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Molecular mechanisms of spatial control of cell differentiation in Drosophila : (which may or may not be basically the same in vertebrates)
A) Spatial determination is accomplished in a series of different molecular signalling steps; B) At each step, transcription is controlled by the proteins coded for by the genes of the previous step. (those of the first step are mostly maternal!) C) Many of the m-RNA transcripts vary in concentration from one area to another in highly regular patterns (as detected by in situ hybridization)
D) Mutations in a given gene generally result in a failure of formation of whatever structure(s) The main families of these (fly) genes are these: 1) Polarity genes (examples include bicoid, nanos, etc.) extreme mutants have tails at both ends 2) Gap genes (krupple) extreme mutants have both sets of end structures, but lack middle structures. 3) Pair-rule genes (even-skipped, fushi terazu) mutants are missing every other segment 4) Segment polarity genes (engrailed) part of every segment is replaced by a mirror image of the rest. 5) Homeotic segment selector genes: mutations cause (certain) imaginal discs to differentiate into structures normally formed only by the imaginal disc of a different segment. In antennapedia, the antenna disc forms a leg instead; in bithorax, haltere discs form an extra pair of wings.
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(Please ignore where it says figure 15.14 etc. these refer to an earlier textbook) Bicoid RNA is transcribed by maternal nurse cells, and nanos RNAs by ovarian follicle cells, & transferred from to the extreme anterior end (bicoid) or extreme posterior end (nanos) of the oocyte! These are therefore said to be maternal effect genes. Translation of these stored RNAs result in diffusion gradients of the proteins (as drawn above) in the uncleaved early embryo. The bicoid and nanos proteins are transcription factors, and can bind selectively to the promoter regions of certain other genes, stimulating the transcription of some and inhibiting the transcription of others. They can also affect translation of certain messengers (for example, Translation of the hunchback gene m-RNA is selectively inhibited by the nanos protein). Combinations of stimulations and inhibitions of transcription cause the spatial distributions of the many different gap genes.
Likewise, the krupple protein (like all the other gap gene proteins) is itself a transcription factor: it binds selectively to sites in the promoter regions of still other genes, thus turning them "on" or "off" Imagine you are a cell somewhere inside a developing fly embryo, trying to decide which cell type to differentiate into! How to do it? Simply measure the concentrations of all these different gene products at your location. That's the idea. An analogy would be if you were lost in a city where there were dozens of chemical factories, paper mills, perfume manufacturers, cedar wood sawmills, etc. The combinations of smells at each location could tell you exactly where you were located. The role of your nose is played by the binding specificities for transcription factors of the DNA base sequences in the promoter sequences for the structural genes of the various luxury proteins. Got it? There are two sets of pair-rule genes: a primary set (including "even-skipped") and a secondary set (including fushi terazu). The messengers of both sets appear in 7 stripes! Normal embryos develop 14 segments, but mutants of either class of pair-rule genes fail to develop every other segment. For example, embryos mutated in the fushi terazu gene, form only segments #2,#4,#6,#8,#10,#12 and #14. Guess which segments fail to form as a result of mutations in the gene called "even-skipped"! Genes with bases sequences too similar to be a coincidence have been found in zebra fish embryos, and shown to be expressed in every other pair of somites! Segment polarity genes, such as "engrailed". Mutations in these genes cause part of every segment is replaced by a mirror image of the remainder of the segment. Transcription of the engrailed gene itself is stimulated by the proteins of both the primary and the secondary pair rule genes - hence the 14 bands! The engrailed gene product seems to help create or maintain the boundaries between "parasegments". Homeotic selector genes: Mutations cause certain imaginal discs differentiate to form what should have been formed by the imaginal disc of a different segment. The difference between follicle cells and nurse cells . In the ovaries of humans, frogs, flies, sea urchins, flies etc. the developing oocytes are surrounded and supported by normal-sized cells, called follicle cells. Oocytes are essentially buried in masses of follicle cells. In vertebrates (including humans) the process of release of oocytes from the ovary is by means of a blister-like fluid swelling among follicle cells adjacent to each oocyte.
These blister-like swellings are called "follicles". Nurse cells are something different, although they also assist in the development of oocytes. The development of each fly oocyte includes a sequence of four mitotic (nuclear divisions) in which the cells do not separate. This produces a cell with 16 nuclei. One of these 16 becomes the nucleus of the oocyte, and the other 15 the nuclei of "nurse cells"; So the 15 nurse cells still have cytoplasmic connections to the oocyte; and cytoplasm from these 15 nurse cells flows into the oocyte.
No kind of vertebrate has nurse cells, but many invertebrates do. Notice, however, that fly ovaries also have follicle cells, that surround the oocyte, in addition to having nurse cells. In other words, nurse cells are not something they have instead of follicle cells. Another important special feature of fly development (and of many other insects, but NO vertebrates), are imaginal discs , which are ~20 pairs of infoldings in the embryonic skin
When a maggot undergoes metamorphosis and becomes a fly (or a caterpillar metamorphoses into a butterfly): More about homeotic mutations : These cause one imaginal disc to develop into a structure that normally develops from a different imaginal disc
Examples include formation of a leg where the antenna should be: "antennapedia" The adjective "homeotic" was coined by Bateson in the 1890s, to refer to abnormalities like canine teeth developing where molars should be. But the first homeotic mutation (and therefore gene) was discovered by Bridges in Thomas Hunt Morgan's lab in 1915 (contrary to what the textbook implies; "mutant" dates from 1901 In flies, all these genes that produce homeotic effects when mutated mapped to the third chromosome, in two separate clusters of genes.
A TANTALIZING FACT WAS DISCOVERED!
The genes nearer the 3' end affected anterior development; This phenomenon is called "colinearity " Probably the mechanism will turn out to be very important and interesting, and there are many hypotheses on the subject. All the genes whose mutation can produce homeotic changes contained certain a 180 DNA base sequence, almost the same in each. This is called the "homeobox "
Obviously it codes for a 60 amino acid length of protein (180 divided by 3 is 60). These proteins all turn out to be transcription factors (function is to bind to DNA wherever there are certain base sequences), and the homeodomain itself includes the main part of the protein that binds to the DNA. Although flies have two clusters of such homeobox genes, most multicellular animals (even including most other insects) have equivalents of these same homeobox genes in one cluster. Flies are unusual in that the cluster got divided into two.
In frogs, mice, and humans, there are FOUR clusters of homeobox genes There are thirteen parologous groups, in the sense that each #4 is rather similar in molecular structure to each of the other number fours. In this sense, mice don't have an a8 or an a12.
3' 1 2 3 4 5 6 7 8 9 10 11 12 13 5'It turns out that the genes for many other transcription factors also contain homeoboxes. For example, bicoid contains a homeobox. Nevertheless, one does not say that bicoid is a hox gene. The name hox gene refers specifically to these ones that are in the clusters, and are members of one of the 13 parologous groups. In developing embryos of vertebrates, each hox gene is expressed just in certain parts of the embryo, along the anterior posterior axis
Gene a1 gets expressed further anterior than gene a2; With only one or 2 exceptions, the anterior borders of the parts of the embryo where each hox gene is expressed are in the same order as the locations of the genes on the chromosome.
As was mentioned before, this phenomenon is called colinearity. Actually, nobody knows why colinearity occurs, either in a functional or a causal sense. In fly embryos, the proteins coded for by the other 4 families of genes (like bicoid, gap genes, etc.) are supposed to control which homeobox gene is expressed where.
But mammals and other vertebrates don't have an equivalent to bicoid nor direct equivalents to gap genes; maybe we have equivalents to some pair-rule genes, (to control somites)
Retinoic acid can shift the boundaries where hox genes are transcribed.
Because retinoic acid can change where in the embryo these and other genes are transcribed, it can therefore cause major birth defects ; it is a teratogen Many people suspect that retinoic acid may serve the a-p control function that the bicoid protein, and the gap proteins supposedly serve in flies. A hypothesis that occurred to me (but that I have never published) is that perhaps vertebrate cells can (somehow) detect whether neighboring cells are expressing the same hox genes as they are, and react to differences, for example, if a cell expressing only #1 finds itself directly in contact with cells expressing #3, then the cells along this boundary activate #2 If cells behaved that way, then you could start with a cell expressing #1 up against cells expressing #13. Then all the others would automatically get filled in. In embryology, a key question is often whether patterns arise from local responses to some gradient or other large-scale variable or whether patterns result from the sum of many close-range stimuli. The cellular automata computer program that was studied in one of the laboratories demonstrates that large scale patterns can be produced by lots of short range effects.
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If a "knock-out is made for Hox #3 (for example) then the cells that should be expressing 1, 2 and 3
can't tell the difference whether they might instead be the cells that only should express Hox 1 and Hox 2. Therefore the phenotype is that two adjacent regions form the SAME set of structures; Specifically, the most anterior segment that normally expresses #3 FAILS TO FORM and is replaced by a DUPLICATED (extra) version of the structures that normally form in front of the anterior border of expression of the missing (knocked-out) hox gene.
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