DISSECTING A CHICK EMBRYO 15
The use of chicken embryo tostudy the early and later stages of development of vertebrates hasproven very important. The hardened eggshell to within which theembryo develops is the natural incubator or culture dish. Byunderstanding the early embryogenesis, our understanding of celldivision and the whole anatomy is enhanced. Multicellular organismsdo share similarities in the pattern of development. The similaritiesbecome clearer in the closely related groups. When a hole is madethrough the eggshell, several manipulations can be done to the embryoto study the development patterns. The coordination of tissues tocontrol the development of organs and the test of a specific moleculeto mediate the interaction has been made possible by the ‘Cutand Paste’ experiment, which has yielded positive results.
One of the development patterns,which can be studied through dissection of an embryo of a chick, isneurulation. In the central nervous system, there exist a largenumber of neuroglial cells called astrocytes. The true shape of thesecells is unknown till today. Some are star-like, others show thereal marker glial fibrillary acidic protein (GFAP) and others lackbrain capillaries (Chung, & Andrew, 2012). When microglial cells,neurons, and oligodendrocytes are removed from the brain, thepopulation of cell that remains is what is called astrocytes.
At some points in the process ofdevelopment of the nervous system, the functional connectivity, andthe shape of the neurons is determined by a complicated network ofevents, resulting in complex neuron circuit. Phosphatidylinositol3-kinases (PI3K) are main enzymes, which contribute to comingtogether of neurons to form a functional circuit.Phosphatidylinositol 3, 4, 5-trisphosphate (PIP3) is formed when PI3Kis activated. PIP3 levels are controlled through the work ofPI3-phosphatase by a process known as de-phosphorylation. The processis mediated by PTEN (tensin homolog and phosphatase deleted onchromosome ten).
Functions of astrocytes
Astrocytes are believed to have many functions.
They perform the function ofbuilding up of the brainarchitecture. Thisthey do by dividing the gray matter of the parenchyma into structuralunits, which are relatively independent.
They are involved in the supply of glutamine for the maintenance of glutamatergic neurotransmission
Intercellular calcium cellformation is also a role played by astrocytes. Those astrocytesinvolved in tripartite synapses are tied by gap junction leading to anetwork capable of supporting large-scale brain integrative functionsfrom the changing glucose delivery to a cognitive informationsynthesis (Hall, 2014).
This project will narrow down toneurulation and secondary neurulation, within the chick’s embryoand it will try to answer the following questions,
What functions do laminin and fibronectin do during the formation of the secondary neural tube?
At what level of development does the secondary neural tube basement membrane start to form, and when does its formation complete?
What is the difference in the deposition of lamina and fibronectin between the completed neural tube and mesenchymal arrangement?
Getting the Embryos
Eggs were obtained from afertilized chicken. The dissecting tools and the eggs were rinsed inan ethanol solution of 70% concentration to prevent bacteria and toensure sterility. Using a pair of forceps, the eggs were punctured toallow the embryo to be exposed. Kimwipes are then used to absorbexcessive albumin, and at the top of the embryo, a 3mm Whatman filterpaper placed. The embryo is then released from the yolk usingscissors. The removed embryo is then put in 60mm dish with ChickRinger’s saline (Colas, & Schoenwolf, 2014).
The embryos are then placed undera dissecting microscope to remove the membrane and the yolk. Thetransfer of the embryos again took place from the paper ring to a 4%paraformaldehyde fixative on ice in a 60mm dish. This allowed thestabilization of the tissues of the embryo. In the fixative solution,the embryo stayed for 20minutes from which they are transferred toalcohol solution for storage.
The Dehydration and Embedding Process
Take the embryos and deep in aseries of ethanol to dehydrate them. Embed the embryos in a paraffinwax to allow for sectioning so that finer details of the tissue ofthe tail and secondary neurulation can be studied. Using ethanol ofconcentrations (50%, 70%, 95% and two 100%), dip the embryos for 5minutes to begin the dehydration process (Chung, & Andrew, 2012).
Submerge the embryos in a 1:1ethanol: Protocol solution for 5 minutes, then 15-minute incubationat a temperature 55-58°C in the embedding oven at 100% Protocolsolution. Incubate the embryos overnight in a 1:1 Protocol: paraffinwax solution, then followed by an overnight incubation in 100%paraffin wax, all in the embedding oven. Pasteur pipettes used tomove the wax in and out of the glass containers were incubated aswell to ensure uniformity in temperature with the wax.
Sectioning of the Embryos
Remove the embryos from theembedded oven so that wax can harden incase the embryoshave completely embedded in the wax. By the use of a razor blade aBunsen burner and a metal spatula, trim the paraffin-embedded embryoand mount on the wooden block for sectioning. For a very smooth andstraight sectioning, a trapezoidal-shaped block was made. For thesafety of the ribbons before the start of sectioning, a microscopicinspection of the razor blade was done to prevent nicks. An adhesivecalled Histogram is used to cover the slides with the sections toensure that the sections remain with the slide after the wax has beenremoved.
Place The Historic-covered slideson a slide warmer at a temperature of 42-43°C, and monitor it sothat it does not go above 45°C. Distilled water was the boiling ofdistilled water is to expel air bubbles. By use of a Pasteurpipette, add water to the slides. The ribbons of the sectionalribbons are put on the slide and properly arranged. After filling theslide with the section,a Pasteur pipette is used to remove water as the slide continues todry while on the warmer and therefore a thorough adhesion of thesections to the slides.
Chick Embryo Sections Interpretation
Presentation photographs havetwo colors:immunolocalized protein/lamininis indicated by red-staining. The blue staining representshematoxylin with a specific end goal to expand and better imagine thered recoloringand to give tissues setting.
“Laminin Staining in Tail Bud of Stage 14 Embryo.”
In the diagram bellow, A:demonstrates the tail bud ofphase 14 incipientorganism, recolored with hostile to laminin. The tail bud mesenchymecontains light laminin recoloring in the mesenchymal inside, whilethere is concentrated laminin recoloring toward the border of themesenchyme. This recoloring is found more in the central district ofthe mesenchymal border. There is a great deal less recoloring in thedorsal area where neural peak cells will later develop. The redrecoloring design in this figure is by all accounts encompassing thephones instead of inside them. This proposes the laminin present inthis segment is a piece of the extracellular grid outside theepithelial cells instead of being situated inside the cytosol.
B: Demonstratinga great deal more focused laminin recoloring toward the border of themesenchyme as opposed to in its inside. Instead of being concentratedonthe ventral border, laminin is currently present all around themesenchymal edge. This edge recoloring is not persistent in thisfigure, recommending that laminin isnot yet shown in acontinuous cellar membrane development around the neural tubeepithelium. Additionally, isolation and polarization of cells arebeginning to happen in this area, and a neural tube lumen seems, byall accounts, to be available in a simplestructure.
C:Demonstrates the last area from of the embryo stage 14, foremost tothe past two areas. The neural tube lumen is bigger and moreproclaimed, similar to the polarization of the cells into epithelialtissue around the lumen. The edge recoloring is more thought andceaseless than that found in the past areas. This proposes laminin isavailable in a continuous storm cellar membrane around the basal faceof the neural tube epithelium. It is still truant, in any case, fromthemost dorsal areaswhere there is still no reasonable division of an outskirt. Cellshape recommends proceeding with mesenchymal character.
In light of the substantialmeasure of this tissue dorsal to the lumina in photos B and C, wetheorize that these segments might be a part of what is known as the"cover zone." The cover zone is a portion of the neuraltube where the two neural tubes shaped by means of essential andauxiliary neurulation cover amid improvement. As expressed in thewriting survey, essential and auxiliary neurulation result in onenonstop lumen. In the cover zone, the essential lumen is shapeddorsal to the auxiliary lumen,and they both blend to frame the nonstop lumen later being developed.If separating was proceeded with anteriorly in this incipientorganism, an essential neural tube lumen would show up in the dorsalmass of cells, and afterward,a blendof the two lumina would be seen later in the improvement of thisfetus (Gilbert, & Singer, 20011).
“Stage 14 Embryo Fibronectin Staining.”
Despite the fact that notappeared in a figure, an alternate slide of the same embryo wasrecolored with B3/D6 and was inspected keeping in mind the end goalto think about the example of fibronectin and laminin amid auxiliaryneurulation. Polarization and arrangement of epithelial tissue areseen, alongside the isolation of cells to make various holes, whichwould in the long run mix and frame the auxiliary neural tube laterbeing developed. Not at all like laminin, which was available on theedge of the mesenchyme in the figures, has fibronectin showed upamong cells inside to the border. This internal area is more criticalthan what was found in the laminin areas.
Additionally, the fibronectinrecoloring is gathered more in the dorsal district of the neuraltube. This grouping of recoloring may go with the start of neuralpeak cells, which rise up out of an epithelial to mesenchymal changeon the dorsal side. Because of constraints of our procedure, standout area was envisioned with hostile to fibronectin recoloring.Consequently,the outcomes are not definitive with one and only segment to giveinformation.
Stage 18 Embryo Fibronectin Staining
The diagram bellow demonstrates asegment through a phase 18 developing life, in a foremost locale ofthe storage compartment neural tube, where essential neurulationhappens. The segment is a result of essential neurulation, in lightof the fact that a notochord is available. As expressed in thewriting audit, essential neurulation is instigated by the notochord,while auxiliary neurulation is most certainly not.
In this photo, fibronectin isdiscovered plentifully around the notochord, the dorsal aortae, andalong the dorsolateral part of the dermomyotome.Around the neural tube, nevertheless, the recoloring is not as smoothor extreme. Fibronectin is available at less extraordinary levels allthrough the mesenchyme and has a more fibrillar structure aroundthere of the area. Strangely, fibronectin recoloring is amassed atthe dorsal part of the neural tube, where neural peak cells arestarting to experience epithelial/mesenchymal moves. These areas forfibronectin are surely understood amid essential neurulation, and ourperceptions are in accord.
“Stage 18 Embryo Secondary Neurulation Succession.”
The diagram bellow demonstrates asuccession of five segments taken from a progression of twenty fromthe same stage 18 developing life. The succession begins from thetail bud and moves anteriorly.
A:Shows mesenchymal tissue at the back tip of the tail bud, not yetseparated into the neuralepithelium. Note that this picture is amplified 20X, as opposed to10X. In the photograph, inside, limited ranges of fibronectinrecoloring can be seen. The inside zones of recoloring give off animpression of being to some degree arbitrary in the example,and there is almost no recoloring on the border of the mesenchyme.
The most serious fibronectinrecoloring in this area has all the earmarks of being intracellular,instead of extracellular proposing that fibronectin is available onthe phones yet not yet emitted into the extracellular network. Thesecells might be discharging fibronectin. Nevertheless, the relativemeasure of fibronectin recoloring contrasted with alternate areas inthis arrangement is practically nothing.
C:Demonstrates a segment foremost to the past one, and elements alittle neural epithelium encompassed by mesenchyme. There isadditionally a little cavitation present amidst the neuralepithelium. We noticed that standoutcavity was seen amid the improvement of this optional neural tube.The recoloring design in the mesenchyme is available in the samearbitrary example found in the past areaand is not seen in bottomless sums in this locale. The mostfibronectin recoloring found in this area is connected with thelittle neural epithelium, not the mesenchyme. It is connected withthe fringe zones of the epithelium, and barely any recoloring isfound onthe inside ofthe epithelial cells. The recoloring is additionally not firmlyorchestrated around the neural tube epithelium.
E:Demonstrates another segment that is more foremost. Here aconsiderably bigger and even more vertically stretched optionalneural tube is seen. Once more, cavitations are found in themesenchyme, however despite everything they don`t give off animpression of being a piece of creating neuroepithelium. The mostthought fibronectin recoloring is in this district where the luminaare framing in the mesenchymal tissue. As found in the past area, theluminin and recoloring around them seem, by all accounts, to beirregular. There is light recoloring around the neural tube, and itis more amassed in the dorsal, neural peak cell area.
Laminin role in chicks` secondary Neurulation
Laminin is an importantextracellular glycoprotein in scaffolding and structure in about alltissues. The molecules frame basement membranes that are found inepithelial tissues` basal surfaces. Notwithstanding numerousdifferent capacities, the basement membrane gives a framework towhich the epithelial cells follow and organize.
Staining of laminin gave ageneral pattern during the entire process: laminin was available atlower intensities in the inside of the tail bud and at focused levelson the border. This example did not change in the examined areas.Hence, we presume that laminin is most gathered at the border of theneural tube with a specific end goal to advise different cells wherethe "edge" isand which way is outward. The low levels of recoloring present in themesenchymal inside demonstrate that all phones contain laminin intheir extracellular networks however not as much as the phones thatline the outside of the neural tube. Laminin marks the edge of theneural tube, and the cells there evidently emit it directionally: tothe basal face of the epithelium.
The directional discharge maylikewise assume a part in the polarizationof mesenchymal cells into the epithelium. Laminin is emitted to thebasal face of the epithelial tissue, where it polymerizes with otherlattice atoms to shape a basement membrane. As already talked aboutin the survey of writing, basement membranes give a platform to whichcells follow. On account of the neuroepithelium, cells dischargelaminin toward the border of mesenchyme. This permits mesenchymalcells to stick to the recently framed basement membrane, energize,and shape the auxiliary neural tube. The development of differentlumina would require a more unpredictable instrument.Nonetheless, itappears to be possible that the essential components are working.
Fibronectins` role in chicksSecondary Optional Neurulation
Fibronectin secreted from cellsand included in sorting out of the extracellular lattice, and thedevelopment, movement, and situating of cells. Amid neurulation,fibronectin is particularly required in neural peak cell movement andthe arrangement of the basement membrane. Amid the succession ofauxiliary neurulation concentrated on, fibronectin was available ingenerally inexhaustible sums in the neural peak cell district, aroundthe neural tube, and all through the mesenchyme (Hamburger,&Hamilton, 2012).
There are numerous similitudesbetween the mouse and the chick statement of fibronectin amidoptional neurulation. At the back tip of the tail bud, scatteredfibronectin recoloring was found, and this recoloring was foundparallel to the midline of the mesenchyme. In further foremostdistricts, a persistent limit of fibronectin recoloring wasdiscovered encompassing the neuroepithelium. This ceaselessrecoloring was particularly thick at the dorsolateral outskirt of theneuroepithelium: the neural peak cell district. Our discoveries wereconcordant with these: we discovered expanded recoloring in theneural peak cell locale however we likewise discovered obviouslybounteous measures of fibronectin in the mesenchymal areas on eitherside of the neural tube.
The staining example found inthese segments was especially similar to the one found in this study:a ceaseless ring around the neural tube with fixation at the neuralpeak cell district. This example recommends that fibronectin beidentified with basement membrane arrangement.However, even morestraightforwardly required in neural peak cell relocation andseparation.
Results demonstrate no patternchange of laminin through optional neurulationand it is most amassed at the edge of the mesenchymal and neuraltube. This recommends laminin might be included in defining the limitof the mesenchyme and neural tube. Its directional statement maylikewise permit the mesenchymal cells to enrapture and stick to thestorm cellar membrane shaped by laminin to frame the optional neuraltube. In spite of the fact that fibronectin was discoveredencompassing the auxiliary neural tube, it was more packed in theneural peak cell area.
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