Autonomous specification leads to what kind of development? Regulative,and then the last one we discussed was the syncytial specification. So I told you syncytiummeans having a lot of nuclei in a common cytoplasm. One cell having multiple nuclei, so thatis the situation in the Drosophila embryo.
Initially, you have nuclear divisions without cytokinesis generating the kind of structures thatyou see here in the slide, and there we talked about the specification is based on where thegiven nucleus is located and why that is important because you have a concentration gradientset up there. Some components are more concentrated in the anterior, and then it slowlydecreases as you move towards the posterior and vice versa for some other components. Thisconcentration gradient is going to be the topic today.(Refer Slide Time: 01:36)
Some molecules function primarily based on their concentration gradient.(Refer Slide Time: 01:41)
That is what this French flag model by Wolpert tries to explain. So Wolpert has another well-known textbook on developmental biology. So this Scott Gilbert, I feel, is more rather better
organized for our purposes, and therefore we follow that, but there is another equally goodbook that is by Wolpert. So this is the same case as we learned in a different context, whereunderlying mesenchyme instructs the epidermis above it during skin development.Depending upon the mesenchyme that is being transplanted with an epidermis, you mighteither form claws or feathers and so on. Then we also saw the Newt and the frogtransplantation where the mouthparts being swapped. So if you remember that, you will findthis easy to understand. So the keyword, before even we start the class, is this morphogen.
So morphogens are molecules whose activity is directly dependent on their concentrationgradient. At different concentrations, they will have a different effect, so that is the hallmarkof a morphogen. So morphogen will not have the same effect at different concentrations. Soin today’s class, let us see the importance of the gradient and the concentration of amorphogen. This French flag model by Wolpert tries to explain the same.
Here in this French flag, you assume that the concentration of a given morphogen is highestnear the left end, and then it slowly goes down as you move away in the X-axis from theorigin. At different concentrations, it will induce a different fate for those cells.
So please assume that the whole flag is the morphogenetic field and let us say a group of cellsthat have the highest concentration is going to make the blue pattern. Then the cells in themiddle of the field where the concentration is intermediate will make the white pattern of theflag. Then when you have a very low concentration of morphogen like below the whitethreshold, it is going to make the red pattern. So now, we are going to do a transplantationexperiment of the American flag. So let us take this part with the horizontal stripes from theAmerican flag and transplant it to the blue part in the French flag and vice versa. We aremerely swapping the parts here.(Refer Slide Time: 05:08)
The result is shown here. So, we take cells from the American flag and put it in the corner inthe French flag, where it is going to sense the morphogen at its highest concentration. So thecells will not make the blue part of the French flag; instead, whatever instruction it alreadyhas based on that, it will make the structure as you see in the cartoon. If you consider this flag
as an embryo, in that part, it would have made the stars and blue color background, and thatis what it is going to do here. Similarly, when you take the cells from the French flag and putit here in the American flag, it is not going to make the horizontal portion; instead, it sensesthat the gradient is lower, and it makes the vertical strips of the French flag. So this is theFrench flag model that explains how morphogen gradients work.
So again, you can compare this with the fate specification during vulva development. So theP6.p cell that gets the highest amount of RTK signaling acquires the primary fate. If the sameP6.p cell gets slightly less signal when placed adjacent to the P4.p position, it will acquire thesecondary fate, and further away, it will acquire tertiary fate. If the tertiary fate cells werebrought to the highest concentration of signaling from the anchor cell, then it is going toacquire the primary fate.
So that is what you see here, so what it means is all these cells have a certain developmentalpotential, on receiving the right instruction, they could make this blue color tissue or thewhite color tissue or the red color tissue. So that instruction is determined by the gradient ofthis morphogen. So the highest morphogen means the same cells that make blue whentransplanted to another place with the lowest concentration gradient will make the red. Thisred and blue pattern making instruction is there in the French flag embryo, but that is notthere in the American flag embryo, which also responds to the same morphogen gradient.Still, its genetic programming that has happened in earlier steps instructs what it is supposedto make. Reciprocal transplant develops according to their final positions in the donor flag, sothis is the idea of morphogen and morphogen gradient. It is essential because it plays a lot ofroles in developmental biology.
So the critical point to remember is, morphogens are molecules whose activity depends ontheir concentration; at different concentrations, they will have different developmentaloutcomes. The fate they specify will be different at different concentrations. Morphogenmeans concentration-specific developmental specification.(Refer Slide Time: 10:03)
So here is an experiment to show how the morphogen gradient works. So this is done on theZebrafish embryo. So in the cartoon (A), in the surface ectodermal cells, you inject the nodalmRNA. So this is one of the morphogens here, so nodal protein’s gradient is essential. In thelateral view, it is seen on the top, and in the dorsal view, you see it on the surface. So fromthe center to one end, there is a concentration gradient of nodal.
So when you inject about 4 picograms (pg) of nodal mRNA into this cell and the nodalprotein produced diffuses across. When it reaches a certain gradient, it is going to induce acertain downstream target like the floating head, which is going to be induced and becomethe notochord. The adjacent cells to the right get much less nodal protein; thus, they startexpressing no-tail, which will become the muscles. So no-tail is the name of a protein. So, theone that receives the highest concentration is going to express goosecoid, and that will makethe head mesoderm. If these cells were swapped, then according to the concentration of thenodal protein, their fates will be swapped as well. So that is experimentally demonstrated inthe subsequent sections here. If you have far less nodal mRNA injected in these cells, theydirectly activate no-tail.
These are not difficult to understand; if you want to think chemically; think about the affinitybetween the protein and the DNA sequence. Suppose an enhancer-binding happens only at ahigher concentration, and if the affinity is low, then those enhancers will not be active at lowconcentration. The relative affinities can explain this; you do not need to invoke morecomplex chemical principles here. If you inject a whole lot, whether absolute value or relativevalue, here you are giving a really large amount (D). Like six or sixty does not matter, this
ten times difference does not matter, and all of them will activate goosecoid. At a highconcentration, they are all going to respond to it.(Refer Slide Time: 12:57)
So now we are going to do more testing, like controls to ensure, whatever we are interpretingon those experimental results are correct.
So, one thing to remember is induction and competence. So, not any cell is going to respondto the nodal protein. If you have a different set of cells that are not going to express the nodalreceptor, they are unlikely to respond to this concentration gradient (A). They lackcompetence. So only the diffusion of the morphogen is required and not adjacent cells; like,for example, in (A), the cell that underwent some developmental alteration and, as a result,existed as different kinds of cells will not respond to the signal. So they are not interferingwith the nodal diffusion. These competent cells in (B), when the concentration comes to theright range, become green and purple cells.
So the diffusion matters; the intervening cells, whatever they are, it does not matter. If wetake a different cell with the appropriate receptor (C) and put it at any position in this field ofthe nodal gradient, then appropriately, it should respond, and that is what you see with thistransplanted cell becoming green. To confirm that this is all nodal dependent not because youpricked a cell with a needle, you add lacz mRNA, and nothing happens. So the effect is onlywith the nodal. This is how you do control experiments.
In (B), the neighboring cells are not the cause for the activation of the floating head in thosecells. It is the nodal that diffuses and induces those cells to express the floating head. This isan important concept in developmental biology—morphogen and morphogen gradient andhow they activate a different set of genes and, therefore different fate specifications.(Refer Slide Time: 16:26)
So far, we have covered the very basic concepts and principles, now let us understand the
development of a particular system. Drosophila embryonic body patterning is one of the well-understood topics in developmental biology. So over the next two or three classes, we will
learn in detail about it.
To begin with, let us understand the organism. So we are going to start with the fly. So all ofus have seen insects, not particularly fruit fly but other insects like a beetle, etc. They all havevery similar body plans like the head, the thoracic and abdominal regions. They also have sixlegs and four wings etc. So Drosophila has two wings (figure a), and therefore this is adipteran insect.
So today, we are going to look at the abdomen region of Drosophila. The abdomen consist oftwo structures called the ovaries (figure b), and that is our topic interest today. So if you takethem out, they look like the one in figure c in the slide. So each ovary will have these kinds ofstructures called ovariole, as shown in the slide (one of the ovarioles is peeled out from theovary). Many ovarioles together form the ovary, and one of them is shown here in gooddetail. If you go to one end of an ovariole, you will see a structure shown in the slide (rightside bottom one).
In that, you have these somatic cells and inside that lies two germline stem cells GSCs. TheseGSCs divide asymmetrically; one cell will be closer to this area called the germline stem cellniche. The other cell is going to be away from it, and that is enough to start the asymmetry.So the one that is staying near the niche receives the niche signaling to remain as a stem cell,and the other that moved away differentiates to become a gamete.
So if you remember, Dpp is a member of the BMP family involved in TGF-β signaling inDrosophila. So the cells that receive Dpp will be GSC, and the one that does not receive Dppwill differentiate to make the oocyte. So that starting cell we call as cystoblast. Thiscystoblast will undergo four mitotic divisions to form sixteen cells with incompletecytokinesis; thus, they are linked. So these sixteen interconnected cells together we call acyst. So the developing cyst moves away and forms an egg chamber. So the place where theGSCs divide and cystoblast formation happens is a germarium.
So this is the following order: ovary, ovariole, germarium, and from germarium, you getthese cysts. As I mentioned earlier, cysts are one of the daughters of the germline stem cell;after that initial division, this cell undergoes four successive mitotic divisions withincomplete cytokinesis, and that is shown in the next slide.(Refer Slide Time: 22:44)
After the first and second division, cells 1 and 2 stay connected. Then these cells divide toform cells 3 and 4. Since cell 3 came from cell 1 and cell 4 came from cell 2; they remain
connected. Since, the cells 3 and 4 came from other cells, they are not connected. So thispattern continues till they form the sixteen cells.So there is this red color present in these dividing cells in figure (A), I will come back to it;let us first finish the cell division.(Refer Slide Time: 23:19)
As they keep dividing, they end up forming an interconnected set of sixteen cells where ifyou see the two cells 1 & 2 have the maximum connection. They are connected to four morecells while the others are connected to either one or two cells. So, the one that has the fourconnections will get the maximum cytoplasmic output from the other cells. Imagine thetranscription and protein production that happens from the nuclei of other cells; all will bemoving towards these cells. Finally, either one the two becomes the big cell (figure B), whichis the oocyte; the rest of the fifteen cells will become the nurse cells.
So let us go back to the germarium. The cell that will become the cystoblast is also called theoogonium (figure B). So, that oogonium undergoes the cell divisions and becomes bigger andbigger. The cytoplasmic output from all these fifteen other nuclei are all connected throughthese things called the ring canals. They all deposit the material in the oocyte, and the oocyteeventually grows larger. So I feel this understanding is essential to understand the patterningof the oocyte, embryo, etc.
So now, let us see about the red color, which is shown here as yellow (figure C). So this is amicrotubule kind of material called the fusome. This fusome expands through the ring canal;the arrow indicates from which cell it is coming. Figure C is a cross-section stained with actin
to mark the outside red color, and this yellow is the fusome. So this fusome helps in thetransport of materials to the oocyte. It has a protein called spectrin, so the fusome structure ismade up of these proteins called spectrin.
Now let us focus on what happens to this oocyte. In most organisms, the front-back, top-bottom asymmetry takes place in the zygote. For example, in C. elegans, it does not happen
in the oocyte. So oocyte is symmetrical when the sperm enters, the site of sperm entry marksthe posterior (same happens in humans), leading to a cytoplasmic rearrangement in theembryo. When the embryo divides into AB and P1, P1 gets the posterior cytoplasm, and ABgets the anterior cytoplasm; therefore, their fates are determined accordingly. So, theasymmetric distribution of components within the cytoplasm starts after fertilization in manyorganisms, but in Drosophila, this asymmetry starts even before fertilization.
In Drosophila, it starts earlier in the oocyte itself. So even before the sperm arrives, theoocyte is already preparing itself for the next generation. So, therefore, we are going to lookat how this oocyte forms its top-bottom and front-back.(Refer Slide Time: 28:49)
We have an understanding of this structure (A); now, I am going to introduce a set of somaticcells that surrounds the entire egg chamber called the follicle cells. These cells are presentthroughout the oocyte development, starting from the germarium. So please do not confusethem with nurse cells. Nurse cells are of GSC origin, which helps in nursing the developingoocyte. These follicle cells are not committed to any particular kind of follicle cell.
So now, let us see how the oocyte makes its top-bottom and its front-back. These nurse cellstransport many proteins and mRNA into the oocyte and one of them is a protein calledGurken. So the gurken mRNA that gets transported into the oocyte ends up localizingbetween the oocyte nucleus and these follicle cells.
So in this particular stage, the oocyte nucleus ends up being closer to the follicle cells thanthe nurse cells. The gurken mRNA that comes between these follicle cells and this nucleusthrough, yet unknown mechanism, gets translated only here. The green color dots are theGurken protein. So that is the starting of everything, this Gurken being made between theoocyte nucleus and these follicle cells.
So during these cell divisions, due to the orientation of the spindle, the nucleus ends up beingcloser to the posterior. So the Gurken protein made here is received by a receptor on thesefollicles called the torpedo receptors. That signaling ends up making them as terminal folliclecells, and now these cells are committed. Before this Gurken-Torpedo signaling, all thesefollicle cells are equal, but after the signal, these are now specified into terminal follicle cells.Now, these follicle cells will send signals back, as we saw in reciprocal induction andcompetence.(Refer Slide Time: 32:58)
And that signal is unknown, but as a consequence, the microtubules organizes itself in aparticular orientation. That unknown signaling from this terminal follicle cells brings thisprotein Par-1, partitioning defect protein in the C. elegans, its ortholog in Drosophila.
Remember, we have learned this Par-1; I did explain this. So we learned this while we werelearning about mutagenesis and mapping. In that context, I said one of the ways to confirmthe final ORF is by injecting the wild-type copy DNA or by injecting antisense RNA. Whilemapping one particular gene, they could not rescue the mutant with the wild-type DNA;instead, when they gave antisense, and as the control, the sense, and both affected. And thegene that they were mapping is par-1, partitioning defective.
So this Par-1 gets localized due to this terminal follicle cells signaling to the posterior in theoocyte (figure C). That reinforces this microtubule orientation, plus end to the one side andminus-end to the other side. So now, we could start calling the ends as the posterior andanterior. So you are going to have a minus-end at the anterior and a plus-end at the posterior.Now these microtubules that look like rail or road can be used for transporting mRNA andproteins. So the motor proteins kinesin and dynein latch onto it; for example, the oskarmRNA move towards the plus end in association with kinesin I, and dynein moves to theanterior carrying the bicoid mRNA.
And the Oskar that gets to the posterior gets translated there into Oskar protein, then thatagain starts setting up the posterior cytoplasm called pole plasm. And this Bicoid that goes tothe anterior end determines the anterior specification. During all these events, the size of theoocyte changes because nurse cells are continuously providing the required material, and as aresult, the oocyte cytoplasm grows and enlarges (D). In the oocyte, now the Bicoid will havea concentration gradient from anterior to posterior. Similarly, Oskar will have the oppositegradient. So this is how the front and back of the oocyte gets formed.
So it all starts with gurken being translated between the nucleus and this Gurken signaling thefollicle cells. As a result, they become terminal follicle cells, and only these terminal folliclecells produce the signal that organizes these microtubules. These microtubules areresponsible for this Bicoid and Oskar asymmetric localization. One thing that reinforces thismicrotubule formation and the terminal follicle signaling is the Par-1 localizing to theposterior. That Par-1 localization to the posterior again reinforces this microtubuleorientation. That microtubule orientation is critical for Bicoid going to the anterior and Oskargoing to the posterior and the rest of the anterior-posterior is a consequence of these two.
So in tomorrow’s class, we will see how the top and bottom are determined. So we have seenthe front and back, so we need to know the asymmetry between the top and bottom.