Developing Chick Brain Research Articles

Histo-anatomical mutilations of developing chick brain induced by in-ovo fluoride and bifenthrin exposure.

Comparative developing brain histo-anatomical pathologies of Fluoride ions and Bifenthrin in-ovo exposures were explored in the golden black variety of domestic chick. Three exposure groups were -the Vehicle control group (Vg); Fluoride (F) group and the Bifenthrin (Bn) group each with forty fertilized eggs and received their respective group treatment at zero day of incubation. Embryos were extracted, dissected from head region and the embryonic whole brains were recovered after 14 days of incubation. The embryonic brains were preserved in bouin fixative for 24h for further studies. The morphological results show the atrophied and hypertrophied embryonic brain in F and Bn groups respectively as compared to Vg group. The toxicological signs of encephalic anatomy and histology of F and Bn exposure were the enlarged third ventricles, optocoeles and arachnoid mater, encephalic spongiosis and decreased neuroglial density. The morphometric data showed significant decrease (p≤0.05) in mean weight and density of whole brain in F and Bn groups compared Vg. The mean length and width of whole brain in F were significantly lower than that of the Bn and Vg. whereas, the mean breadth of third ventricle in Bn remained significantly lower than F and Vg groups. On the other hand, the mean breadth of optocoele and fourth ventricle in F and Bn groups remained significantly higher than Vg. Conversely the mean optic lobe wall thickness in F remained significantly lower than Bn and Vg. Additionally, the mean neuronal density in diencephalon, optic lobe and cerebellum in F group and Bn group remained significantly (p≤0.05) lower than Vg. Results show that low dose in-ovo fluoride or bifenthrin exposure may cause neuro-developmental abnormalities in the developing chick embryos indicating that the Fluoride-ions and Bifenthrin harbor strong developmental neurotoxic capacity.

ChHDAC11 mRNA Expression During Prenatal and Postnatal Chicken (Gallus gallus) Brain Development

Background: Histone deacetylation plays an essential role in transcriptional regulation of cell cycle progression and other evolutionary processes. Several results confirm the importance of the latest found HDAC11 gene to deacetylate histone core in neurons and their supportive cells in developing the vertebrate Central Nervous System (CNS). Objectives: This study investigates the HDAC11 potential role in early chicken CNS development by studying its mRNA expression profile which may have unique means in studying human subjects. Materials & Methods: Chicken HDAC11 RNAs were reverse transcribed to cDNAs, and the amount of chHDAC11 transcripts was measured by ΔCT mean calculation using the real-time quantitative PCR method. One-way ANOVA and Duncan’s analysis (SigmaStat software version 4.0) were used to test the statistical significance of the results. The levels of significance were set at P≤0.05. Quantitative data are presented as Mean±SD. Results: The amount of HDAC11 mRNAs gradually increases, at least 2-3 times, from as early as day 14 (E14/HH40) of prenatal cortex formation to day P0 (E20=HH45) and continue to increase to day 40 in both cortical and hippocampal regions of the postnatal chicken brain during development (*P≤0.05). HDAC11 mRNA is not only expressed in the postnatal cortex and hippocampi regions but also—for the first time—in the developing brain during the prenatal period. Conclusion: Our results show a possibly important role for the latest found HDAC11 conserved gene in the development of vertebrates’ embryonic brain, which in turn may have a significant impact on understanding human brain development.

Immunohistochemistry and In Situ Hybridization in the Developing Chicken Brain.

One of the first steps in studies of gene function is the spatiotemporal analysis of patterns of gene expression. Indirect immunohistochemistry is a method that allows the detection of a protein of interest by incubating a histological section with an antibody or antiserum raised against the protein, and then localizing this primary antibody with a tagged secondary antibody. To determine the cellular source of a protein of interest, or if a specific antibody is not available, specific transcripts can be localized using in situ hybridization. A histological section is incubated with a labeled RNA probe that is complementary to the target transcript; after hybridization with the target transcript the labeled RNA probe can be identified with an antibody. Here we describe materials and methods used to perform basic indirect immunohistochemistry and in situ hybridization on frozen sections through the developing chicken brain, emphasizing controls and potential problems that may be encountered.

Gene Silencing in Chicken Brain Development.

Despite the development of brain organoids and neural cultures derived from iPSCs (induced pluripotent stem cells), brain development can only be studied in an animal. The mouse is the most commonly used vertebrate model for the analysis of gene function because of the well-established genetic tools that are available for loss-of-function studies. However, studies of gene function during development can be problematic in mammals. Many genes are active during different stages of development. Absence of gene function during early development may cause aberrant neurogenesis or even embryonic lethality and thus prevent analysis of later stages of development. To avoid these problems, precise temporal control of gene silencing is required.In contrast to mammals, oviparous animals are accessible for experimental manipulations during embryonic development. The combination of accessibility and RNAi- or Crispr/Cas9-based gene silencing makes the chicken embryo a powerful model for developmental studies. Depending on the time window during which gene silencing is attempted, chicken embryos can be used in ovo or ex ovo in a domed dish for easier access during later stages of development. Both techniques allow for precise temporal control of gene silencing during embryonic development.

The value of scaffolds

Brain Development![Figure][1] Neurons migrate to form surface layers of the chick brain. PHOTO: F. GARCIA-MORENO ET AL. CELL REP. 22 , 96 (2018) The brain is built by groups of neurons that migrate and interdigitate to form layers and circuits. This process varies in different phyla of animals. Garcia-Moreno et al. draw lessons from the development of the chick brain to understand what makes the mammalian brain distinctive. In mammals, excitatory glutamatergic neurons born deep in the brain migrate radially to the cortex, whereas inhibitory GABAergic interneurons born elsewhere migrate tangentially across the cortex. And, like the external scaffolds on a building under construction, some glutamatergic neurons migrate tangentially, instruct organization, then disappear. The developing chick brain, although it has tangentially migrating interneurons, lacks the tangentially migrating transient neurons. Cell Rep. 22 , 96 (2018). [1]: pending:yes

Segmentation of the chick central and peripheral nervous systems.

The chick embryo has provided a prominent model system for the study of segmental patterning in the nervous system. During early development, motor and sensory axon growth cones traverse the anterior/rostral half of each somite, so avoiding the developing vertebral components and ensuring separation of spinal nerves from vertebral bones. A glycoprotein expressed on the surface of posterior half-somite cells confines growth cones to the anterior half-somites by a contact repulsive mechanism. Hindbrain segmentation is also a conspicuous feature of chick brain development. We review how its contemporary analysis was initiated in the chick embryo, and the advantages the chick system continues to provide in its detailed elucidation at both molecular and neural circuit levels.

Does Early Environmental Complexity Influence Tyrosine Hydroxylase in the Chicken Hippocampus and "Prefrontal" Caudolateral Nidopallium?

In adult chickens, the housing system influences hippocampal morphology and neurochemistry. However, no work has been done investigating the effects of the early life environment on chicken brain development. In the present study, we reared 67 commercial laying hens (Gallus gallus domesticus) in two environments that differed in the degree of complexity (aviary or cage system). These two groups were further divided into two age groups. At 20 weeks of age, 18 aviary-reared birds and 15 cage-reared birds were humanely euthanized and their brains dissected. At 24 weeks of age, a further 16 brains from aviary-reared birds and 18 brains from cage-reared birds were collected. These brains were prepared for immunohistochemical detection of tyrosine hydroxylase (TH), the rate-limiting enzyme in the biosynthesis of dopamine, in the hippocampus and the caudolateral nidopallium (NCL). There were no differences between the treatment groups in TH staining intensity in the hippocampus or the NCL. In the medial hippocampus, the right hemisphere had higher TH staining intensity compared to the left hemisphere. The opposite was true for the NCL, with the left hemisphere being more strongly stained compared to the right hemisphere. The present study supports the notion that the hippocampus is functionally lateralized, and our findings add to the body of knowledge on adult neural plasticity of the avian brain.

Regulations of glycolipid: XI. glycosyltransferase (GSL: GLTs) genes involved in SA-LeX and related GSLs biosynthesis in carcinoma cells by Biosimilar apoptotic agents: potential anticancer drugs.

New biosimilar apoptotic agents identified during our recent studies can be employed as a new generation of anti-cancer drugs after being properly delivered to the patients by a proper drug delivery system. These agents regulate at gene level as evidenced by both translational and transcriptional studies. In order to study glyco-gene regulation, we used four clonal metastatic cancer cells of colon and breast cancer-tissue origin (Colo-205, SKBR-3, MDA-468, and MCF-7). The glyco-genes for synthesis of SA-Lex and SA-Lea (which contain N-acetylglucosamine, sialic acid, and fucose) in these cells was modulated differentially at various phases induced by l-PPMP, d-PDMP (inhibitor of glucosylceramide biosynthesis), Betulinic Acid (a triterpinoid isolated from the bark of certain trees and used for cancer treatment in China), Tamoxifen (a drug in use in the west for treatment of early stages of the disease in breast cancer patients), and cis-platin (an inhibitor of DNA biosynthesis used for testicular cancer patients) when used for induction of apoptosis in the above mentioned cell lines. Biosimilarities of these chemicals reside in their killing abilities of these highly metastatic cells by apoptosis. Within 2–6 h, transcriptional modulation of a number of glyco-genes was observed by DNA micro-array (containing over 340 glyco-genes attached to the glass cover slips) studies. Under a long incubation time (24–48 h) almost all of the glyco-gene products, glycolipid: glycosyltransferases (GLTs) were downregulated. The cause of these glyco-gene regulations during apoptotic induction in metastatic carcinoma cells is unknown and needs future investigations for further explanations. At least two GSL-GLT activities (GLTs) of Basu-Roseman pathway2 catalyzing the biosynthesis of GD1a and GD3 gangliosides and more than five GSL: GLTs in the SA-LeX pathway2 have been regulated. Most of these glyco-genes are expressed in the early stages (7–17 days) of embryonic chicken-brain development and lowered in the adult stage. However, the mechanism of regulation of the enzymatic activities of these GLTs in the synthesis of SA-LeX (human cancer cell marker) is unknown. Our recent attempt of in vitro Glyco-gene regulation in apoptotic metastatic cells may lead us to explain the in vivo Glyco-gene regulation in normal or diseased animal organs.

Entropy-Complexity Characterization of Brain Development in Chickens

Electroencephalography (EEG) reflects the electrical activity of the brain, which can be considered chaotic and ruled by a nonlinear dynamics. Chickens exhibit a protracted period of maturation, and this temporal separation of the synapse formation and maturation phases is analogous to human neural development, though the changes in chickens occur in weeks compared to years in humans. The development of synaptic networks in the chicken brain can be regarded as occurring in two broadly defined phases. We specifically describe the chicken brain development phases in the causality entropy-complexity plane H × C, showing that the complexity of the electrical activity can be characterized by estimating the intrinsic correlational structure of the EEG signal. This allows us to identify the dynamics of the developing chicken brain within the zone of a chaotic dissipative behavior in the plane H × C.

Polarized light microscopy in reproductive and developmental biology

The polarized light microscope reveals orientational order in native molecular structures inside living cells, tissues, and whole organisms. It is a powerful tool used to monitor and analyze the early developmental stages of organisms that lend themselves to microscopic observations. In this article, we briefly discuss the components specific to a traditional polarizing microscope and some historically important observations on: chromosome packing in the sperm head, the first zygote division of the sea urchin, and differentiation initiated by the first asymmetric cell division in the sand dollar. We then introduce the LC-PolScope and describe its use for measuring birefringence and polarized fluorescence in living cells and tissues. Applications range from the enucleation of mouse oocytes to analyzing the polarized fluorescence of the water strider acrosome. We end with new results on the birefringence of the developing chick brain, which we analyzed between developmental stages of days 12-20.

Molecular cloning and characterization of chicken neuronal intermediate filament protein α‐internexin

α-Internexin is one of the neuronal intermediate filament (IF) proteins, which also include low-, middle-, and high-molecular-weight neurofilament (NF) triplet proteins, designated NFL, NFM, and NFH, respectively. The expression of α-internexin occurs in most neurons as they begin differentiation and precedes the expression of the NF triplet proteins in mammals. However, little is known about the gene sequence and physiological function of α-internexin in avians. In this study we describe the molecular cloning of the mRNA sequence encoding the chicken α-internexin (chkINA) protein from embryonic brains. The gene structure and predicted amino acid sequence of chkINA exhibited high similarity to those of its zebrafish, mouse, rat, bovine, and human homologs. Data from transient-transfection experiments show that the filamentous pattern of chkINA was found in transfected cells and colocalized with other endogenous IFs, as demonstrated via immunocytochemistry using a chicken-specific antibody. The expression of chkINA was detected at the early stage of development and increased during the developmental process of the chicken. chkINA was expressed widely in chicken brains and colocalized with NF triplet proteins in neuronal processes, as assessed using immunohistochemistry. We also found that chkINA was expressed abundantly in the developing cerebellum and was the major IF protein in the parallel processes of granule neurons. Thus, we suggest that chkINA is a neuron-specific IF protein that may be a useful marker for studies of chicken brain development.

BrdU Birth Dating Can Produce Errors in Cell Fate Specification in Chick Brain Development

Birth dating neurons with bromodeoxyuridine (BrdU) labeling is an established method widely employed by neurobiologists to study cell proliferation in embryonic, postnatal, and adult brain. Birth dating studies in the chick dorsal telencephalon and the mammalian striatum have suggested that these structures develop in a strikingly similar manner, in which neurons with the same birth date aggregate to form "isochronic clusters." Here we show that isochronic cluster formation in the chick dorsal telencephalon is an artifact. In embryos given standardly employed doses of BrdU, we observed isochronic clusters but found that clusters were absent with BrdU doses close to the limits of detection. In addition, in situ hybridization experiments established that neurons in the clusters display errors in cell type specification: BrdU cell clusters in nidopallium adopted a mesopallial neuronal fate, mesopallial clusters were misspecified as nidopallial cells, and in some instances, the BrdU clusters failed to express neuronal differentiation markers characteristic of the dorsal telencephalon. These results demonstrate that the chick dorsal telencephalon does not develop by isochronic cluster formation and highlight the need to test the integrity of BrdU-treated tissue with gene expression markers of regional and cell type identity.

Dynamic mRNA distribution pattern of thyroid hormone transporters and deiodinases during early embryonic chicken brain development

Maternal thyroid hormones (THs) are important in early brain development long before the onset of embryonic TH secretion, but information about the regulation of TH availability in the brain at these early stages is still limited. We therefore investigated in detail the mRNA distribution pattern of the TH activating type 2 and inactivating type 3 deiodinases (D2 and D3) and the TH transporters, organic anion transporting polypeptide 1c1 (Oatp1c1) and monocarboxylate transporter 8 (Mct8), in chicken embryonic brain as well as in retina and inner ear from day 3 to day 10 of development. Oatp1c1, Mct8 and D3 are expressed in the choroid plexus and its precursors allowing selective uptake of THs at the blood–cerebrospinal fluid-barrier with subsequent inactivation of excess hormone. In contrast, the developing blood–brain-barrier does not express Oatp1c1 or Mct8 but appears to be a site for TH activation by D2. Expression of D3 in several sensory brain centers may serve as protection against premature TH action. Expression of D2 and Mct8 but not D3 in the developing pituitary gland allows accumulation of active THs even at early stages. Mct8 is widely expressed in gray matter throughout the brain. This is the first comprehensive study on the dynamic distribution pattern of TH-transporters and deiodinases at stages of embryonic brain development when only maternal THs are available. It provides the essential background for further research aimed at understanding early developmental processes depending on maternal THs.

Distinct patterns of human medulloblastoma dissemination in the developing chick embryo nervous system

Medulloblastoma (MB) is the most common malignant primary brain tumor in children. Aggressive tumors that disseminate along the leptomeninges carry extremely poor prognoses. Mechanisms that predict dissemination are poorly understood. Our objective was to develop a reliable and reproducible model to study MB dissemination. We have created a chicken-human xenograft to study features of MB with leptomeningeal dissemination. Human MB cell lines (D283, Daoy), primary human MB cells (SF8113), and primary genetic mouse model (Math1cre:SmoM2 flox/flox) MB cells were either transfected to express green fluorescent protein (GFP) or were labeled with a membrane permeable green fluorescent probe. Cells were then injected as aggregates or implanted as pellets into the developing chicken brain immediately after neural tube closure at embryonic day 2 (E2). Most embryos were harvested three days after implantation (E5) though some were harvested up to E15. The developing brain was analyzed via whole mount fluorescent imaging and tissue section immunohistochemistry. Human and mouse MBs survived in the developing chicken central nervous system (CNS). They exhibited distinct patterns of incorporation and dissemination into the CNS that were consistent with observed phenotypes of the corresponding human patient or mouse host. Specifically, metastatic D283 cells disseminated along the leptomeninges whereas Daoy, primary mouse MB, and primary human MB cells did not. This work supports an avian-human xenograft as a successful model to study patterns of MB dissemination. Our model provides a basis for manipulating cell signaling mechanisms to understand critical targets involved in MB dissemination.

Expression profile and thyroid hormone responsiveness of transporters and deiodinases in early embryonic chicken brain development

We used the chick embryo to study the mechanisms regulating intracellular TH availability in developing brain. TH-transporters OATP1C1 and MCT8, and deiodinases D1, D2, and D3 were expressed in a region-specific way, well before the onset of endogenous TH secretion. Between day 4 and 10 of development MCT8 and D2 mRNA levels increased, while OATP1C1 and D3 mRNA levels decreased. D2 and D3 mRNAs were translated into active protein, while no D1 activity was detectable. Injection of THs into the yolk 24h before sampling increased TH levels in the brain and resulted in decreased OATP1C1 and increased MCT8 expression in 4-day-old embryos. A compensatory response in deiodinase activity was only observed at day 8. We conclude that THs are active in the early embryonic brain and TH-transporters and deiodinases can regulate their availability. However, the absence of clear compensatory mechanisms at day 4 makes the brain more vulnerable for changes in maternal TH supply.

Prenatal Exposure to Chronic Ethanol on N Methyl D Aspartate Receptor Distribution

Problem statement: Effect of chronic exposure to ethanol on NR1 subunit of the NMDA receptor distribution in chick embryo brain chronic ethanol intake leads to the development of dependence on and tolerance to ethanol in both human and animals. The glutamate system plays a major role in mediating ethanol effects on brain and behavior. The aim of the present study was to investigate whether chronic ethanol exposure altered NR1 subunit of N-Methyl-D-Aspartate (NMDA) receptor distribution in the developing chick brain on 10 and 15th day of egg incubation. Approach: Forty fertilized eggs divided in 2 groups, control and chronic. In the chronic group the eggs were chronically exposed to ethanol 10% during 15 day of embryonic period. Immunohistochemical procedure was used for NR1 subunit of NMDA receptor distribution determination. Image analyzer program were used for color analyzing of immunohistochemistry slides. Data were analyzed statistically by Student T test. The level of significance was assumed to be (p<0.05. Results: Immunohistological finding of these experiments indicated that chronic exposure to ethanol significantly (p<0.05) decreased density of NR1 subunit of NMDA receptors in cerebral cortex of chick brain on 10 and 15th day of embryonic stage. But the number of immunolable cell per mm2 of cerebral cortex did not show any difference. Conclusion/Recommendation: Our results indicated that chronic exposure to ethanol decreased NR1 subunit of NMDA receptor distribution in cerebral cortex of chick embryo on 10 and 15th day of embryonic stage. But did not have any effect on number of immunolable cell per mm2 of cerebral cortex.

Quantitative and dynamic expression profile of premature and active forms of the regional ADAM proteins during chicken brain development

The ADAM (A Disintegrin and Metalloprotease) family of transmembrane proteins plays important roles in embryogenesis and tissue formation based on their multiple functional domains. In the present study, for the first time, the expression patterns of the premature and the active forms of six members of the ADAM proteins — ADAM9, ADAM10, ADAM12, ADAM17, ADAM22 and ADAM23 — in distinct parts of the developing chicken brain were investigated by quantitative Western blot analysis from embryonic incubation day (E) 10 to E20. The results show that the premature and the active forms of various ADAM proteins are spatiotemporally regulated in different parts of the brain during development, suggesting that the ADAMs play a very important role during embryonic development.

Autotaxin controls caudal diencephalon‐mesencephalon development in the chick

The diencephalon is the embryonic anlagen of the higher integration centers of the brain. Recent studies have elucidated how the cells in the rostral diencephalon acquire their regional identities. However, the understanding of the mechanisms under which the caudal diencephalon is formed is still limited. Here we focus on the role of Autotaxin (ATX), a lysophospholipid-generating exoenzyme, whose mRNA is detected in the caudal diencephalon. RNA interference against ATX altered the expression pattern of Pax6-regualted genes, Tcf4, Lim1, and En1, implying that ATX is required for the maintenance of the regional identity of the caudal diencephalon and the diencephalon-mesencephalon boundary (DMB). Furthermore, ATX-RNAi inhibited neuroepithelial cell proliferation on both sides of the DMB. We propose a dual role of ATX in chick brain development, in which ATX not only contributes to the formation of caudal diencephalon as a short-range signal, but also regulates the growth of mesencephalon as a long-range signal.

Early embryonic brain development in rats requires the trophic influence of cerebrospinal fluid

Cerebrospinal fluid has shown itself to be an essential brain component during development. This is particularly evident at the earliest stages of development where a lot of research, performed mainly in chick embryos, supports the evidence that cerebrospinal fluid is involved in different mechanisms controlling brain growth and morphogenesis, by exerting a trophic effect on neuroepithelial precursor cells (NPC) involved in controlling the behaviour of these cells. Despite it being known that cerebrospinal fluid in mammals is directly involved in corticogenesis at fetal stages, the influence of cerebrospinal fluid on the activity of NPC at the earliest stages of brain development has not been demonstrated. Here, using “in vitro” organotypic cultures of rat embryo brain neuroepithelium in order to expose NPC to or deprive them of cerebrospinal fluid, we show that the neuroepithelium needs the trophic influence of cerebrospinal fluid to undergo normal rates of cell survival, replication and neurogenesis, suggesting that NPC are not self-sufficient to induce their normal activity. This data shows that cerebrospinal fluid is an essential component in chick and rat early brain development, suggesting that its influence could be constant in higher vertebrates.

Early Formation of a GFAP-Positive Cell Population in the Ventricular Zone during Chicken Brain Development

It is believed that neurons are generated near the surface of the embryonic cerebral ventricles, whereas glial cell proliferation occurs at sites distant to the ventricles. There is still uncertainty concerning the developmental stages when glial and neuronal cell lines diverge in the proliferative zone. The purpose of this study was to determine whether at early stages of chicken brain development during neurogenesis, cells from the astrocytic lineage are present in relevant amounts, where they are located in the neural tube, and to what extent brain regional differences exist. In this report, we demonstrate that specific markers for astrocytes [glial fibrillary acidic protein (GFAP) and glutamate-aspartate transporter (GLAST)] are expressed and translated during early neurogenesis in the developing chicken embryo at day 4 in the investigated brain regions. Between days 4 and 7, GFAP expression declines, and increases again after day 7. This profile is not entirely paralleled by GLAST expression, suggesting a later acquisition of this functional glial property. Comparison of different brain regions revealed that caudal parts of the developing chicken central nervous system are delayed in development with respect to the switch from neuronal to glial genesis. In conclusion, we show that gliogenesis occurs much earlier in the embryonic chicken brain than hitherto assumed.