Over-expression of DMRT1 induces the male pathway in embryonic chicken gonads.

DMRT1 encodes a conserved transcription factor with an essential role in gonadal function. In the chicken, DMRT1 in located on the Z sex chromosome and is currently the best candidate master regulator of avian gonadal sex differentiation. We previously showed that knockdown of DMRT1 expression during the period of sexual differentiation induces feminisation of male embryonic chicken gonads. This gene is therefore necessary for proper testis development in the chicken. However, whether it is sufficient to induce testicular differentiation has remained unresolved. We show here that over-expression of DMRT1 induces male pathway genes and antagonises the female pathway in embryonic chicken gonads. Ectopic DMRT1 expression in female gonads induces localised SOX9 and AMH expression. It also induces expression of the recently identified Z-linked male factor, Hemogen (HEMGN). Masculinised gonads show evidence of cord-like structures and retarded female-type cortical development. Furthermore, expression of the critical feminising enzyme, aromatase, is reduced in the presence of over-expressed DMRT1. These data indicate that DMRT1 is an essential sex-linked regulator of gonadal differentiation in avians, and that it likely acts via a dosage mechanism established through the lack of global Z dosage compensation in birds.

Identification, expression, and regulation of anti-Müllerian hormone type-II receptor in the embryonic chicken gonad.

Anti-Müllerian hormone (AMH) signaling is required for proper development of the urogenital system in vertebrates. In male mammals, AMH is responsible for regressing the Müllerian ducts, which otherwise develop into the fallopian tubes, oviducts, and upper vagina of the female reproductive tract. This role is highly conserved across higher vertebrates. However, AMH is required for testis development in fish species that lack Müllerian ducts, implying that AMH signaling has broader roles in other vertebrates. AMH signals through two serine/threonine kinase receptors. The primary AMH receptor, AMH receptor type-II (AMHR2), recruits the type I receptor, which transduces the signal intracellularly. To enhance our understanding of AMH signaling and the potential role of AMH in gonadal sex differentiation, we cloned chicken AMHR2 cDNA and examined its expression profile during gonadal sex differentiation. AMHR2 is expressed in the gonads and Müllerian ducts of both sexes but is more strongly expressed in males after the onset of gonadal sex differentiation. In the testes, the AMHR2 protein colocalizes with AMH, within Sertoli cells of the testis cords. AMHR2 protein expression is up-regulated in female embryos treated with the estrogen synthesis inhibitor fadrozole. Conversely, knockdown of the key testis gene DMRT1 leads to disruption of AMHR2 expression in the developing seminiferous cords of males. These results indicate that AMHR2 is developmentally regulated during testicular differentiation in the chicken embryo. AMH signaling may be important for gonadal differentiation in addition to Müllerian duct regression in birds.

A new ex vivo model of the bone tissue response to the hyperglycemic environment - The embryonic chicken femur organotypic culture in high glucose conditions.

Diabetes mellitus (DM) embrace a group of chronic metabolic conditions with a high morbidity, causing deleterious effects in different tissues and organs, including bone. Hyperglycemia seems to be one of the most contributing etiological factors of bone-related alterations, altering metabolic functionality and inducing morphological adaptations. Despite the established models for the assessment of bone functionality in hyperglycemic conditions, in vitro studies present a limited representativeness given the imperfect cell-cell and cell-matrix interactions, and restricted three-dimensional spatial arrangement; while in vivo studies raise ethical issues and offer limited mechanistic characterization, given the modulatory influence of many systemic factors and/or regulatory systems. Accordingly, the aim of this study is to establish and characterize an innovative ex vivo model of the bone tissue response to hyperglycemia, reaching hand of the organotypic culture of embryonic chicken femurs in high glucose conditions, showcasing the integrative responsiveness of the model regarding hyperglycemia-induced alterations. A thorough assessment of the cellular and tissue functionality was further conducted. Results show that, in high glucose conditions, femurs presented an increased cell proliferation and enhanced collagen production, despite the altered protein synthesis, substantiated by the increased carbonyl content. Gene expression analysis evidenced that high glucose levels induced the expression of pro-inflammatory and early osteogenic markers, further impairing the expression of late osteogenic markers. Furthermore, the tissue morphological organization and matrix mineralization were significantly altered by high glucose levels, as evidenced by histological, histochemical and microtomographic evaluations. Attained data is coherent with acknowledged hyperglycemia-induced bone tissue alterations, validating the models' effectiveness, and evidencing its integrative responsiveness regarding cell proliferation, gene and protein expression, and tissue morpho-functional organization. The assessed ex vivo model conjoins the capability to access both cellular and tissue outcomes in the absence of a systemic modulatory influence, outreaching the functionality of current experimental in vitro and in vivo models of the diabetic bone condition.

The relationship between liver-kidney impairment and viral load after nephropathogenic infectious bronchitis virus infection in embryonic chickens.

To examine the relationship of impairments of the liver and kidney with viral load after nephropathogenic infectious bronchitis virus (NIBV) infection in embryonic chickens, 120 specific-pathogen-free Leghorn embryonated chicken eggs were randomly divided into two groups (infected and control), with three replicates per group and 20 eggs in each replicate. The eggs in the infected and control groups were challenged with 0.2 mL of 105.5 ELD50 NIBV and sterile saline solution, respectively. The embryonic chickens' plasma and liver and kidney tissues were collected at 1, 3, and 5 days post-inoculation (dpi), the liver and kidney functional parameters were quantified, and the tissue viral loads were determined with real-time PCR. The results showed that plasma potassium, sodium, chlorine, magnesium, calcium, and phosphorus levels were increased. The infected group exhibited significantly higher plasma uric acid, blood urea nitrogen, and creatinine levels than the control group at 3 dpi. The plasma concentrations of aspartate aminotransferase and alanine aminotransferase were significantly increased in the infected group. The total protein, albumin, and globulin levels in the infected group were significantly lower than those in the control group. The liver-kidney viral load in the infected group peaked at 3 dpi, at which time the kidney viral load was significantly higher than that of the liver. Our results indicated that NIBV infection caused liver and kidney damage in the embryonic chickens, and the results also demonstrated that the liver and kidney damage was strongly related to the tissue viral load following NIBV infection in embryonic chickens.

Genetic Manipulation of the Avian Urogenital System Using In Ovo Electroporation.

One of the advantages of the avian embryo as an experimental model is its in ovo development and hence accessibility for genetic manipulation. Electroporation has been used extensively in the past to study gene function in chicken and quail embryos . Readily accessible tissues such as the neural tube, somites, and limb bud, in particular, have been targeted. However, more inaccessible tissues, such as the embryonic urogenital system , have proven more challenging to study. Here, we describe the use of in ovo electroporation of TOL2 vectors or RCASBP avian viral vectors for the rapid functional analysis of genes involved in avian sex determination and urogenital development . In the context of the developing urogenital system , these vectors have inherent advantages and disadvantages, which will be considered here. Either vector can both be used for mis-expressing a gene and for targeting endogenous gene knockdown via expression of short hairpin RNAs (shRNAs). Both of these vectors integrate into the genome and are hence spread throughout developing tissues. Going forward, electroporation could be combined with CRISPR/Cas9 technology for targeted genome editing in the avian urogenital system .

Identifying and targeting determinants of melanoma cellular invasion.

Epithelial-to-mesenchymal transition is a critical process that increases the malignant potential of melanoma by facilitating invasion and dissemination of tumor cells. This study identified genes involved in the regulation of cellular invasion and evaluated whether they can be targeted to inhibit melanoma invasion. We identified Peroxidasin (PXDN), Netrin 4 (NTN4) and GLIS Family Zinc Finger 3 (GLIS3) genes consistently elevated in invasive mesenchymal-like melanoma cells. These genes and proteins were highly expressed in metastatic melanoma tumors, and gene silencing led to reduced melanoma invasion in vitro. Furthermore, migration of PXDN, NTN4 or GLIS3 siRNA transfected melanoma cells was inhibited following transplantation into the embryonic chicken neural tube compared to control siRNA transfected melanoma cells. Our study suggests that PXDN, NTN4 and GLIS3 play a functional role in promoting melanoma cellular invasion, and therapeutic approaches directed toward inhibiting the action of these proteins may reduce the incidence or progression of metastasis in melanoma patients.

Hair cell regeneration or the expression of related factors that regulate the fate specification of supporting cells in the cochlear ducts of embryonic and posthatch chickens.

Hair cells in posthatch chickens regenerate spontaneously through mitosis or the transdifferentiation of supporting cells in response to antibiotic injury. However, how embryonic chicken cochleae respond to antibiotic treatment remains unknown. This study is the first to indicate that unlike hair cells in posthatch chickens, the auditory epithelium was free from antibiotic injury (25-250 mg gentamicin/kg) in embryonic chickens, although FITC-conjugated gentamicin actually reached embryonic hair cells. Next, we examined and counted the cells and performed labeling for BrdU, Sox2, Atoh1/Math1, PV or p27(kip1) (triple or double labeling) in the injured cochlea ducts after gentamicin treatment at 2 h (h), 15 h, 24 h, 2 days (d), 3 d and 7 d after BrdU treatment in posthatch chickens. Our results indicated that following gentamicin administration, proliferating cells (BrdU+) were labeled for Atoh1/Math1 in the damaged areas 3d after gentamicin administration, whereas hair cells (PV+) renewed through mitosis (BrdU+) or direct transdifferentiation (BrdU-) were evident only after 5 d of gentamicin administration. In addition, Sox2 expression was up-regulated in triggered supporting cells at an early stage of regeneration, but stopped at the advent of mature hair cells. Our study also indicated that p27(kip1) was expressed in both hair cells and supporting cells but was down-regulated in a subgroup of the supporting cells that gave rise to hair cells. These data and the obtained dynamic changes of the cells labeled for BrdU, Sox2, Atoh1/Math1, PV or p27(kip1) are useful for understanding supporting cell behaviors and their fate specification during hair cell regeneration.

Developmental expression of Kv1 voltage-gated potassium channels in the avian hypothalamus.

Specialized hypothalamic neurons integrate the homeostatic balance between food intake and energy expenditure, processes that may become dysregulated during the development of diabetes, obesity, and other metabolic disorders. Shaker family voltage-gated potassium channels (Kv1) contribute to the maintenance of resting membrane potential, action potential characteristics, and neurotransmitter release in many populations of neurons, although hypothalamic Kv1 channel expression has been largely unexplored. Whole-cell patch clamp recordings from avian hypothalamic brain slices demonstrate a developmental shift in the electrophysiological properties of avian arcuate nucleus neurons, identifying an increase in outward ionic current that corresponds with action potential maturation. Additionally, RT-PCR experiments identified the early expression of Kv1.2, Kv1.3, and Kv1.5 mRNA in the embryonic avian hypothalamus, suggesting that these channels may underlie the electrophysiological changes observed in these neurons. Real-time quantitative PCR analysis on intact microdissections of embryonic hypothalamic tissue revealed a concomitant increase in Kv1.2 and Kv1.5 gene expression at key electrophysiological time points during development. This study is the first to demonstrate hypothalamic mRNA expression of Kv1 channels in developing avian embryos and may suggest a role for voltage-gated ion channel regulation in the physiological patterning of embryonic hypothalamic circuits governing energy homeostasis.

Embryonic Chicken Transplantation is a Promising Model for Studying the Invasive Behavior of Melanoma Cells.

Epithelial-to-mesenchymal transition is a hallmark event in the metastatic cascade conferring invasive ability to tumor cells. There are ongoing efforts to replicate the physiological events occurring during mobilization of tumor cells in model systems. However, few systems are able to capture these complex in vivo events. The embryonic chicken transplantation model has emerged as a useful system to assess melanoma cells including functions that are relevant to the metastatic process, namely invasion and plasticity. The chicken embryo represents an accessible and economical 3-dimensional in vivo model for investigating melanoma cell invasion as it exploits the ancestral relationship between melanoma and its precursor neural crest cells. We describe a methodology that enables the interrogation of melanoma cell motility within the developing avian embryo. This model involves the injection of melanoma cells into the neural tube of chicken embryos. Melanoma cells are labeled using fluorescent tracker dye, Vybrant DiO, then cultured as hanging drops for 24 h to aggregate the cells. Groups of approximately 700 cells are placed into the neural tube of chicken embryos prior to the onset of neural crest migration at the hindbrain level (embryonic day 1.5) or trunk level (embryonic day 2.5). Chick embryos are reincubated and analyzed after 48 h for the location of melanoma cells using fluorescent microscopy on whole mounts and cross-sections of the embryos. Using this system, we compared the in vivo invasive behavior of epithelial-like and mesenchymal-like melanoma cells. We report that the developing embryonic microenvironment confers motile abilities to both types of melanoma cells. Hence, the embryonic chicken transplantation model has the potential to become a valuable tool for in vivo melanoma invasion studies. Importantly, it may provide novel insights into and reveal previously unknown mediators of the metastatic steps of invasion and dissemination in melanoma.

Cultivation of chicken proventricular epithelial cells and their potential for differentiation.

Epithelial cells of chicken proventriculus (glandular stomach) differentiate into two types; luminal and glandular epithelial cells. The molecules regulating the differentiation of proventricular epithelial cells are not well understood. As the first step in screening the molecular determinants involved in the cell differentiation process, we tried to establish an in vitro culture system for isolated proventricular epithelial cells. Various basal media, growth factors and sera were tested. The medium that supports well the proliferation of epithelial cells was composed of Ham's F12 as the basal medium with epidermal growth factor (10 µg/mL), insulin (10 µg/mL), cholera toxin (1 µg/mL) and bovine pituitary extract (100 µg/mL). Fetal calf serum stimulated cell proliferation 1.7-fold, while horse serum was rather toxic. Proventricular epithelial cells proliferated for 3 days, but began to die out within 1 week of culture. Cultured epithelial cells never expressed embryonic chicken pepsinogen (ECPg), a marker gene of glandular epithelial cells, or maintained ECPg expression. The capacity for ECPg expression in cultured epithelial cells was analyzed by recombination with the proventricular mesenchyme and ECPg was detected in epithelial cells cultured up to 3 days. We concluded therefore, that epithelial cells keep the capacity for ECPg expression for 3 days of cultivation and proventricular mesenchymal cells are required for the actual expression of the ECPg gene.