MCDB 423 Lecture 6: Lecture 6

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Molecular, Cellular and Developmental Biology
MCDB 423
John Kuwada

Lecture 6 Slide 1 We discussed the dorsal ventral patterning in the previous lecture (and the paper we will read will continue this) -What happens when cells see SHH (things that will be discussed on the paper) Now we’ll turn to patterning of the anterior and posterior axis We’re gonna consider just AP patterning in the brain This is an early vertebrate brain and is patterned along the AP axis In the most anterior portion, there is the telencephalon followed by the diencephalon and these two together make the forebrain Then it’s the midbrain -In the anterior end of the midbrain, there is the griseum tectalis (GT) which are just nucleus of cells -In the posterior, you get trochlear nucleus which are just motor neurons Then it’s followed by the cerebellum and the hindbrain There is a thin strip of cells that express a secreted protein called FGF8 (fibroblast growth factor) Prior to this type of axis, the nervous system is broken into the 3 sections (shown above) There are genes that are expressed in a patterned way in the AP axis -One particular genes are the engrailed genes 1 and 2 -These are transcriptional factors and expressed highly in the metencephalon but fall off in the midbrain as you go in the anterior direction How does this pattern get formed? As a note, the midbrain-hindbrain boundary is called the isthmus but he’ll refer to it as the mh boundary Slide 2 Recalled that for DV patterning, there’s a gradient that works in opposite directions So maybe the patterning of the AP axis may involve a similar gradient Slide 3 For a lot of reasons, they thought the mh boundary was a key organizing factor and this experiment showed that it actually is These experiments were done in chicks Donor tissue was derived from quail In the control experiment, a piece of the anterior prosencephalon that normally gives rise to the telencephalon was transplanted to the posterior prosencephalon which gives rise to the diencephalon -When you do this, you get a completely normal brain along the AP axis (has no effect) -The expression of engrail is normal too However if you do the same transplant except you take a piece of tissue found in the mh boundary and transplant it into the posterior part of the prosencephalon, what you get is a brain that looks abnormal -You get a telencephalon followed by a cerebellum (which were donor quail derived) and there was an induced midbrain -The midbrain actually had reverse AP polarity -Then there was a normal midbrain with normal AP polarity then the cerebellum and the hindbrain -The diencephalon is hidden by the midbrain -If you look at the expression of engrail 1 and 2, there is normal expression in the mesen section and a reverse in the prosen This indicates that the induced midbrain has reversed AP polarity This tells you that the midbrain-hindbrain boundary can induce posterior prosencephalon cells that normally become diencephalon to become midbrain instead -Furthermore it demonstrates that the mh boundary cells gives rise to the cerebellum slide 4 So what is the inducer? -It must be a gene that is expressed by the mh boundary If you look at the genes expressed early in development, there is a set of genes called otx1 and 2 which are transcriptional factors that are expressed by the prosen and mes and stop right at the mh boundary Gbx2 is expressed in the anterior position of the met -This gives rise to the anterior hindbrain and cerebellum -It’s anterior border is at the mh boundary In both sides of the mhb, there are two strips of cells -On the anterior side, you have wnt1 which are diffusible molecules and fgf8 which is found on the posterior side which is also a secreted diffusible molecule So is fgf8 the inducer? Slide 5 This experiment used a bead of fgf8 Instead of transplanting a region of the mhb, this time the hypothesis was that if fgf8 is the inducer for the midbrain, then we should be able to manipulate fgf8 and manipulate the induction of the midbrain So the experiment: -They took a bead and infused it with fgf8 and implant that bead in the exact place where the mhb tissue was grafted to in the previous experiment -When you do this and assay for engrailed 1 and 2, you see normal expression on the posterior side and in the diencephalon region, you see a reverse gradient of 1 and 2 which is what we saw when we grafted the mhb tissue If you let the brain grow, what you see is a brain that has a telen, fgf bead, a midbrain with reverse AP polarity, another midbrain with normal AP, then the cerebellum and then the hindbrain It was also seen that the host cells immediate to the bead expressed fgf8 and others expressed wint1 -These are the two diffusable secreted proteins that are released in the mhb -So this suggests that fgf8 and wnt1 is important for determining the mhb and determining the kind of inductive effects they have This is what you call a gain of function experiment Slide 6 So what about loss of function? What happens if you remove fgf8 -You can’t do a simple knockout because it plays a role in other parts of development -So you have to do a conditional knockout using the Cre-Lox system So the conditional knockout was made that spared the early expression but knocked it out later in the mhb So when you do that, you get a telen, diance, no midbrain, and no cerebellum -If you use a marker (in this case they use TH because it detects dopamine neurons) you don’t see them for the midbrain and anterior hindbrain -You don’t have to use markers for cerebellum because it’s an obvious structure So you lose the midbrain, cerebellum and the anterior hindbrain when you have no fgf8 The mes expresses otx1 and 2 so cells that express otx1 and 2 are competent to be induced by fgf8 to become the midbrain The anterior met expresses gbx2 and those cells are competent to be induced by fgf8 to become cerebellum and anterior hindbrain Slide 7 The fact that the fgf8 can induce the cerebellum, they took a closer look at the issue They took an fgf8 bead and put it into the middle of the mes If you implant the fgf8 bead in the mesencephalon (the original experiment implanted it in the posterior procenphalone), what you see is a midbrain with normal polarity, a cerebellum and an induced cerebellum -They know it’s induced because if you look at it with markers which are only expressed in the cerebellum (which in this case are punkiji cells, they can be labeled with an antibody slide 8 My answer: B Actual answer: D For the first segment of cells that release fgf8, it will be released to the right of the black bar. This means that you have high concentrations of it there and lower concentrations to the left of the segment and lower concentrations as you go to the right -High concentrations of fgf8 is equal to high concentrations of engrail 1 and 2 and low = low concentrations of 1 and 2 This is why the first one is a regular midbrain because you have low concentration of fgf8 and as you go right, it increases The second is a reversed midbrain because you’re going from a high concentration of fgf8 to a low concentration The third is normal because you’re going from low to high concentration Slide 9 Where fgf8 expressed is important -And since it’s expressed in the mhb, the mhb is important as well So if you look at the transcriptional factors surrounding the mhb, there are two sets: oxtx 1 and 2 are expressed anterior to the mhb and gbx2 is posterior If this is the case, if you manipulate their expression patterns, you should be able to shift the mhb and thereby change the AP patterning of the brain One of the easiest thing to do is do a double knockout of both factors -But if you do this, it leads to early lethality So instead of making a conditional knockout, maybe the two genes which are very similar in structure and generate proteins that are very similar to each other are redundant -So if we can decrease the number of wild type alleles then maybe we can spare the early function of otx genes but not the brain function To do this, they looked at a double heterozygote of otx1 and 2 and found that i
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