is associated with the development of non-alcoholic steatohepatitis-related fibrosis. However, underlying mechanisms are not well understood. The present study investigated the role of inducible nitric oxide synthase (iNOS) in cholesterol-induced liver fibrosis by feeding wild-type (WT) and iNOS-deficient mice with control or high-cholesterol diet (HCD) for 6 weeks. WT mice fed with HCD developed greater liver fibrosis, compared with iNOS-deficient mice, as evident by Sirius red staining and higher expression levels of profibrotic genes. Enhanced liver fibrosis in the presence of iNOS was associated with hypoxia-inducible factor-1α stabilization, matrix metalloproteinase-9 expression, and enhanced hepatic DNA damage. The profibrotic role of iNOS was also demonstrated in vivo using a selective inhibitor of iNOS as well as in vitro in a rat liver stellate cell line (HSC-T6). In conclusion, these findings suggest that iNOS is an important mediator in HCD-induced liver fibrosis.

The involvement of collagen triple helix repeat containing 1 in muscular dystrophies.

Fibrosis is the main complication of muscular dystrophies. We identified collagen triple helix repeat containing 1 (Cthrc1) in skeletal and cardiac muscles of mice, representing Duchenne and congenital muscle dystrophies (DMD and CMD, respectively), and dysferlinopathy. In all of the mice, Cthrc1 was associated with high collagen type I levels; no Cthrc1 or collagen was observed in muscles of control mice. High levels of Cthrc1 were also observed in biopsy specimens from patients with DMD, in whom they were reversibly correlated with that of ß-dystroglycan, whereas collagen type I levels were elevated in all patients with DMD. At the muscle sites where collagen and Cthrc1 were adjacent, collagen fibers appeared smaller, suggesting involvement of Cthrc1 in collagen turnover. Halofuginone, an inhibitor of Smad3 phosphorylation downstream of the transforming growth factor-ß signaling, reduced Cthrc1 levels in skeletal and cardiac muscles of mice, representing DMD, CMD, and dysferlinopathy. The myofibroblasts infiltrating the dystrophic muscles of the murine models of DMD, CMD, and dysferlinopathy were the source of Cthrc1. Transforming growth factor-ß did not affect Cthrc1 levels in the mdx fibroblasts but decreased them in the control fibroblasts, in association with increased migration of mdx fibroblasts and dystrophic muscle invasion by myofibroblasts. To our knowledge, this is the first demonstration of Cthrc1 as a marker of the severity of the disease progression in the dystrophic muscles, and as a possible target for therapy.

HREM, RNAseq and Cell Cycle Analyses Reveal the Role of the G2/M-Regulatory Protein, WEE1, on the Survivability of Chicken Embryos during Diapause.

Avian blastoderm can enter into diapause when kept at low temperatures and successfully resume development (SRD) when re-incubated in body temperature. These abilities, which are largely affected by the temperature and duration of the diapause, are poorly understood at the cellular and molecular level. To determine how temperature affects embryonic morphology during diapause, high-resolution episcopic microscopy (HREM) analysis was utilized. While blastoderms diapausing at 12 °C for 28 days presented typical cytoarchitecture, similar to non-diapaused embryos, at 18 °C, much thicker blastoderms with higher cell number were observed. RNAseq was conducted to discover the genes underlying these phenotypes, revealing differentially expressed cell cycle regulatory genes. Among them, WEE1, a negative regulator of G2/M transition, was highly expressed at 12 °C compared to 18 °C. This finding suggested that cells at 12 °C are arrested at the G2/M phase, as supported by bromodeoxyuridine incorporation (BrdU) assay and phospho-histone H3 (pH 3) immunostaining. Inhibition of WEE1 during diapause at 12 °C resulted in cell cycle progression beyond the G2/M and augmented tissue volume, resembling the morphology of 18 °C-diapaused embryos. These findings suggest that diapause at low temperatures leads to WEE1 upregulation, which arrests the cell cycle at the G2/M phase, promoting the perseverance of embryonic cytoarchitecture and future SRD. In contrast, WEE1 is not upregulated during diapause at higher temperature, leading to continuous proliferation and maladaptive morphology associated with poor survivability. Combining HREM-based analysis with RNAseq and molecular manipulations, we present a novel mechanism that regulates the ability of diapaused avian embryos to maintain their cytoarchitecture via cell cycle arrest, which enables their SRD.

Storage temperature dictates the ability of chicken embryos to successfully resume development by regulating expression of blastulation and gastrulation genes.

The avian embryo has a remarkable ability that allows it to suspend its development during blastulation for a long time at low temperatures, and to resume normal development when incubated. This ability is used by poultry hatcheries to store eggs prior to incubation. We have previously found that this ability correlates with the temperature during storage; embryos recover much better following prolonged storage at 12°C rather than at 18°C. However, the molecular and cellular mechanisms underlying these differences are poorly understood. To successfully resume development following storage, the embryo has to shift from the blastulation phase to gastrulation. Several genes are known to partake in the blastulation-to-gastrulation transition under normal conditions, such as the pluripotency-related genes Inhibitor of DNA Binding 2 (ID2) and NANOG that are expressed during blastulation, and the gastrulation-regulating genes NODAL and Brachyury (TBXT). However, their expression and activity following storage is unknown. To elucidate the molecular mechanisms that initiate the ability to successfully transit from blastulation to gastrulation following storage, embryos were stored for 28 days at 12°C or 18°C, and were assessed either prior to incubation, 12, or 18 h of incubation at 37.8°C. Immediately following storage at 18°C group showed remarkable impaired morphology compared to the blastoderm of the 12°C group and of non-stored control embryos. Concurrently with these, expression of ID2 and NANOG was maintained following storage at 12°C similar to the control group, but was significantly reduced upon storage at 18°C. Nevertheless, when the 18°C-stored embryos were incubated, the morphology and the reduced genes were reverted to resemble those of the 12°C group. At variance, key gastrulation genes, NODAL and its downstream effector Brachyury (TBXT), which were similarly expressed in the control and the 12°C group, were not restored in the 18°C embryos following incubation. Notably, ectopic administration of Activin rescued NODAL and TBXT expression in the 18°C group, indicating that these embryos maintain the potential to initiate. Collectively, this study suggests a temperature-dependent mechanisms that direct the transition from blastulation to gastrulation. These mechanisms promote a successful developmental resumption following prolonged storage at low temperatures.

Hsp90 and angiogenesis in bone disorders--lessons from the avian growth plate.

Thiram-induced tibial dyschondroplasia (TD) and vitamin-D deficiency rickets are avian bone disorders of different etiologies characterized by abnormal chondrocyte differentiation, enlarged and unvascularized growth plates, and lameness. Heat-shock protein 90 (Hsp90) is a proangiogenic factor in mammalian tissues and in tumors; therefore, Hsp90 inhibitors were developed as antiangiogenic factors. In this study, we evaluated the association between Hsp90, hypoxia, and angiogenesis in the chick growth plate. Administration of the Hsp90 inhibitor to TD- and rickets-afflicted chicks at the time of induction resulted in reduction in growth-plate size and, contrary to its antiangiogenic effect in tumors, a major invasion of blood vessels occurred in the growth plates. This was the result of upregulation of the VEGF receptor Flk-1, the major rate-limiting factor of vascularization in TD and rickets. In addition, the abnormal chondrocyte differentiation, as characterized by collagen type II expression and alkaline phosphatase activity, and the changes in hypoxia-inducible factor-1α (HIF-1α) in both disorders were restored. All these changes resulted in prevention of lameness. Inhibition of Hsp90 activity reduced growth-plate size, increased vascularization, and mitigated lameness also in TD chicks with established lesions. In summary, this is the first reported demonstration of involvement of Hsp90 in chondrocyte differentiation and growth-plate vascularization. In contrast to the antiangiogenic effect of Hsp90 inhibitors observed in mammals, inhibition of Hsp90 activity in the unvascularized TD- and rickets-afflicted chicks resulted in activation of the angiogenic switch and reinstated normal growth-plate morphology.

Fibrosis inhibition and muscle histopathology improvement in laminin-alpha2-deficient mice.

In muscular dystrophies (MD) the loss of muscle and its ability to function are associated with fibrosis. We evaluated the efficacy of halofuginone in reducing fibrosis in the dy(2J)/dy(2J) mouse model of congenital MD. Mice were injected intraperitoneally with 5 microg of halofuginone 3 times a week for 5 or 15 weeks, starting at the age of 3 weeks. Halofuginone caused a reduction in collagen synthesis in hindlimb muscles. This was associated with reductions in the degenerated area, in cell proliferation, in the number of myofibers with central nuclei, with increased myofiber diameter, and with enhanced motor coordination and balance. Halofuginone caused a reduction in infiltrating fibroblasts that were located close to centrally nucleated myofibers. Our results suggest that halofuginone reduced the deleterious effects of fibrosis, thus improving muscle integrity. Halofuginone meets the criteria for a novel antifibrotic therapy for MD patients.

Early pathological signs in young dysf-/- mice are improved by halofuginone.

Dysferlinopathies are a non-lethal group of late-onset muscular dystrophies. Here, we evaluated the fusion ability of primary myoblasts from young dysf-/- mice and the muscle histopathology prior to, and during early stages of disease onset. The ability of primary myoblasts of 5-week-old dysf-/- mice to form large myotubes was delayed compared to their wild-type counterparts, as evaluated by scanning electron microscopy. However, their fusion activity, as reflected by the presence of actin filaments connecting several cells, was enhanced by the antifibrotic drug halofuginone. Early dystrophic signs were already apparent in 4-week-old dysf-/- mice; their collagen level was double that in wild-type mice and continued to rise until 5 months of age. Continuous treatment with halofuginone from 4 weeks to 5 months of age reduced muscle fibrosis in a phosphorylated-Smad3 inhibition-related manner. Halofuginone also enhanced myofiber hypertrophy, reduced the percentage of centrally nucleated myofibers, and increased muscle performance. Together, the data suggest an inhibitory effect of halofuginone on the muscle histopathology at very early stages of dysferlinopathy, and enhancement of muscle performance. These results offer new opportunities for early pharmaceutical treatment in dysferlinopathies with favorable outcomes at later stages of life.

High-resolution episcopic microscopy enables three-dimensional visualization of plant morphology and development.

The study of plant anatomy, which can be traced back to the seventeenth century, advanced hand in hand with light microscopy technology and relies on traditional histologic techniques, which are based on serial two-dimensional (2D) sections. However, these valuable techniques lack spatial arrangement of the tissue and hence provide only partial information. A new technique of whole-mount three-dimensional (3D) imaging termed high-resolution episcopic microscopy (HREM) can overcome this obstacle and generate a 3D model of the specimen at a near-histological resolution. Here, we describe the application of HREM technique in plants by analyzing two plant developmental processes in woody plants: oil secretory cavity development in citrus fruit and adventitious root formation in persimmon rootstock cuttings. HREM 3D models of citrus fruit peel showed that oil cavities were initiated schizogenously during the early stages of fruitlet development. Citrus secretory cavity formation, shape, volume, and distribution were analyzed, and new insights are presented. HREM 3D model comparison of persimmon rootstock clones, which differ in their rooting ability, revealed that difficult-to-root clones failed to develop adventitious roots due to their inability to initiate root primordia.

Hydroxy group requirement for halofuginone-dependent inhibition of muscle fibrosis and improvement of histopathology in the mdx mouse model for Duchenne muscular dystrophy.

In Duchenne muscular dystrophy (DMD), the progressive loss of muscle and its ability to function is associated with significant fibrosis, representing the major disease complication in patients. Halofuginone, a halogenated analog of the naturally occurring febrifugine, has been shown to prevent fibrosis in various animal models, including those of muscular dystrophies. Here, two optically active enantiomers of deoxyhalofuginone - a halofuginone analogue in which the hydroxy group in position 3 was removed from the piperidinyl entity - were evaluated with respect to their effect on muscle histopathology in mdx mice. Male mdx mice were treated with either deoxyhalofuginone (as single enantiomers or in racemic form), or halofuginone, for 10 weeks, starting at the age of 4 weeks. Halofuginone caused a significant reduction in total collagen content, degenerative areas, as well as in utrophin and phosphorylated-Smad3 levels in the mdx diaphragms. However, neither the deoxyhalofuginone enantiomers, nor its racemic form had any effect on these parameters. A positive effect of the deoxyhalofuginone (+)-enantiomer was observed on myofiber diameters; however, it was lesser than that of halofuginone. It is concluded that the hydroxy group plays a key role in halofuginone's effects related to fibrosis in DMD, and points towards the transforming growth factor ß/Smad3 signaling pathway being involved in this inhibition. Elucidation of the structure-function relationship of halofuginone, in relation to inhibiting fibrosis in muscular dystrophies, is of the utmost importance for creating the next generation of anti-fibrotic therapies that will be more efficacious and less toxic, hence improving life quality of patients.

A proof of concept study demonstrating that environmental levels of carbamazepine impair early stages of chick embryonic development.

Carbamazepine (CBZ) is an anticonvulsant drug used for epilepsy and other disorders. Prescription of CBZ during pregnancy increases the risk for congenital malformations. CBZ is ubiquitous in effluents and persistent during wastewater treatment. Thus, it is re-introduced into agricultural ecosystems upon irrigation with reclaimed wastewater. People consuming produce irrigated with reclaimed wastewater were found to be exposed to CBZ. However, environmental concentrations of CBZ (µgL-1) are magnitudes lower than its therapeutic levels (µgml-1), raising the question of whether and how environmental levels of CBZ affect embryonic development. The chick embryo is a powerful and highly sensitive amniotic model system that enables to assess environmental contaminants in the living organism. Since the chick embryonic development is highly similar to mammalians, yet, it develops in an egg, toxic effects can be directly analyzed in a well-controlled system without maternal influences. This research utilized the chick embryo to test whether CBZ is embryo-toxic by using morphological, cellular, molecular and imaging strategies. Three key embryonic stages were monitored: after blastulation (st.1HH), gastrulation/neurulation (st.8HH) and organogenesis (st.15HH). Here we demonstrate that environmental relevant concentrations of CBZ impair morphogenesis in a dose- and stage- dependent manner. Effects on gastrulation, neural tube closure, differentiation and proliferation were exhibited in early stages by exposing embryos to CBZ dose as low as 0.1µgL-1. Quantification of developmental progression revealed a significant difference in the total score obtained by CBZ-treated embryos compared to controls (up to 5-fold difference, p<0.05). Yet, defects were unnoticed as embryos passed gastrulation/neurulation. This study provides the first evidence for teratogenic effect of environmental-relevant concentrations of CBZ in amniotic embryos that impair early but not late stages of development. These findings call for in-depth risk analysis to ensure that the environmental presence of CBZ and other drugs is not causing irreversible ecological and public-health damages.

Prevention of muscle fibrosis and improvement in muscle performance in the mdx mouse by halofuginone.

Fibrosis is a known feature of dystrophic muscles, particularly the diaphragm, in the mdx mouse. In this study we evaluated the effect of halofuginone, a collagen synthesis inhibitor, on collagen synthesis in various muscles of young wild-type (C57/BL/6J) and mdx mice. Halofuginone prevented the age-dependent increase in collagen synthesis in the diaphragms of mdx with no effect on wild-type mice (n = 5 for each time point). This was associated with a decrease in the degenerated areas and number of central nuclei. Halofuginone also inhibited collagen synthesis in cardiac muscle. Moreover, enhanced motor coordination, balance and improved cardiac muscle function were observed implying reduced muscle injury. Halofuginone inhibited Smad3 phosphorylation downstream of TGFbeta in the diaphragm and cardiac muscles, in C2 cell line and in primary mouse myoblast cultures representing various muscular dystrophies. We suggest that via its effect on Smad3 phosphorylation, halofuginone inhibits muscle fibrosis and improves cardiac and skeletal muscle functions in mdx mice.

Inhibition of fibroblast to myofibroblast transition by halofuginone contributes to the chemotherapy-mediated antitumoral effect.

Stromal myofibroblasts play an important role in tumor progression. The transition of fibroblasts to myofibroblasts is characterized by expression of smooth muscle genes and profuse synthesis of extracellular matrix proteins. We evaluated the efficacy of targeting fibroblast-to-myofibroblast transition with halofuginone on tumor progression in prostate cancer and Wilms' tumor xenografts. In both xenografts, low doses of halofuginone treatment, independent of the route of administration, resulted in a trend toward inhibition in tumor development. Moreover, halofuginone synergizes with low dose of docetaxel in prostate cancer and vincristine and dactinomycin in Wilms' tumor xenografts, resulting in significant reduction in tumor volume and weight comparable to the effect observed by high doses of the respective chemotherapies. In prostate cancer and Wilms' tumor xenografts, halofuginone, but not the respective chemotherapies, inhibited the synthesis of collagen type I, alpha-smooth muscle actin, transgelin, and cytoglobin, all of which are characteristics of activated myofibroblasts. Halofuginone, as the respective chemotherapies, increased the synthesis of Wilms' tumor suppressor gene product (WT-1) and prostate apoptosis response gene-4 (Par-4), resulting in apoptosis/necrosis. These results suggest that targeting the fibroblast-to-myofibroblast transition with halofuginone may synergize with low doses of chemotherapy in achieving a significant antitumoral effect, avoiding the need of high-dose chemotherapy and its toxicity without impairing treatment efficacy.

The Tomato BLADE ON PETIOLE and TERMINATING FLOWER Regulate Leaf Axil Patterning Along the Proximal-Distal Axes.

Leaf axil patterning occurs concomitantly with leaf development and takes place at the boundary zone which demarcates the initiating leaf primordium from the shoot apical meristem. Subsequent growth and differentiation result in establishment of the axillary meristem and abscission zone (AZ) along the proximal-distal axis of the leaf axil, yet the molecular mechanisms that regulate these events are poorly understood. We studied the role of the tomato BLADE ON PETIOLE (SlBOP) boundary gene family on the development of the leaf axil using BOP-silenced plants as well as BOP-mutated lines. We show that silencing of the tomato SlBOP gene family affects patterning of the leaf axil along the proximal-distal axis, manifested by dispositioning of the AM and abnormal development of the adjacent tissue resulting in lack of a functional leaf AZ. Dissection of the role of each of the three tomato SlBOPs by analysis of single, double and triple null-mutants demonstrated that SlBOP2 is the dominant gene in leaf axil patterning, but does not rule out involvement of SlBOP1 and SlBOP3 in correct AM positioning. We further studied the potential role of TERMINATING FLOWER (TMF), a transcription factor which was previously shown to interact with SlBOPs, in leaf axil patterning using TMF mutant tomato lines. The results suggest that similar to SlBOP2, TMF is involved in leaf axil proximal-distal patterning and AZ development.

Identification of Trophectoderm-Derived Cripto as an Essential Mediator of Embryo Implantation.

Cripto-1 (TDGF1) is a multifunctional signaling factor that stimulates cellular effects, including proliferation, migration, survival, epithelial-to-mesenchymal transition, and angiogenesis, to regulate embryogenesis, tissue homeostasis, and tumorigenesis. Those cell behaviors are also associated with implantation of the embryo into the uterine wall, and this led us to investigate the role of embryo-derived Cripto in embryo attachment and implantation. In this study, we show that Cripto and its signaling mediator GRP78 are uniquely localized to embryo implantation sites. We knocked down Cripto expression specifically in trophoblast cells and found that this resulted in a corresponding decrease in the levels of its downstream signaling mediators, phosphorylated (phospho-)SMAD2, phospho-SRC, phospho-extracellular signal-regulated kinase, and phospho-AKT, which are also known mediators of embryo implantation. We then transplanted Cripto knockdown and control embryos into uteri of pseudopregnant female mice and found that embryos with Cripto-depleted trophoblast cells had dramatically impaired capacity to attach to the uterine wall when compared with controls. This loss of appropriate embryo attachment following Cripto knockdown in trophoblast cells was associated with abnormally enlarged implantation sites that were almost completely devoid of microvessels. A role for Cripto in embryo implantation was further supported by our demonstration that attachment of trophoblast-derived spheroids to endometrial cells in vitro was stimulated by Cripto treatment and diminished by treatment with either of two mechanistically distinct Cripto blocking agents. Collectively, our findings identify Cripto as a novel and critical embryo attachment factor and suggest that modulation of Cripto signaling may have significant therapeutic potential for the treatment of infertility and other related disorders.

Roles of the cilium-associated gene CCDC11 in left-right patterning and in laterality disorders in humans.

Axial determination occurs during early stages of embryogenesis. Flaws in laterality patterning result in abnormal positioning of visceral organs, as manifested in heterotaxy syndrome, or complete left-right inversion as in situs inversus totalis. These malformations are often associated with ciliopathies, as seen in primary ciliary dyskinesia. We have recently described a novel mutation in the Coiled-Coil Domain-Containing 11 (CCDC11) gene associated with laterality disorders in a consanguineous family of Arab-Muslim origin with two affected siblings presenting with diverse phenotypes, one with heterotaxy syndrome and the other with non-primary ciliary dyskinesia situs inversus totalis. This study further characterizes the roles of CCDC11 and the implications of the identified mutation on left-right axial patterning in patient-derived cells and in the frog embryo as a model organism. We analyzed patient-derived cells and manipulated Ccdc11 levels in Xenopus laevis frog embryos. Cilia length in patient cells was longer than in controls, and CCDC11 was localized to the centriole and the actin cytoskeleton. Mutated truncated protein accumulated and was also localized to the centriole and actin cytoskeleton. In frog embryos, Ccdc11 was regulated downstream of FoxJ1, and overexpression of the full-length or truncated protein, or downregulation of the gene resulted in severe disruption of embryonic left-right axial patterning. Taken together, our initial description of the deleterious mutation in CCDC11 in patients, the current results and more recent supportive studies highlight the important role of CCDC11 in axial patterning.

Localized LoxL3-Dependent Fibronectin Oxidation Regulates Myofiber Stretch and Integrin-Mediated Adhesion.

For muscles to function, myofibers have to stretch and anchor at the myotendinous junction (MTJ), a region rich in extracellular matrix (ECM). Integrin signaling is required for MTJ formation, and mutations affecting the cascade lead to muscular dystrophies in mice and humans. Underlying mechanisms for integrin activation at the MTJ and ECM modifications regulating its signaling are unclear. We show that lysyl oxidase-like 3 (LoxL3) is a key regulator of integrin signaling that ensures localized control of the cascade. In LoxL3 mutants, myofibers anchor prematurely or overshoot to adjacent somites, and are loose and lack tension. We find that LoxL3 complexes with and directly oxidizes Fibronectin (FN), an ECM scaffold protein and integrin ligand enriched at the MTJ. We identify a mechanism whereby localized LoxL3 secretion from myofiber termini oxidizes FN, enabling enhanced integrin activation at the tips of myofibers and ensuring correct positioning and anchoring of myofibers along the MTJ.

Identification of the Long-Sought Leptin in Chicken and Duck: Expression Pattern of the Highly GC-Rich Avian leptin Fits an Autocrine/Paracrine Rather Than Endocrine Function.

More than 20 years after characterization of the key regulator of mammalian energy balance, leptin, we identified the leptin (LEP) genes of chicken (Gallus gallus) and duck (Anas platyrhynchos). The extreme guanine-cytosine content (∼70%), the location in a genomic region with low-complexity repetitive and palindromic sequence elements, the relatively low sequence conservation, and low level of expression have hampered the identification of these genes until now. In vitro-expressed chicken and duck leptins specifically activated signaling through the chicken leptin receptor in cell culture. In situ hybridization demonstrated expression of LEP mRNA in granular and Purkinje cells of the cerebellum, anterior pituitary, and in embryonic limb buds, somites, and branchial arches, suggesting roles in adult brain control of energy balance and during embryonic development. The expression patterns of LEP and the leptin receptor (LEPR) were explored in chicken, duck, and quail (Coturnix japonica) using RNA-sequencing experiments available in the Short Read Archive and by quantitative RT-PCR. In adipose tissue, LEP and LEPR were scarcely transcribed, and the expression level was not correlated to adiposity. Our identification of the leptin genes in chicken and duck genomes resolves a long lasting controversy regarding the existence of leptin genes in these species. This identification was confirmed by sequence and structural similarity, conserved exon-intron boundaries, detection in numerous genomic, and transcriptomic datasets and characterization by PCR, quantitative RT-PCR, in situ hybridization, and bioassays. Our results point to an autocrine/paracrine mode of action for bird leptin instead of being a circulating hormone as in mammals.