Faculty of Biological Sciences, University of Leeds

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Evolutionary Developmental Biology Lecture 12

Epigenetic cascades and pattern (OVERHEAD)

So it looks as if a single epigenetic process, coupled with threshold responses not only initiates differentiation and morphogenesis of the nervous system, but also integrates the differentiation and morphogenesis of all the primary organ systems within the embryo. This concept of an epigenetic cascade is a useful one, - a single morphogenetic event setting off a string of others. If the epigenetic cascade could be modified to include or exclude certain events then the structure formed might be modified.

Epigenetic cascades (OVERHEAD)

Despite the utility of the concept we know very little about epigenetic cascades, and have only worked out the detail of a few of them. Besides the development of the embryonic axis we know something about the eye and ear, the teeth of fetal mice and the mesonephric kidney of the newt.

Induction of the sense organs

1. the amphibian eye

The classical story of the development of the eye is often quoted as an example of embryonic induction. In the future head the neural ectoderm is specified to form the three major divisions of the brain by the notochord. The forebrain has two fates (OVERHEAD), part of it develops into the cerebral hemispheres and the remainder grows out as optic lobes. These grow towards the overlying ectoderm, becoming optic vesicles and finally forming the optic cup. in the optic cup several different cell types differentiate, neural retina, pigmented retina, iris. Once to optic vesicle has contacted the overlying ectoderm the vesicle induces the ectoderm to bud off a lens vesicle which then rounds up and differentiates into the lens. The lens acts on the ectoderm overlying it to induce the transparent cornea instead of opaque skin.

So here is an elegant cascade, with one structure (the optic vesicle) inducing another (the lens) which in turn induces another (the cornea).

It is also a textbook interpretation of the truth. There are, in fact, many species of anuran and urodele amphibians in which the lens can form in the absence of the optic cup, prior interactions of the ectoderm with endoderm and/or mesoderm being sufficient. There are genera within which the eye is induced by the optic cup in some species but self differentiates in others. There is at least one species (Rana esculentia) where the lens self differentiates but the optic cup can still induce lens differentiation from mesoderm. We shall return to the evolutionary significance of this variation between related species later.

2. the amphibian ear

The situation here is slightly different from that of the eye. Instead of a cascade of different inducers producing different tissues we see here a number of inducors co-operating to produce a single structure, the cartilaginous ear capsule or otic vesicle. In Ambystoma punctata cranial ectoderm in the region of the hindbrain is induced to form an otic vesicle in two major steps. During early to mid neurulation cranial mesoderm induces cranial ectoderm. As this wanes in mid neurulation a second inductive wave is produced by the hindbrain. (OVERHEAD) Once the otic vesicle has formed it acts inductively on adjacent mesoderm to form the cartilaginous otic capsule.

Timing is obviously important in this sort of multi-stage process, and this is regulated by the formation of cellular contacts, including gap junctions, which appear only during the phase of induction. What regulates the timing of these cellular contacts is another question. Timing is also regulated by the acquisition of ectodermal competence to respond to induction. Ectoderm from the early neural tube will not respond to inductive influences produced by the hindbrain of late nerulae and ectoderm from beyond neurulation doesn't respond either.

Structures other than sense organs

1. Mammalian tooth induction.

Teeth (OVERHEAD) are composite structures made up of the outer white enamel which covers the teeth above the gums and the inner dentine, a different mineralised tissue forming the root and interior of the teeth. Dentine and enamel are extracellular products of two different types of cell, the ameloblasts (enamel) and odontoblasts (dentine).

Ameloblasts arise from epithelium, the oral epithelium. Odontoblasts arise in the neural crest. The neural crest loses many special populations of cells which migrate off to different places within the body: the pre-odontoblasts are one such group which migrate into the developing oral cavity and hence along the oral epithelium.

Odontoblasts are formed from these cells after a series of epigenetic interactions with the overlying epidermis. The critical factor is that the mesenchymal cells stop migrating and settle down against the buccal epithelium at the locations of the future teeth. We might like to ask ourselves why they stop precisely in those positions, but we shall come back to the whole question of spatial analysis later.

Dental mesenchyme (OVERHEAD) induces adjacent oral epithelium to proliferate (interaction 1). Dental epithelium then induces adjacent mesenchyme to proliferate to form a dental papilla (2). The dental papilla then induces the epithelium to form an enamel organ. If these two are artificially or experimentally separated from each other at this stage no further development occurs. If left in association the enamel organ induces the dental papilla to differentiate as preodontoblasts, which develop into odontoblasts (4). The odontoblasts and the predentine deposited upon them induces the preameloblasts to become ameloblasts and to deposit enamel (5,6). Again nothing further occurs if the pre-ameloblasts and pre-odontoblasts are separated.

So in both the ear and the tooth we have cascades of interactions following one another and building up a complex whole. In these examples all cascade steps are positive and promote differentiation or development. This is not necessarily always so.

2. The newt kidney

In the kidney of the newt (OVERHEAD) another complex series of interactions is known which starts in the early gastrula and continues late into larval life. These associations are, as one would expect between an epithelium and mesenchyme. At first there is an interaction between endoderm and gut mesoderm, then nephric ducts and kidney mesenchyme. Notochord also acts as a positive inducer. Somitic and lateral plate mesoderm however, initially stimulate, then inhibit, and the neural tube has a small inhibitory role throughout. The largest inhibitor is, however, the neural crest.

So how does this relate to the sort of thing we saw in the tooth? It's more complex: it isn't a sequence of one step interactions: it isn't simple series of ping pong interactions, it is a complex interplay of inhibitory as well as stimulatory events. What we have described as induction, and perhaps seen as a simple switching on, is in fact a complex sequence of interacting processes which may switch the induction off as well as on. On balance, over time they will result in the induction of a kidney: but there is enormous scope for tinkering. If we changed things a bit we might end up with a completely different organ!

On a larger scale - Oral and buccal development in the newt.

Lets now try to see how whole regions of embryos, rather than individual organ systems are formed. In the mouth region of the newt four distinct cell populations, (lateral mesoderm, the prechordal plate, the neural crest and the pharyngeal ectoderm (OVERHEAD) are all acting together to form mesenchymes and epithelia which interact together to make teeth, cartilages, bones, loose connective tissue and the various epithelia around the buccal cavity. Each element has its own epigenetic cascade, as described before, but the cascades are integrated: if not we might find a random mixture of structures such as that seen in a teratoma, where recognisable organs are formed in no particular order or arrangement.

In the newt (and other animals) the vomer, palatine, dentary and splenial bones (in the face) will not form until the trabecular and Meckel's cartilages (the cartilages of the first arch) have formed. Teeth will not form unless jaws are present. These are just a few examples of a widespread phenomenon: this interaction between cascades is important in both phylogeny and evolution. We can see immediately the importance of building the right things in the right order in an embryo, but it is just as important to see that this sort of constraint, the dependence of one system on another is what limits evolutionary possibilities. We can't have three eyes, or a nose on top of our head because these changes would influence other systems to an extent that would make the whole process of development untenable.

The tetrapod limb (OVERHEAD).

Once we can begin to understand the way in which regions like the buccal region develop we ought to be able to look at rather bigger bits, like the limbs.

We don't need to go into great detail, but the limb is a good example of morphogenesis, depending as it does on interactions between an epidermis and underlying mesoderm. Also limbs are quite variable structures: so as well as the way limbs develop lets look at some of the ways in which they might vary.

In all vertebrates the limb is organised first as a simple limb field in the flank. That is to say limb development is limited to a couple of regions, one anterior and one posterior (OVERHEAD). This wasn't always the case: some of the first fishes to have paired fins (which we can regard as homologous to limbs) had up to seven pairs (OVERHEAD).

In the putative limb regions a ridge of epidermis, the apical ectodermal ridge, develops as a line of rather tall cells (OVERHEAD). Beneath the ridge mesenchyme accumulates, setting up a classical ectoderm/mesoderm situation. Within the ridge dorsal - ventral and anterior - posterior axis quickly develop (experimentally verified by rotating cut off tips and seeing how they develop (OVERHEAD) and the limb characteristically becomes a footplate at the end of a narrower leg. The mesenchyme in the limb is complex in origin. In chicks it is relatively easy to show (by experiments using grafts from quail, which have a characteristic nucleolus, so that cells are easily identified) that the cells which will form the muscles originate in the somites, whilst skeletal and fibroblastic cells come from more lateral mesoderm. Innervation is rather late and segmental, but the number of segments involved in a limb, and which ones they are, is rather variable.

There is a general hypothesis, due to Saunders and Zwilling, that the apical ectodermal ridge is maintained by a mesodermal maintenance factor (OVERHEAD). The general characteristics of the limb are controlled by the mesoderm. Early in development a zone of polarising activity, or ZPA, develops on the posterior side of the limb bud. This has many interesting features, such as involvement with many theories of development, position effect, progress zones, retinoic acid etc. At present lets just regard it as an organiser region specifying a-p pattern.

 

All the cells in a developing embryo are part of a pattern, and each one has to be in the right place at the right time.

The early development of the egg was a good example of this. Whether regulatory or determinate the mechanism ensures a pattern (the blastula) with the right cells in the right place. In the determinate egg this is quite literal, cell 86 or whatever has to be there else the embryo won't have a kidney or a gonad. In the regulatory system the precise instructions have yet to be issued, but the right sort of cell, perhaps in terms of size, shape, stickiness, motility etc. is in the right area of the developing embryo. The influences which govern this early phase of pattern formation comes more or less passively from the distribution to daughter cells and clones of cytoplasmic determinants from the oocyte, from localised signalling via cell to cell contact and interactions from cell behaviour.

The blastomeres and their derivatives in fact behave as if they have knowledge of their positions in time and space. Horstadius (1973) showed that the cleavage patterns of sea urchin embryos follow a strict internal clock that seems to run on absolute time. Position sense is even more complex: the right cells end up in the right place. Wolpert (1969) suggested that cells have positional information. If a cell has positional information it must:

1. register its position in an embryo according to a set of signalling systems then remember this information

2. Allow subsequent cell behaviour to be governed by this information.

 

In everyday language a cell is given a password at a certain point or time, and the password determines the fate of the cell.

An alternative view is that there is a clock in the cell, and that at a given elapsed time from zero the cell records the values of external signals and these form the password.

In either case the cells must be responding to a pattern set up within the embryo. Or more accurately a series of patterns existing one after the other, and each generated by the one before.

This is difficult stuff, so let's relax bit with the idea of patterns and how they work.

Pattern (OVERHEAD) is the spatial relationships within a developing form. An arm and a leg contain much the same tissues, x % bone, y% cartilage, z% muscle, but a given structure is recognisable as an arm or a leg. The pattern is different, although similar. So how are similar cells arranged into dissimilar patterns, and why is one pattern preferred for the arm and another for the leg. And again why and how is the pattern in an aardvark different from that in a sloth?

We can suggest five main types of patterns (OVERHEAD)

1. Unit generated patterns.(OVERHEAD) The arhytypical unit generated pattern is that seen in an inorganic crystal. Here ions and atoms are held together by valency forces. The shape generated is very precise and crystals can be at least as large as cells. The secret is in the very precise arrangements of components and the valency forces which hold them together. But it doesn't need to be that uniform or that precise: globular protein molecules with two asymmetrical sticky patches on the surface shaken together would form a helix. If the units were cylinders, like insulin, the unit would be a four lobed fibre. Fibres can be monotype, made up of one sort of molecule, like collagen, or polytypic, like muscle fibres with actin and myosin. It is easy to see that a suitable unit might make up into a sheet.

2. Instruction generated patterns (OVERHEAD) It is also possible to make a structure from a series of units plus a plan. Think of a bricklayer with a pile of bricks and a plan building a house. The shape of the house is not governed by the shape of the bricks, but by the instructions in the plan. In this case the structure is generated diachronically i.e. slowly, but similar patterns can be generated very quickly. When the Sergeant-major shouts 'fall in' (instructions) a structure made up of individual soldiers (bricks) is generated very rapidly.

3. Template generated systems (OVERHEAD) In biological systems it seems most likely that a modification of the house building set-up is likely to occur. Instead of a scaled plan the architect could peg out the pattern of the house directly on the ground. This is just the same as a dressmakers pattern. The template is a structure made of dissimilar materials on or around which the structure is formed. Of course there is a philosophical catch in this: the template is itself a pattern - how do we generate it?

Again templates can be synchronic or diachronic. A diachronic example might be the formation of a hen's egg. Here the various layers of the egg are laid down sequentially by different regions of the oviduct. More usually they are synchronous. More important however is that the hen's egg is a non copy of the template. Nowhere in the hen is there the equivalent of a dressmakers pattern for an egg: nowhere is there an egg mould. The dorsal lip of the blastopore induces a neural tube: a neural tube is not a dressmakers pattern of a blastopore lip. So templates can produce non-copies as well as copies.

4. Condition generated patterns. (OVERHEAD) These are not related to the substance from which they are made, nor do they involve a set of instructions, but arise from the interactions of a series of spatially distributed conditions. Lets start with a cylinder of tissue and impose the following conditions

1. the cells are capable of differentiating into precartilage cells

2. when the density of these cells reaches a certain level they die

3. the threshold for death varies linearly.

Mesoderm differentiates into cartilage under the influence of oxygen concentration. Therefore a rod of precartilage cells will appear in the centre of the cylinder. At the end with the lowest death threshold cells will begin to die. If we impose another condition, that the cylinder is flattened a little at the end the rod of cartilage will in fact eventually split into two. What have we made? a limb with femur, tibia and fibula.

5. Positional Information. Wolpert applied this concept to the limb in order to demonstrate pattern generation (OVERHEAD). If a line of cells differentiates according to a gradient so that one third is red, one third white, one third blue the length of the line is unimportant. This might be applied to the various regions of the limb: however the number of thresholds for colour or region change is large.

In a structure like a limb different pattern generating methodologies will be acting at the same time. Collagen fibres in the extracellular matrix will be assembling because of the shape of tropocollagen molecules, protein will be forming on RNA templates and RNA will be forming on DNA templates in every cell. Mesoderm and ectoderm will be interacting to form non-copy template patterns.

Where is the pattern?

The pattern forming properties might be intrinsic, or extrinsic. Cells might have properties which predispose them to make patterns (like our proto-limb) or they might be responding to some sort of stimulus from elsewhere. If the stimulus is internal do all cells know about the pattern, or only some of them? We can simplify matters a little by sticking with our limb. Limbs have a limited number of tissue types. We can rule out the circulatory system because that develops late, after the patterns have been established. For the same reason the nervous system can be ruled out: perfectly shaped limbs can be formed with no innervation. The skin and its precursor the ectoderm is not responsible. The ectoderm, of course, interacts with the mesoderm to produce the outgrowth of the limb, but transplanting ectoderm from forelimb to hindlimb still leaves a hindlimb, and vice versa. So it looks as if the pattern lies in the skeleton or the muscles. Both skeleton and muscles form patterns and the patterns are related to each other: specific bones carry specific muscles. Is this the same pattern? In the region of the digits all the relevant cells are skeletogenic - no muscles, so the skeletogenic cells have the pattern: we could argue that the skeleton cells only have the pattern and impose this upon the incoming muscle cells. Or is it the other way around?

We don't really know, but at least we are narrowing the field. We do know that both types of cell form condensations. Are condensations the result of cell behaviour or receipt of a signal? In other words what sorts of behaviour do cells show to other cells?


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