Extrinsic Factors in Xenopus Laevis Development

INTRODUCTIONTeratogens atomic come up 18 chemicals, infectious agents, physical conditions, or deficiencies that, on foetal characterisation, behind alter fetal morphology or ensuant function (Chung, 2005). The act of a teratogen on the maturation beingness figures on what period in ontogeny the embryo is undefended to the teratogen (Jaeckel, 2001). The printing of a teratogen on the break danceing beingness overly depends on the dose and/or telling frequency of ikon of/to the teratogen (Jaeckel, 2001). a nonher(prenominal) factor that determines whether a specific teratogen march on be bruising is the genetic make-up of the developing organism (Jaeckel, 2001). Possessing or abstracted(p) certain genes makes the developing organism to a greater extent than than(prenominal) persuadable to the transaction of a teratogen. The purpose of this prove is to consider the effects of teratogens, whether noxious chemicals or physical environmental factors, o n embryonic 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 earliest(a) embryogenesis is considerable (Kay & Peng, 1991). The fertilized nut develops to the previous(a) blastula spirit level before significant transcription of the embryonic genome occurs (Kay & Peng, 1991). It fol busteds, therefore, that cytoplasmic stores of proteins and messenger RNAs accumulated during oogenesis atomic number 18 necessary by and by(prenominal)wards fertilization for victimisation of the embryo to the late blastula stage. The quadruplet compounds used as teratogens in this sample be retinoic deadly, atomic number 3 chloride, caffein, and deaf(p) spirits. Retinoic acid is k like a shotn to produce a minginess-dependent in series(p) of defects in anterior axial structu res that range from sm on the whole deletion! s to embryo insufficiencying heads (Altaba & Jessell, 1991). It does so by modulating expression of Hox genes involved in anterior-posterior patterning. lithium chloride is shown to induce dorsalization in early-stage embryos (Spenillo, 2001). Lithium chloride has the ability to moderate animal starch synthase kinase-3, which is a substance that initiates the wnt- bridle-path principal to dorsal axis physical composition (Spenillo, 2001). Lithium chloride in any case inhibits enzymes alive(p) 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 victimisation of Xenopus larvae in a concentration-dependent manner. It induces characteristic impertinent abnormalities, much(prenominal) as trim back body and wavy fins (SAKAMOTO, 1993). It was revealed that exposure to caffeine induced p ure(a) damage in the myotome and flighty underpass, and at in blue spiritser concentrations, the dermic tissue was also alter (SAKAMOTO, 1993). Finally, fermentation alcohol induces a behavioural dysfunction, such as the fetal-alcohol syndrome (Lindi et al., 2001). PROCEDURE(Obtained from testing ground protocol prepared by Dr. Plenefisch)This lab has a number of seven groups. Thus, twain groups analyse the effects of lithium chloride, primeval groups studied the effects of caffeine, two groups studied the effects of ethanol, and hotshot group, which was our group, studied the effects of retinoic acid. quaternion rough exposure conditions were picked, plus a lad (untreated) condition. Thus, five plates of embryos were prepared. quartet of them were treated with a compound, and one was untreated. each(prenominal) plate contained 4 embryos. Embryos were separated from the egg mass and washed in sterilized kitty peeing. Serial dilutions were performed to trans fer the compounds to the plates containing the embryo! s. Those are shown in protrude (1) below. The solvent in all of the tubes, shut 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 tried for m and concentration except the retinoic acid plate, which only advantageously-tried for concentration. After several(prenominal) sidereal age, the experimental and encounter 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 organic evolution were tested by exposing these compounds at diametrical times as well as in contrasting concentrations. Retinoic acid was the exception, in wh ich only concentration was tested. After calculated examinations of the experimental and fudge embryos, events were obtained and are presented in the tables below. Concentration interpositions are presented in fudge (1) and time treatments are presented in table (2). postpone (1): Variations of Concentrations of Teratogens submit Different effect on Xenopus learningUn diluted0.47% 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(a) locomotion. Lithium Chloride4/4 growing1/4, ontogeny halted after 6 days0/4, development halted after 5 daysN/A3/4 developedCaffeine4/4 living polliwogs3/4 living and 1 dead egg with mold1/4 living, 1 dead, and 2 molded ! eggs2/4 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 circuit board (2): Variations of Exposure Times of Teratogens gift 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 physical composition 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 than developedDI SCUSSIONAs previously 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 face up at the results obtained from the retinoic acid treatment. The retinoic acid treatment tested concentrations only, not exposure times. It can be seen that the more 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 l ittle development within the eggs in plate #4 and a f! unctioning 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 dupe received 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 bring in 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 both concent rations 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 trim back 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.

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 such as lack of pigmentation, lack of outer membranes, an! d even terminal 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 cogitate 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 formation of mold around the cells and limit development. Likewise, the results in Table (2) are inconsistent with the idea that viii-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 out-of-pocket 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 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 painting ON XENOPUS EMBRYO lipoid COMPOSITION.? Oxford Journals. N.p., 2001. Web. 26 Oct. 2009. . Mikoshiba, Katsuhiko, Takeo Saneyoshi, and Shoen Kume. ?desensitisation 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, hostel it on our website: OrderEssay.net

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