1. We learned about morphogen gradients and how a dominant negative receptor was
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1. We learned about morphogen gradients and how a dominant negative receptor was used in the neural tube to provided evidence that Shh functions as a a) Consider an organism that is very similar to Chick, but develops seven digits instead of three in their wing (Figure). Shh localization experiments have shown a gradient of Shh protein in the early limb bud (Figure, orange dots are Shh proteins). Describe an experiment using the dominant negative Patched receptor that would test whether Shh functions as a morphogen gradient in the limb of this species. (Note that a common way to express genes in the chick is to transduce with a virus expressing a gene of interest). Be sure to be specific about timing and localization during your experiment. Explain the predicted result if Shh is functioning as a morphogen (i.e. the resulting digit patterning) and precisely why the kev result supports a mophogen gradient mechanism rather than a signal relay. 1 2 5 4 Digits b) Would it be possible to use the same dominant negative receptor strategy to study whether the BMP gradients function as morphogens in the Xenopus? Can dominant negative receptors be used similarly for Bicoid in early Drosophila embryos? Explain your answer.Explanation / Answer
a.) Morphogens are thise molecules that act in na gradient specific manner to perform various crucial functions in development. The major work of morphogens has been done in Drsoophila embryonic development and limb development in mammals in well studied example of morphogen gradient.
In case of chick linb development, the formation of three digits involve gradient of shh morphogen which in a gradient specific manner defines which digit would act as first, second and third. In a simpler manner, if we try to understand this concept, the more the intensity of shh, the digit formed will be third, the middle it is the digit formed will be second and least or no amount, the digit formed will be 3rd. The manner in which shh works is a normal signal transduction pathway. Shh binds to its receptor patched in the cell, which allows smothened to go inside the cell and allow gli to go inside the nucleus to activate Hh regulated genes.
So if we have an organism like chick with 7 digits, and shh works in it or not the best experiment to work on is using a dominant negative of patched which implies that patched wont be expressing in the cells, so if at all shh plays a part in limb formation it will not be able to act as the receptor is not present. But this has to be managed tenporally because in case of chick when digit formation begans shh is present but as an embryo grows and digits are formed, it moves away from shh expression. Sio we will have to study what time of this embryo development digits are formed and try to use dominant negative expression of patched at that particular stage. For example:
1.) if digits start forming at Stage 6, digit 1 is formed at stage 6, 2 at stage 7, 3 at stage 8 and so on.
2.) We will have to use patched dominant negative at stage 12...to check what dies digit formation look like, if 1,2,3 digits are formed and there is defect in digits 4,,5 and digits 6 and 7 are not formed at all, it clearly suggests that shh was playing a role, similarly using patched doiminant negative at various staged from stage 6-12 can give us a proper understanding of what role does shh plays.
Things like digit formation prefers to use morphigen gradient beacuse the formation of digits requires a same fundamentals but different requirements at the different time points. So signaling relays will use more energy and can be more complicated may be requiring more time also.
b.) Yes it is possible to use dominant negative strategy for BMP gradient in Xenopus, but the only condition is the gradient of morphogens should give result to differnet but similar organs eg digits or it should be possible to modulate differnt cells in which BMP acts as a morphogen both spatially and temporally.
For Drosophila, Bicoid is a RNA, it does not bind to any receptor, so dominant negative strategy cannot be used to understand Bicoid function or expression pattern in Drosophila.
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