The Curious Case of Avian Sex Determination.

Ioannidis and colleagues show that the gene DMRT1 is the master regulator of testis development in the chicken. Yet, remarkably, when this gene is deleted in genetic males and gonads form ovaries, the body remains male. This debunks the notion that somatic sex is driven primarily by hormones in birds.

HoxB genes regulate neuronal delamination in the trunk neural tube by controlling the expression of Lzts1.

Differential Hox gene expression is central for specification of axial neuronal diversity in the spinal cord. Here, we uncover an additional function of Hox proteins in the developing spinal cord, restricted to B cluster Hox genes. We found that members of the HoxB cluster are expressed in the trunk neural tube of chicken embryo earlier than Hox from the other clusters, with poor antero-posterior axial specificity and with overlapping expression in the intermediate zone (IZ). Gain-of-function experiments of HoxB4, HoxB8 and HoxB9, respectively, representative of anterior, central and posterior HoxB genes, resulted in ectopic progenitor cells in the mantle zone. The search for HoxB8 downstream targets in the early neural tube identified the leucine zipper tumor suppressor 1 gene (Lzts1), the expression of which is also activated by HoxB4 and HoxB9. Gain- and loss-of-function experiments showed that Lzts1, which is expressed endogenously in the IZ, controls neuronal delamination. These data collectively indicate that HoxB genes have a generic function in the developing spinal cord, controlling the expression of Lzts1 and neuronal delamination.

The homeobox gene TGIF1 is required for chicken ovarian cortical development and generation of the juxtacortical medulla.

During early embryogenesis in amniotic vertebrates, the gonads differentiate into either ovaries or testes. The first cell lineage to differentiate gives rise to the supporting cells: Sertoli cells in males and pre-granulosa cells in females. These key cell types direct the differentiation of the other cell types in the gonad, including steroidogenic cells. The gonadal surface epithelium and the interstitial cell populations are less well studied, and little is known about their sexual differentiation programs. Here, we show the requirement of the homeobox transcription factor gene TGIF1 for ovarian development in the chicken embryo. TGIF1 is expressed in the two principal ovarian somatic cell populations: the cortex and the pre-granulosa cells of the medulla. TGIF1 expression is associated with an ovarian phenotype in estrogen-mediated sex reversal experiments. Targeted misexpression and gene knockdown indicate that TGIF1 is required, but not sufficient, for proper ovarian cortex formation. In addition, TGIF1 is identified as the first known regulator of juxtacortical medulla development. These findings provide new insights into chicken ovarian differentiation and development, specifically cortical and juxtacortical medulla formation.

Optogenetic techniques for understanding the gut peristalsis during chicken embryonic development.

Gut peristaltic movements transport ingested materials along the gut axis, which is critical for food digestion and nutrient absorption. While a large amount of studies have been devoted to analyzing the physiological functions of peristalsis in adults, little is known about how the peristaltic system is established during embryogenesis. In recent years, the chicken developing gut has emerged as an excellent model, in which specific sites along the gut axis can be genetically labeled enabling live imaging and optogenetic analyses. This review provides an overview of recent progress in optogenetic studies of gut peristalsis. Analyses with an improved channelrhodopsin-2 variant demonstrated that the peristalsis can artificially be generated in the developing gut. These studies unveiled novel functional coordination between different regions along the gut axis. In addition, imaging with GCaMP6s, a genetically encoded calcium indicator, enabled a fine mapping of developmental changes in the peristaltic patterns as Ca2+ signals. These advanced techniques will broaden our knowledge of how embryonic peristalsis is established at the cellular and molecular level, leading to the understanding of physiological and pathological processes in adult peristalsis.

Inflammatory responses and barrier disruption in the trachea of chicks following Mycoplasma gallisepticum infection: a focus on the TNF-α-NF-κB/MLCK pathway.

Mycoplasma gallisepticum (MG) can induce persistent inflammatory damage to the tracheal mucosa of poultry and cause chronic respiratory diseases in chickens. To further investigate the mechanism of MG-induced injury to the tracheal mucosa, we used chick embryo tracheal organ culture (TOC) as a model to study the invasion and reproduction of MG, the effect of MG on tracheal morphology, and the potential factors that promote MG tissue invasion. The results showed that MG infection significantly damaged the tracheal epithelial structure and weakened tracheal epithelial barrier function; MG also increased the occurrence of bacterial displacement, with a significant (p < 0.05) increase in the bacterial load of the infected TOCs at 5 and 7 days post-infection. In addition, MG significantly (p < 0.05) increased the expression levels of inflammatory cytokines, such as TNF-α, interleukin-1ß (IL-1ß), and IL-6, and activated the NF-κB signalling pathway, leading to increased nuclear translocation of NF-κB p65. Simultaneously, the map kinase pathway (MAPK) was activated. This activation might be associated with increased myosin light chain (MLC) phosphorylation, which could lead to actin-myosin contraction and disruption of tight junction (TJ) protein function, potentially compromising epithelial barrier integrity and further catalysing MG migration into tissues. Overall, our results contribute to a better understanding of the interaction between MG and the host, provide insight into the mechanisms of damage to the tracheal mucosa induced by MG infection, and provide new insights into the possible pathways involved in Mycoplasma gallisepticum infection in vivo.

Comamonas testosteroni as the cause of mortality in embryonated chicken eggs of breeding broiler hens in Costa Rica.

Mortality of chicken embryos and first-week chickens was reported in a commercial incubator company in Costa Rica. Six 1-day-old Cobb chickens and twenty-four embryonated chicken eggs were examined in the Laboratory of Avian Pathology and the Laboratory of Bacteriology of the National University of Costa Rica. Twelve dead-in-shell embryos showed maceration and were immersed in a putrid, turbid, slightly thick brown liquid. Additionally, the other 12 embryonated eggs had milky yellow-orange content. The livers of those embryos had congestion, haemorrhages and multifocal cream foci of necrosis. Granulocytic infiltration was observed in the bursa of Fabricius, myocardium, liver, lung and kidney. Livers and egg yolks from six embryonated chickens and all 1-day-old chickens were aseptically collected and cultured. In addition, tissues from six better conserved embryos and all 1-day-old chickens were fixed in buffered formalin and embedded in paraffin. Biochemical and molecular tests identified Comamonas testosteroni as the cause of the early, middle and late embryo mortality. As all the eggshells from the sampled embryonated eggs were dirty with soiled a fecal matter, contamination after manipulating the eggs was considered the source of infection. C. testosteroni is an environmental microorganism that has rarely been reported to cause human disease. To our knowledge, this is the first report of C. testosteroni causing mortality in a hatchery. Cleaning and disinfection using ozone were implemented in the hatchery to eliminate the embryo mortality associated with C. testosteroni.

Primary sex determination in birds depends on DMRT1 dosage, but gonadal sex does not determine adult secondary sex characteristics.

In birds, males are the homogametic sex (ZZ) and females the heterogametic sex (ZW). Primary sex determination is thought to depend on a sex chromosome gene dosage mechanism, and the most likely sex determinant is the Z chromosome gene Doublesex and Mab-3-Related Transcription factor 1 (DMRT1). To clarify this issue, we used a CRISPR-Cas9-based monoallelic targeting approach and sterile surrogate hosts to generate birds with targeted mutations in the DMRT1 gene. The resulting chromosomally male (ZZ) chicken with a single functional copy of DMRT1 developed ovaries in place of testes, demonstrating the avian sex-determining mechanism is based on DMRT1 dosage. These ZZ ovaries expressed typical female markers and showed clear evidence of follicular development. However, these ZZ adult birds with an ovary in place of testes were indistinguishable in appearance to wild-type adult males, supporting the concept of cell-autonomous sex identity (CASI) in birds. In experiments where estrogen synthesis was blocked in control ZW embryos, the resulting gonads developed as testes. In contrast, if estrogen synthesis was blocked in ZW embryos that lacked DMRT1, the gonads invariably adopted an ovarian fate. Our analysis shows that DMRT1 is the key sex determination switch in birds and that it is essential for testis development, but that production of estrogen is also a key factor in primary sex determination in chickens, and that this production is linked to DMRT1 expression.

Developmental 6:2 FTCA exposure impairs renal development in chicken embryos via IGF signaling.

6:2 fluorotelomer carboxylic acid (6:2 FTCA) is a perfluorooctanoic acid (PFOA) substitute, which is supposedly less accumulative and toxic than PFOA. However, 6:2 FTCA is structurally similar to PFOA, and there had already been reports about its toxicities comparable to PFOA. The aim of the current study is to assess potential effects of developmental exposure to 6:2 FTCA on the development of kidney in chicken embryo and to investigate underlying mechanism. Fertile chicken eggs were exposed to 1.25 mg/kg, 2.5 mg/kg or 5 mg/kg doses of 6:2 FTCA, or 2 mg/kg PFOA, then incubated to hatch. Serum and kidney of hatchling chickens were collected. Blood urea nitrogen (BUN) and creatinine (Cre) levels were measured with commercially available kits. Morphology of kidney was assessed with histopathology. To further reveal molecular mechanism of observed endpoints, IGF signaling molecules were assessed in the kidney samples with qRT-PCR, results indicated that IGFBP3 is a potentially crucial molecule. Lentiviruses overexpressing or silencing IGFBP3 were designed and applied to enhance/suppress the expression of IGFBP3 in developing chicken embryo for further verification of its role in the observed effects. Disrupted nephron formation, in the manifestation of decreased glomeruli number/area and increased serum BUN/Cre levels, was observed in the animals developmentally exposed to 6:2 FTCA. Correspondingly, IGF signaling molecules (IGF1, IGF1R and IGFBP3) were affected by 6:2 FTCA exposure. Meanwhile, overexpression of IGFBP3 effectively alleviated such changes, while silencing of IGFBP3 mimicked observed effects. In conclusion, developmental exposure to 6:2 FTCA is associated with disrupted chicken embryo renal development, in which IGFBP3 seems to be a remarkable contributor, suggesting potential health risks for human and other species. Further risk assessments and mechanistic works are necessary.

Retinoic Acid-Mediated Control of Energy Metabolism Is Essential for Lung Branching Morphogenesis.

Lung branching morphogenesis relies on intricate epithelial-mesenchymal interactions and signaling networks. Still, the interplay between signaling and energy metabolism in shaping embryonic lung development remains unexplored. Retinoic acid (RA) signaling influences lung proximal-distal patterning and branching morphogenesis, but its role as a metabolic modulator is unknown. Hence, this study investigates how RA signaling affects the metabolic profile of lung branching. We performed ex vivo lung explant culture of embryonic chicken lungs treated with DMSO, 1 µM RA, or 10 µM BMS493. Extracellular metabolite consumption/production was evaluated by using 1H-NMR spectroscopy. Mitochondrial respiration and biogenesis were also analyzed. Proliferation was assessed using an EdU-based assay. The expression of crucial metabolic/signaling components was examined through Western blot, qPCR, and in situ hybridization. RA signaling stimulation redirects glucose towards pyruvate and succinate production rather than to alanine or lactate. Inhibition of RA signaling reduces lung branching, resulting in a cystic-like phenotype while promoting mitochondrial function. Here, RA signaling emerges as a regulator of tissue proliferation and lactate dehydrogenase expression. Furthermore, RA governs fatty acid metabolism through an AMPK-dependent mechanism. These findings underscore RA's pivotal role in shaping lung metabolism during branching morphogenesis, contributing to our understanding of lung development and cystic-related lung disorders.

Dysregulated VEGF/VEGFR-2 Signaling and Plexogenic Lesions in the Embryonic Lungs of Chickens Predisposed to Pulmonary Arterial Hypertension.

Plexiform lesions are a hallmark of pulmonary arterial hypertension (PAH) in humans and are proposed to stem from dysfunctional angioblasts. Broiler chickens (Gallus gallus) are highly susceptible to PAH, with plexiform-like lesions observed in newly hatched individuals. Here, we reported the emergence of plexiform-like lesions in the embryonic lungs of broiler chickens. Lung samples were collected from broiler chickens at embryonic day 20 (E20), hatch, and one-day-old, with PAH-resistant layer chickens as controls. Plexiform lesions consisting of CD133+/vascular endothelial growth factor receptor type-2 (VEGFR-2)+ angioblasts were exclusively observed in broiler embryos and sporadically in layer embryos. Distinct gene profiles of angiogenic factors were observed between the two strains, with impaired VEGF-A/VEGFR-2 signaling correlating with lesion development and reduced arteriogenesis. Pharmaceutical inhibition of VEGFR-2 resulted in enhanced lesion development in layer embryos. Moreover, broiler embryonic lungs displayed increased activation of HIF-1α and nuclear factor erythroid 2-related factor 2 (Nrf2), indicating a hypoxic state. Remarkably, we found a negative correlation between lung Nrf2 activation and VEGF-A and VEGFR-2 expression. In vitro studies indicated that Nrf2 overactivation restricted VEGF signaling in endothelial progenitor cells. The findings from broiler embryos suggest an association between plexiform lesion development and impaired VEGF system due to aberrant activation of Nrf2.

Effects of Resveratrol on In Vivo Ovarian Cancer Cells Implanted on the Chorioallantoic Membrane (CAM) of a Chicken Embryo Model.

Ovarian cancer poses a significant threat to patients in its advanced stages, often with limited treatment options available. In such cases, palliative management becomes the primary approach to maintaining a reasonable quality of life. Therefore, the administration of any medication that can benefit patients without a curative option holds potential. Resveratrol, a natural compound known for its in vitro anticancer activities, has generated contrasting results in vivo and human studies. In this study, we aimed to assess the anticancer effects of resveratrol on ovarian cancer cells grown on the chorioallantoic membrane (CAM) of chicken embryos. Two ovarian cancer cell lines, OVCAR-8 and SKOV-3, were cultured in collagen scaffolds for four days before being implanted on the CAM of chicken embryos on day 7. Different doses of resveratrol were applied to the CAM every two days for six days. Subsequently, CAM tissues were excised, fixed, and subjected to histological analysis. Some CAM tumours were extracted to analyse proteins through Western blotting. Our findings indicate that specific doses of resveratrol significantly reduce angiogenic activities, pNF-κB levels, and SLUG protein levels by using immunohistochemistry. These results suggest that resveratrol may have the potential to impact the behaviour of ovarian cancer CAM tumours, thereby warranting further consideration as a complementary treatment option for women with incurable ovarian cancer.

Determination of the embryotoxic effects of propofol injected into eggs on the cerebellum and spinal cord using histologic methods: an animal study.

Background/aim: This study aims to determine the possible embryotoxic effects of propofol on the cerebellum and spinal cord using fertile chicken eggs. Materials and methods: A total of 430 fertile eggs were divided into 5 groups: control, saline, 2.5 mg.kg-1, 12.5 mg.kg-1, and 37.5 mg.kg-1 propofol. Injections were made immediately before incubation via the air chamber. On the 15th, 18th, and 21st day of incubation, 6 embryos from each group were evaluated. Serial paraffin sections taken from the cerebellum and spinal cord were stained with hematoxylin-eosin, Kluver-Barrera, toluidine blue, and periodic acid-Schiff's reaction. The outer granular layer and total cortex thickness were measured, and the linear density of the Purkinje cells was determined. The ratios of the substantia grisea surface area to the total surface area of the spinal cord were calculated. The transverse and longitudinal diameters of the canalis centralis were also assessed. Results: No structural malformation was observed in any embryos examined macroscopically. No significant difference was observed between the groups in terms of development and histologic organization of the cerebellum and spinal cord. However, on the 15th, 18th, and 21st day, the outer granular layer (p < 0.001 for all days) and the total cortex thickness (p < 0.01, p < 0.001, and p < 0.001, respectively) decreased significantly in different propofol dose groups in varying degrees in the cerebellum. Similarly, in the spinal cord, there were significant changes in the ratios of the substantia grisea surface area to the total surface area (p < 0.01 and p < 0.001, respectively). Conclusion: It was concluded that the in-ovo-administered propofol given immediately before incubation has adverse effects on the developing cerebellum and spinal cord. Therefore, it is important for anesthesiologists always to remain vigilant when treating female patients of childbearing age.

Oestrogen in the chick embryo can induce chromosomally male ZZ left gonad epithelial cells to form an ovarian cortex that can support oogenesis.

In chickens, the embryonic ovary differentiates into two distinct domains before meiosis: a steroidogenic core (the female medulla), overlain by the germ cell niche (the cortex). The differentiation of the medulla is a cell-autonomous process based on chromosomal sex identity (CASI). In order to address the extent to which cortex differentiation depends on intrinsic or extrinsic factors, we generated models of gonadal intersex by mixing ZW (female) and ZZ (male) cells in gonadal chimeras, or by altering oestrogen levels of ZW and ZZ embryos. We found that CASI does not apply to the embryonic cortex. Both ZW and ZZ cells can form the cortex and this can happen independently of the phenotypic sex of the medulla as long as oestrogen is provided. We also show that the cortex-promoting activity of oestrogen signalling is mediated via estrogen receptor alpha within the left gonad epithelium. However, the presence of a medulla with an 'intersex' or male phenotype may compromise germ cell progression into meiosis, causing cortical germ cells to remain in an immature state in the embryo.

In-ovo echocardiography for application in cardiovascular research.

Preclinical cardiovascular research relies heavily on non-invasive in-vivo echocardiography in mice and rats to assess cardiac function and morphology, since the complex interaction of heart, circulation, and peripheral organs are challenging to mimic ex-vivo. While n-numbers of annually used laboratory animals worldwide approach 200 million, increasing efforts are made by basic scientists aiming to reduce animal numbers in cardiovascular research according to the 3R's principle. The chicken egg is well-established as a physiological correlate and model for angiogenesis research but has barely been used to assess cardiac (patho-) physiology. Here, we tested whether the established in-ovo system of incubated chicken eggs interfaced with commercially available small animal echocardiography would be a suitable alternative test system in experimental cardiology. To this end, we defined a workflow to assess cardiac function in 8-13-day-old chicken embryos using a commercially available high resolution ultrasound system for small animals (Vevo 3100, Fujifilm Visualsonics Inc.) equipped with a high frequency probe (MX700; centre transmit: 50 MHz). We provide detailed standard operating procedures for sample preparation, image acquisition, data analysis, reference values for left and right ventricular function and dimensions, and inter-observer variabilities. Finally, we challenged incubated chicken eggs with two interventions well-known to affect cardiac physiology-metoprolol treatment and hypoxic exposure-to demonstrate the sensitivity of in-ovo echocardiography. In conclusion, in-ovo echocardiography is a feasible alternative tool for basic cardiovascular research, which can easily be implemented into the small animal research environment using existing infrastructure to replace mice and rat experiments, and thus, reduce use of laboratory animals according to the 3R principle.

Hexafluoropropylene oxide tetramer acid (HFPO-TeA)-induced developmental toxicities in chicken embryo: Peroxisome proliferator-activated receptor Alpha (PPARα) is involved.

Hexafluoropropylene oxide tetramer acid (HFPO-TeA) is an emerging environmental contaminant, with environmental presence but limited toxicological information. To investigate its potential developmental toxicities, various doses of HFPO-TeA exposure were achieved in chicken embryos via air cell injection, and the exposed embryos were incubated until hatch. Within 24 h of hatch, the hatchling chickens were assessed with electrocardiography and histopathology for toxicological evaluation. For mechanistic investigation, in ovo silencing of PPARα was achieved via lentivirus microinjection, then the morphological/functional endpoints along with protein expression levels of PPARα-regulated genes were assessed. HFPO-TeA exposure in chicken embryo resulted in developmental cardiotoxicity and hepatotoxicity. Specifically, decreased right ventricular wall thickness, increased heart rate and hepatic steatosis were observed, whereas silencing of PPARα resulted in alleviation of observed toxicities. Western blotting for EHHADH and FABPs suggested that developmental exposure to HFPO-TeA effectively increased the expression levels of both targets in hatchling chicken heart and liver tissue samples, while PPARα silencing prevented such changes, suggesting that PPARα and its downstream genes are playing critical roles in HFPO-TeA induced developmental toxicities.

Developmental toxicity in early chicken embryos on exposure to an organophosphorus flame retardant, tris(2-chloroisopropyl) phosphate.

Tris(2-chloroisopropyl) phosphate (TCIPP) is an organophosphate flame retardant detected in the environment and eggs, feathers, and livers. Early-developmental-stage avian embryos are vulnerable to the toxic effects of chemicals. However, studies on the specific effects of TCIPP on avian embryonic development are limited. We aimed to investigate the toxicity of TCIPP in early chicken embryos using a previously developed shell-less incubation system. Fertilized chicken (Gallus gallus domesticus) eggs (n = 220) were exposed to 50 or 500 nmol TCIPP/(g egg) or dimethyl sulfoxide (DMSO) as a vehicle control on Day 0 of incubation. Development of 198 embryos was monitored from Days 3-9 of incubation, and 22 embryos on Day 4 and 74 embryos on Day 9 were dissected. Messenger RNA expression levels for several genes were measured in embryos on Day 4. Both TCIPP-exposed groups showed a significant reduction in survival rate. Imaging analyses revealed significant decreases in body length, head and bill length, eye diameter, and forelimb and hindlimb length in both TCIPP-treated groups. TCIPP exposure significantly impaired the development of extraembryonic blood vessels and the production of red blood cells. A TCIPP-dose-dependent decreasing trend in heart rate was observed on Days 4-7. The somitic angle increased significantly on Days 4-6, and embryos with curved somites showed cleavage in the back and gaps between somites, resulting in asymmetrical somite formation. A significant correlation was found between the somitic angle and FGF8 expression levels, suggesting that TCIPP exposure affects somite formation through an altered FGF-signaling pathway. Embryos with somitic deformities in TCIPP-exposed groups had significantly reduced survival rates, indicating that abnormal segment formation directly increased mortality. Finally, eye weight and ocular luminosity values were significantly reduced, suggesting that TCIPP may also affect eye development. Overall, these findings highlight severe toxic effects of TCIPP on avian embryonic development, including in vascularization, cardiac function, and somite and ocular development.

Preliminary Study on Expression and Function of the Chicken W Chromosome Gene MIER3 in Embryonic Gonads.

The sex chromosomes of birds are designated Z and W. The male is homogamous (ZZ), and the female is heterogamous (ZW). The chicken W chromosome is a degenerate version of the Z chromosome and harbors only 28 protein-coding genes. We studied the expression pattern of the W chromosome gene MIER3 (showing differential expression during gonadogenesis) in chicken embryonic gonads and its potential role in gonadal development. The W copy of MIER3 (MIER3-W) shows a gonad-biased expression in chicken embryonic tissues which was different from its Z copy. The overall expression of MIER3-W and MIER3-Z mRNA and protein is correlated with the gonadal phenotype being higher in female gonads than in male gonads or female-to-male sex-reversed gonads. Chicken MIER3 protein is highly expressed in the nucleus, with relatively lower expression in the cytoplasm. Overexpression of MIER3-W in male gonad cells suggested its effect on the GnRH signaling pathway, cell proliferation, and cell apoptosis. MIER3 expression is associated with the gonadal phenotype. MIER3 may promote female gonadal development by regulating EGR1 and αGSU genes. These findings enrich our knowledge of chicken W chromosome genes and support a more systematic and in-depth understanding of gonadal development in chickens.

Assessment of the effect of kinetin against formic acid toxicity in chicken embryo model.

The current study was designed to investigate the in ovo injection of formic acid (FA) on hatchability rate (HR; Experiment 1) and the potential ameliorative role of kinetin concurrent with FA on biochemical parameters of hatched broilers (Experiment 2). In Experiment 1, live embryonated eggs (n = 280; Day 4 of incubation) were in ovo injected with 0.03, 0.06, 0.125, 0.25, 0.50, 1, 2, 4, 8, 16 and 32 m m FA. In Experiment 2, intra-yolk-sac administration of toxic doses of FA (2 m m) concurrent with kinetin at 50, 100 or 200 µ m were evaluated on hatched embryos. The amount of malondialdehyde (MDA), total antioxidant capacity (TAC), total nitrate-nitrite (TNN), total lipid hydroperoxide (TLHP) and superoxide dismutase (SOD) activity was measured in serum, liver, heart and brain tissues. The results revealed that injection of 2 mM FA significantly increased mortality compared to the control group (p < 0.05). Concurrent administration of 50 or 100 µ m kinetin + 2 m m FA increased HR to 10% and 20% compared to the FA-alone-treated group, respectively. Intra-yolk-sac-received FA group showed greater amounts of MDA, TLHP and TNN and lesser amounts of TAC and SOD activity in serum and tissue samples of liver, heart and brain compared to control groups (p < 0.001). In comparison to the FA-alone-treated group, all doses of kinetin were able to increase the TAC levels in serum and tissue samples when administered concurrently with FA. The doses of 50 and 100 µ m kinetin were efficacious to ameliorate the toxic role of FA injection on SOD activities (p < 0.001). Co-injection of 100 µ m kinetin plus FA significantly reduced the amounts of MDA, TNN and TLHP in measured samples compared to the FA-alone-injected group (p < 0.001). Our results indicated that kinetin (especially at 100 µ m doses) would ameliorate the toxic effects of FA on developing live chicken embryos.

Quantitative proteomics analysis of chicken embryos reveals key proteins that affect right gonadal degeneration in females.

In birds, embryonic gonads of females develop in a way different from mammals, with the left one develops into a functional ovary, while the right one degenerates during embryogenesis. Here, we examined the proteomics profiles of the female and male left and right gonads at embryonic day 6.5 (E6.5) with the label free tandem mass spectrometry proteomics technique. The relative protein abundance of the left and right gonads of female and male embryos was determined to identify their differential proteins. Overall, a total of 7726 proteins were identified, of which 79 and 54 proteins were significantly different in female and male right gonads compared with female left gonads and male left gonads respectively. Bioinformatics analysis showed that the proteins DMRT1, ZFPM2, TSHZ3 were potentially associated with the degeneration of the right gonads in female embryos. The proteomics in this study provide clues for further elucidation of the pathways of sex determination, sex differentiation, and right gonadal degeneration in birds.

Fine-tuning of the PAX-SIX-EYA-DACH network by multiple microRNAs controls embryo myogenesis.

MicroRNAs (miRNAs), short non-coding RNAs, which act post-transcriptionally to regulate gene expression, are of widespread significance during development and disease, including muscle disease. Advances in sequencing technology and bioinformatics led to the identification of a large number of miRNAs in vertebrates and other species, however, for many of these miRNAs specific roles have not yet been determined. LNA in situ hybridisation has revealed expression patterns of somite-enriched miRNAs, here we focus on characterising the functions of miR-128. We show that antagomiR-mediated knockdown (KD) of miR-128 in developing chick somites has a negative impact on skeletal myogenesis. Computational analysis identified the transcription factor EYA4 as a candidate target consistent with the observation that miR-128 and EYA4 display similar expression profiles. Luciferase assays confirmed that miR-128 interacts with the EYA4 3'UTR. In vivo experiments also suggest that EYA4 is regulated by miR-128. EYA4 is a member of the PAX-SIX-EYA-DACH (PSED) network of transcription factors. Therefore, we identified additional candidate miRNA binding sites in the 3'UTR of SIX1/4, EYA1/2/3 and DACH1. Using the miRanda algorithm, we found sites for miR-128, as well as for other myogenic miRNAs, miR-1a, miR-206 and miR-133a, some of these were experimentally confirmed as functional miRNA target sites. Our results reveal that miR-128 is involved in regulating skeletal myogenesis by directly targeting EYA4 with indirect effects on other PSED members, including SIX4 and PAX3. Hence, the inhibitory effect on myogenesis observed after miR-128 knockdown was rescued by concomitant knockdown of PAX3. Moreover, we show that the PSED network of transcription factors is co-regulated by multiple muscle-enriched microRNAs.