Rapid and efficient enhancer cloning and in vivo screening using the developing chick embryo.
Ontology highlight
ABSTRACT:
SUBMITTER: Williams RM
PROVIDER: S-EPMC8121703 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Synovial joints are among the most important structures that give us complex motor abilities as humans. Degenerative joint diseases, such as arthritis, cause loss of normal joint functioning and affect over 40 million people in the USA and approximately 350 million people worldwide. Therapies based on regenerative medicine hold the promise of effectively repairing or replacing damaged joints permanently. Here, for the first time, we introduce a model for synovial joint regeneration utilizing the chick embryo. In this model, a block of tissue that contains the prospective elbow is excised, leaving a window with strips of anterior and posterior tissue intact (window excision, WE). In contrast, we also slice out the same area containing the elbow and the distal piece of the limb is pinned back onto the stump (slice excision, SE). Interestingly, when the elbow is removed via WE, regeneration of the joint takes place, whereas the elbow joint does not regenerate following SE. In order to investigate whether the regeneration response recapitulates the developmental program of forming joints, we used GDF-5 and Autotaxin (Atx) as joint tissue specific markers, and Sox-9 and Col-9 as cartilage markers for in situ hybridization on sections at different time points after WE and SE surgeries. Re-expression of GDF-5 and Atx is observed in the WE samples by 60h after surgery. In contrast, the majority of the samples that underwent SE surgery did not express GDF-5 and Atx. Also, in SE fusion of cartilage elements takes place and the joint interzone does not form. This is indicated by continuous Col-9 expression in SE limbs, whereas Col-9 is downregulated at the joint interzone in the regenerating WE samples. This order and pattern of gene expression observed in regenerates is similar to the development of a joint suggesting that regeneration recapitulates development at the molecular level. This model defines some of the conditions required for inducing joint regeneration in an otherwise nonregenerating environment. This knowledge can be useful for designing new therapeutic approaches for joint loss or for conditions affecting joint integrity in humans.
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Project description:1. The development, localization and heterogeneity of acid phosphatase and a Zn(2+)-activated acid phosphatase in cellular fractions of developing chick liver were studied. 2. Acid phosphatase is distributed abundantly in the particulate and soluble fractions. The soluble fraction is rich in Zn(2+)-activated acid phosphatase, which attains its peak activity at about 15 days of incubation. 3. The particulate acid phosphatase activity is inhibited by fluoride but not by sodium l(+)-tartrate or cysteine. On the other hand, the soluble Zn(2+)-activated acid phosphatase activity is inhibited by sodium l(+)-tartrate and cysteine but not by fluoride. 4. The pH optimum of these two enzymes is similar at about 5.6. 5. The soluble Zn(2+)-activated acid phosphatase activity appears to be thermally stabilized by the treatment with Triton X-100 or bovine serum albumin.
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Project description:1. The changes in the net amounts of retinol, retinyl esters and retinal in both the developing chick embryo and the newly hatched chick were investigated. The embryo requires about 68nmol of the vitamin for its growth, whereas the baby chick requires about 108nmol during the first 7 days after hatching. 2. Retinal was present in the egg in fairly high concentrations at the beginning of the incubation but it virtually disappeared from the extra-embryonic tissue after day 17 of incubation. It was not found in the liver of the embryo or of the newly hatched chick up until day 7.
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Project description:Embryos are growing organisms with highly heterogeneous properties in space and time. Understanding the mechanical properties is a crucial prerequisite for the investigation of morphogenesis. During the last 10 years, new techniques have been developed to evaluate the mechanical properties of biological tissues in vivo. To address this need, we employed a new instrument that, via the combination of micro-indentation with Optical Coherence Tomography (OCT), allows us to determine both, the spatial distribution of mechanical properties of chick embryos, and the structural changes in real-time. We report here the stiffness measurements on the live chicken embryo, from the mesenchymal tailbud to the epithelialized somites. The storage modulus of the mesoderm increases from (176 ± 18) Pa in the tail to (716 ± 117) Pa in the somitic region (mean ± SEM, n = 12). The midline has a mean storage modulus of (947 ± 111) Pa in the caudal (PSM) presomitic mesoderm (mean ± SEM, n = 12), indicating a stiff rod along the body axis, which thereby mechanically supports the surrounding tissue. The difference in stiffness between midline and presomitic mesoderm decreases as the mesoderm forms somites. This study provides an efficient method for the biomechanical characterization of soft biological tissues in vivo and shows that the mechanical properties strongly relate to different morphological features of the investigated regions.
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Project description:Caffeine consumption is worldwide. It has been part of our diet for many centuries; indwelled in our foods, drinks, and medicines. It is often perceived as a "legal drug", and though it is known to have detrimental effects on our health, more specifically, disrupt the normal fetal development following excessive maternal intake, much ambiguity still surrounds the precise mechanisms and consequences of caffeine-induced toxicity. Here, we employed early chick embryos as a developmental model to assess the effects of caffeine on the development of the fetal nervous system. We found that administration of caffeine led to defective neural tube closures and expression of several abnormal morphological phenotypes, which included thickening of the cephalic mesenchymal tissues and scattering of somites. Immunocytochemistry of caffeine-treated embryos using neural crest cell markers also demonstrated uncharacteristic features; HNK1 labeled migratory crest cells exhibited an incontinuous dorsal-ventral migration trajectory, though Pax7 positive cells of the caffeine-treated groups were comparatively similar to the control. Furthermore, the number of neurons expressing neurofilament and the degree of neuronal branching were both significantly reduced following caffeine administration. The extent of these effects was dose-dependent. In conclusion, caffeine exposure can result in malformations of the neural tube and induce other teratogenic effects on neurodevelopment, although the exact mechanism of these effects requires further investigation.
| S-EPMC3314624 | biostudies-literature
Project description:WNT1 and WNT3A drive a dorsal to ventral gradient of ?-catenin-dependent Wnt signaling in the developing spinal cord. However, the identity of the receptors mediating downstream functions remains poorly understood.In this report, we show that the spatiotemporal expression patterns of FZD10 and WNT1/WNT3A are highly correlated. We further show that in the presence of LRP6, FZD10 promotes WNT1 and WNT3A signaling using an 8xSuperTopFlash reporter assay. Consistent with a functional role for FZD10, we demonstrate that FZD10 is required for proliferation in the spinal cord. Finally, by using an in situ proximity ligation assay, we observe an interaction between FZD10 and WNT1 and WNT3A proteins.Together, our results identify FZD10 as a receptor for WNT1 and WNT3A in the developing chick spinal cord.
| S-EPMC4031291 | biostudies-literature
Project description:The relationship between bone formation and vitamin D metabolism was investigated in the developing chick embryo. Fertilized White Leghorn eggs were incubated at 38 degrees C in an incubator for 21 days. The fresh weight and calcium content of embryonic tibiae began to increase at day 12 and attained maximal values at day 19. Bone alkaline phosphatase and citrate decarboxylation activities, both of which represent osteoblastic activity, also began to increase at days 10-12, reached maximal values at day 19 and sharply declined thereafter. Both bone enzyme activities were highly correlated with CA2+-binding activity in the chorioallantoic membrane measured by the Chelex 100 assay. When mesonephric and metanephric homogenates were incubated with 25-hydroxy[3H]cholecalciferol, a marked and concomitant increase occurred in the metanephric 1 alpha- and 24-hydroxylase activity after day 14. The production of 1 alpha, 25-dihydroxycholecalciferol attained a maximal value at day 19 and decreased thereafter, whereas that of 24,25-dihydroxycholecalciferol continued to increase until hatching. The production rate of 1 alpha, 25-dihydroxycholecalciferol by the metanephros coincided with the changes in Ca2+-binding activity in the chorioallantoic membrane and osteoblastic activity. Since both intestinal calcium absorption and bone mineral mobilization do not occur in embryonic life, these results support the idea that 1 alpha, 25-dihydroxycholecalciferol may be involved directly in bone formation or induction of a calcium-binding protein in the chorioallantoic membrane.
| S-EPMC1162722 | biostudies-other
Project description:Leishmaniasis is one of the diverse and neglected tropical diseases. Embryo-toxicity of drugs has always been a major concern. Chick embryo is a preclinical model relevant in the assessment of adverse effects of drugs. The current study aimed to assess embryonic histopathological disorders and amniotic fluid biochemical changes following meglumine antimoniate treatment. The alteration of vascular branching pattern in the chick's extra-embryonic membrane and exploration of molecular cues to early embryonic vasculogenesis and angiogenesis were also quantified. Embryonated chicken eggs were treated with 75 or 150 mg/kg of meglumine antimoniate. Embryo malformations, growth retardation and haemorrhages on the external body surfaces were accompanied by histopathological lesions in the brain, kidney, liver and heart in a dose-dependent manner. Significant rise occurred in the biochemical indices of alkaline phosphatase, aspartate aminotransferase, alanine aminotransferase and amylase in the amniotic fluid. Quantification of the extra-embryonic membrane vasculature showed that the anti-angiogenic and anti-vasculogenic effects of the drug were revealed by a significant decrease in fractal dimension value and mean capillary area. The relative expression levels of vascular endothelial growth factor A and vascular endothelial growth factor receptor 2 mRNA also significantly reduced. Concerns of a probable teratogenicity of meglumine antimoniate were established by data presented in this study. It is concluded that tissue lesions, amniotic fluid disturbance, altered early extra-embryonic vascular development and gene expression as well as the consecutive cascade of events, might eventually lead to developmental defects in embryo following meglumine antimoniate treatment. Therefore, the use of meglumine antimoniate during pregnancy should be considered as potentially embryo-toxic. Hence, physicians should be aware of such teratogenic effects and limit the use of this drug during the growing period of the fetus, particularly in rural communities. Further pharmaceutical investigations are crucial for planning future strategies.
| S-EPMC5969735 | biostudies-literature
Project description:Lineage analysis plays a central role in exploring the developmental potential of stem and progenitor cell populations. In higher vertebrates, a variety of techniques have been used to label individual cells or cell populations, including interspecies grafting, intracellular microinjection, and Cre-mediated recombination. However, these approaches often suffer from difficulties in progenitor cell targeting, low cellular resolution and/or ectopic labeling. To circumvent these issues, here we utilize replication incompetent avian (RIA) retroviruses to deliver combinations of fluorescent proteins into distinct cellular compartments in chick embryos. In particular, RIA-mediated lineage tracing is optimal for long term mapping of dispersing cell populations like the neural crest. Using this tool, we confirm that trunk neural crest cells are multipotent. Furthermore, our RIA vector is engineered to be fully adaptable for other purposes such as cell fate analysis, gene perturbation studies and time-lapse imaging. Taken together, we present a novel approach of multiplex lineage analysis that can be applied to normal and perturbed development of diverse cell populations in avian embryos.
| S-EPMC6487888 | biostudies-literature
Project description:BackgroundLittle is known regarding the molecular pathways that underlie the process of retinal development. The purpose of this study was to identify proteins which may be involved in development of retina. We used a proteomics-based approach to identify proteins that are up- or down-regulated during the development of the embryo chick retina.ResultsTwo-dimensional gel electrophoresis was performed with the retina of embryo chicken, which was obtained from embryos of day 7 (ED7) and of day 11 (ED11). The protein spots showing significant differences were selected for identification by MALDI mass spectrometry. Thirteen proteins were differentially expressed; seven proteins were up-regulated in embryo retina of chicken at ED 11 and six proteins were down-regulated. Significant proteins were also evaluated in embryo day 15 (ED15). Some of identified proteins were known to regulate cell proliferation, cell death, transport, metabolism, organization and extracellular matrix, and others also included novel proteins.ConclusionWe identified thirteen proteins which differentially expressed in embryonal retina of chicken at day 7, as compared to the retina of embryo of day 11. They were various regulatory proteins for cellular signaling.
| S-EPMC2267454 | biostudies-literature