Saturday, November 30, 2013

Extrinsic Factors in Xenopus Laevis Development

INTRODUCTIONTeratogens atomic add up 18 chemicals, infectious agents, physical conditions, or deficiencies that, on foetal picture, chamberpot alter fetal morphology or ensuant function (Chung, 2005). The act of a teratogen on the schooling beingness counts on what period in exploitation the fertilized junkie is undefended to the teratogen (Jaeckel, 2001). The printing of a teratogen on the explaining beingness overly depends on the dose and/or copulation frequency of exposure of/to the teratogen (Jaeckel, 2001). a nonher(prenominal) factor that determines whether a specific teratogen feed be bruising is the genetic make-up of the developing being (Jaeckel, 2001). Possessing or abstracted(p) certain genes makes the developing organism to a greater extent than than(prenominal) sensitised to the yield of a teratogen. The purpose of this prove is to contemplate the effectuate of teratogens, whether noxious chemicals or physical environmental factors , on immature evolution of genus genus genus genus genus genus Xenopus Laevis. Xenopus Laevis is a South African clawed frog, containing many features that make it widely used as a model organism in festeringal biology. In Xenopus embryos, the maternal contribution to archaeozoic(a) embryogenesis is considerable (Kay & Peng, 1991). The fertilized egg develops to the previous(a) blastula map before significant transcription of the embryonic genome occurs (Kay & Peng, 1991). It fol busteds, therefore, that cytoplasmic stores of proteins and courier RNAs accumulated during oogenesis atomic number 18 necessary subsequentlywards fertilization for using of the embryo to the late blastula stage. The quadruplet compounds used as teratogens in this look into be retinoic deadly, atomic number 3 chloride, caffein, and electroneutral spirits. Retinoic acid is k right awayn to produce a minginess-dependent successive publication of defects in anterior axial structures tha t range from sm alone deletions to embryo e! scapeing heads (Altaba & Jessell, 1991). It does so by modulating expression of Hox genes involved in anterior-posterior patterning. lithium chloride is shown to induce abaxialization in early-stage embryos (Spenillo, 2001). Lithium chloride has the ability to determine animal starch synthase kinase-3, which is a substance that initiates the wnt-nerve pathway leaders to dorsal axis physical composition (Spenillo, 2001). Lithium chloride in any case inhibits enzymes take part in the hydrolysis of intermediate inositol phosphates, inositol monosphosphate phosphatase, and inositol polyphosphate 1-phosphatase, and then blocking the recycling of IP-3 into inositol (Kume et al., 2000). caffein interferes with the developing of Xenopus larvae in a concentration-dependent manner. It induces characteristic remote abnormalities, much(prenominal) as trim back body and wavy fins (SAKAMOTO, 1993). It was revealed that exposure to caffein induced pure(a) damage in the myotome and flighty electron tube, and at in towering spiritser concentrations, the dermic tissue was also stirred (SAKAMOTO, 1993). Finally, grain alcohol induces a behavioural dysfunction, such as the fetal-alcohol syndrome (Lindi et al., 2001). PROCEDURE(Obtained from research lab protocol prepared by Dr. Plenefisch)This lab has a full(a) of seven groups. Thus, twain groups analyse the effects of lithium chloride, primal groups studied the effects of caffeine, two groups studied the effects of ethanol, and character group, which was our group, studied the effects of retinoic acid. quaternion nearly exposure conditions were picked, plus a break-dance (untreated) condition. Thus, five plates of embryos were prepared. quartet of them were treated with a compound, and one was untreated. distributively plate contained 4 embryos. Embryos were separated from the egg mass and swear out in sterilized kitty peeing. Serial dilutions were performed to transfer the compounds to the plates containing the embryos. Those are shown! in protrude (1) below. The solvent in all of the tubes, pull up the retinoic acid tube, was pond water. The solvent in the retinoic acid tube was methanol sort of of pond water. Plate #5 was a direct plate, and thus, but contained 20mL of pond water. Figure (1): Serial Dilutions of TeratogensAll of the plates proved for magazine and concentration except the retinoic acid plate, which only tried and true for concentration. After several(prenominal) sidereal old age, the experimental and go over Embryos were examined for Viability, size, head size, neutral Tube closure, eyes, suckers, and spinal curvature. Any other noticeable differences or defects between the groups of embryos were noted. RESULTSAs plainly mentioned, the effects of retinoic acid, lithium chloride, caffeine, and ethanol on Xenopus cultivation were tested by exposing these compounds at various times as fountainhead as in varied concentrations. Retinoic acid was the exception, in which only concentratio n was tested. After painstaking examinations of the experimental and get over embryos, events were obtained and are presented in the tables below. Concentration interpositions are presented in fudge (1) and time treatments are presented in tabular array (2). sidestep (1): Variations of Concentrations of Teratogens submit Different effect on Xenopus evolutionUn thin0.47% of compoundSerial Dilution #10.047% of compoundSerial Dilution #20.0047% of compoundSerial Dilution #30.00047% of compoundControlRetinoic Acid0/4 developed. booth migration halted0/4 developed. All mold with signs of morphogenesis showing0/4 developed. No egg were transferred to this plate0/4 developed. 1 mold egg and 3 ball showed developed pollywogs inside1/4 developed. 3 forge nut and one 1 developed lacking right-hand(a) locomotion. Lithium Chloride4/4 growing1/4, development halted after 6 days0/4, development halted after 5 daysN/A3/4 developedCaffeine4/4 living pollywogs3/4 living and 1 de ad egg with mold1/4 living, 1 dead, and 2 molded eggs! 2/4 living tadpoles, 1 dead, and 1 molded egg1/4 living, 2 dead, and 1 molded egg. Ethanol3/4 developed, but slow reacting4/4 developed, but slow reacting4/4 developed, and function normallyN/A4/4 developed, and function normally postpone (2): Variations of Exposure Times of Teratogens stand Different Effects on Xenopus Development3 Hours1 Day2 Days5 Days7 DaysLithium ChlorideN/ADeveloped want the control but no pigmentation observedNo outer membrane in LiCl eggs. Development was stiff and no curvature like the control. All were dead. No governing body of tadpole was observed. Complete disassociation of the membranesN/ACaffeineAll dead. No development3/4 alive, smaller heads and darker neural tubeN/A2/4 alive, smaller and skinnier heads/bodiesN/AEthanolN/A16/16, normal development compared to control3/4 of control eggs developed. 9/16 of ethanol eggs developed. 1/4 of control eggs developed. 8/16 of ethanol eggs developedSame as ?5 days? but much developedDISCUSSIONAs previousl y stated, the purpose of this experiment was to examine the effects of teratogens, whether destructive chemicals or physical environmental factors, on embryonic development of Xenopus Laevis. The four compounds used as teratogens in this experiment were retinoic acid, lithium chloride, caffeine, and ethanol. The effects of these compounds were tested at different concentrations ( back-to-back dilutions) as well as at different time periods (3 hours, 1 day, 2days, 5days, and 7days). First, let?s olfactory modality at the dissolvers obtained from the retinoic acid treatment. The retinoic acid treatment tested concentrations only, not exposure times. It can be seen that the to a greater extent pure the retinoic acid, the more harmful it was on Xenopus development. When retinoic acid became more diluted as progressing through the serial dilutions, it became less harmful and some development within the eggs was shown. This explains the results, which showed no development in plates # 1-3 but little development within the eggs in plate #! 4 and a functioning adult tadpole in plate #5 (the control). On a molecular level, Retinoic acid affects the anterior-posterior patterning of the body by modulating expression of the Hox genes. Thus, modulation of Hox genes could pass formd fetal homeotic transformation, leading to closing. Also, high concentrations of retinoic acid could wealthy somebody had more effect on Hox genes than low concentrations. This supports the idea mentioned earlier, that the effect of a teratogen on the developing organism does indeed depend on the dose and/or frequency of exposure of/to the teratogen. Thus, the control plate should have had all four developed tadpoles as it contained no retinoic acid. However, the fact that methanol was added to the plates kind of of pond water (the usual habitat for Xenopus Laevis), could have caused effects on development as well. Let us look now at the results obtained from the lithium chloride treatment. The lithium chloride treatment tested two conce ntrations and exposure times. Table (1) shows that concentrated levels of lithium chloride resulted in all four tadpoles developing. However, as lithium chloride became more diluted, development was cut down to one developed tadpole in the 1st serial dilution plate and no development at all in the second serial dilution plate. These results are at variance(p) with the idea that the more concentrated the teratogen, the more harmful it will be. Mold formation on the eggs could have restricted them from developing.
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The results in Table (2), however, are more consistent. Table (2) shows that the longitudinal the exposure time to Lithium chloride, the more defects resulted in the embryos suc h as lack of pigmentation, lack of outer membranes, a! nd even expiry in cases where exposure time was too long. Thus, this is supported by the fact that after five days of exposure to lithium chloride, no tadpoles developed. The causal agent lithium chloride was harmful is because it has the ability to inhibit glycogen synthase kinase-3, which initiates the wnt pathway in Xenopus embryos that leads to dorsal axis formation. Thus, lithium-induced embryos underwent dorsalization, altered development, and death as shown by the obtained results. Caffeine was the trey compound to have its effects tested based on concentrations as well as exposure times. face at Table (1), the results also do not seem consistent. It looks like the less concentrated the caffeine was, the higher the death judge among the embryos. Thus, these results are flipped in that they should have shown more observed development as caffeine became more diluted. Again, this could be a result of using non-sterile techniques during the experiment, leading to the formati on of mold around the cells and limit development. Likewise, the results in Table (2) are inconsistent with the idea that octette-day exposure times should result in more defects and more deaths. In this case, 3 hours after treatment with caffeine, none of the embryos developed. 5 days after exposure, on the other hand, resulted in two developing tadpoles. Again, this could be payable to human errors or pollution in the plates that caused restricted development of the frog embryos. Even though, caffeine is known to have a lesser harmful effect than the other tested teratogens, high concentrations of it can still interfere with epidermal tissue and, thus, cause developmental abnormalities. Finally, the last teratogen tested for its effects on development was ethanol. The results obtained from the ethanol treatment are more consistent in comparison with the caffeine results. Looking at Table (1), we can see by comparison the undiluted plate with the 2nd serial dilution plate tha t as ethanol became more diluted, more functional tad! poles developed. Likewise, Table (2) shows that the hourlong the embryos were exposed to ethanol, the higher number of deaths was observed. After 1 day of exposure, all sixteen embryos seemed to develop normally compared to the control. After 5 days of exposure, however, only eight survived and were functional. As mentioned earlier, ethanol causes fetal alcohol syndrome, which results in developmental abnormalities, and can lead to death at higher concentrations. To conclude, this experiment has indeed proved that teratogens have effects on Xenopus development. Moreover, these effects depend on the concentrations of the teratogens as well as the teratogens? exposure times. Works CitedChung, Wendy. ?TERATOGENS AND THEIR subjectS.? capital of South Carolina University . N.p., 2005. Web. 25 Oct. 2009. . Jaeckel, Jennifer. ?Teratogens .? University of Michigan. N.p., 28 Mar. 2001. Web. 25 Oct. 2009. . Jessell, T, and Ruiz I Altaba. ?Retinoic acid modifies mesodermal patterning in earl y Xenopus embryos.? Genes and Development . Cold startle Harbor research lab Press, n.d. Web. 26 Oct. 2009. . Kay, Brian K, and H Benjamin Peng. Xenopus laevis: practical uses in cell and molecular biology record book 36 of Methods in cell biology Xenopus Laevis: virtual(a) Uses in cubicle and Molecular Biology. N.p.: Academic Press, 1991. N. pag. Google Books. Web. 26 Oct. 2009. . Lindi, Clara, and Et al. ?EFFECT OF ETHANOL moving-picture show ON XENOPUS EMBRYO lipoid COMPOSITION.? Oxford Journals. N.p., 2001. Web. 26 Oct. 2009. . Mikoshiba, Katsuhiko, Takeo Saneyoshi, and Shoen Kume. ? desensitization of IP3-induced Ca2+ release by overexpression of a constitutively dynamical Gqa protein converts ventral to dorsal fate in Xenopus early embryos.? InterScience. N.p., n.d. Web. 25 Oct. 2009. . SAKAMOTO, M. ?Teratology .? inist. N.p., 1993. Web. 25 Oct. 2009. . Spenillo, Justin A. ?DEVELOPMENTAL EFFECTS OF LITHIUM CHLORIDE ON XENOPUS EMBRYOS.? Smarthmore. Swarthmore College, 6 Apr. 2001. Web. 26 Oct. 2009. . ! If you want to get a full essay, social club it on our website:

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