Presentation on theme: "Establishment of the Body Plan in Drosophila -In the early 1980s Christiane Nusslein-Vollhard and Eric Weischaus (top left and middle) were seeking to."— Presentation transcript:
Establishment of the Body Plan in Drosophila -In the early 1980s Christiane Nusslein-Vollhard and Eric Weischaus (top left and middle) were seeking to identify the complete set of genes that are required for proper segmentation of the Drosophila embryo. They treated flies with chemical mutagens and looked for mutants in which embryonic segments were either missing or transformed. - From these screens the two researchers identified the maternal effect, Gap, pair-rule and segment polarity genes that we discussed in the previous lecture. One of the mutant strains that they isolated produced embryos that contained two tail segments (bottom left panel). In this mutant the head is converted into a second tail segment – they called the gene bicaudal. Over the years nearly all of the genes that they identified in the original screens have been shown to be evolutionarily conserved all the way to mammals (including humans). For their efforts both were awarded the 1995 Nobel Prize in Medicine. - During our discussions of cell-cell communication we talked about the Toll, EGF Receptor and Hedgehog signaling pathways. These pathways were discovered in the Vollhard and Weischaus embryonic patterning screens. As we discussed each of these pathways is present in mammals and control a range of processes including the immune response, cell fate specification and tissue growth.
The Bithorax Complex in Drosophila - The 1995 Nobel Prize in Medicine was also awarded to Edward Lewis who is depicted on the top right image. He was awarded the prize for his contributions to our understanding of how homeotic genes established the body plan. In particular he conducted a genetic analysis of the Bithorax Complex - a set of three genes (Ultrabithorax, Abdominal-A and Abdominal-B) that specify fates of the thorasic and abdominal segments in Drosophila. - Mutations that reduce the activity of Ultrabithorax within the third thorasic segment lead to a homeotic transformation of the third throasic segment into a second thorasic segment. The end result is an animal with two thoraxes (hence the name) and four wings (lower left panels). It is worth noting that true flies differ from other insects like dragonflies and butterflies in the number of wings. This is due to differential regulation of Ultrabithorax. If Ultrabithorax is forcibly expressed within the second thorasic segment the wing is converted into a haltere. - As a side note Ed Lewis was a graduate student in the lab of Alfred Sturtevant who himself was a student with Thomas Morgan.
The Antennapedia Complex in Drosophila - Thom Kaufman and Walter Gehring made significant strides in furthering our understanding of how the body plan of Drosophila is established through their studies of the Antennapedia complex. Within this gene cluster lies five homeotic genes – they are called labial (lab), proboscipedia (pb), Deformed (Dfd), Sex comb reduced (Scr) and Antennapedia (Antp). The loss or mis-expression of any member of this complex results in a homeotic transformation of entire segments. - Antp is normally expressed in the segments that give rise to the legs and is absent from the segment that gives rise to the antenna. Loss of Antp expression leads to the transformation of the leg into a antenna. In contrast, expression of Antp within the antennal segment results in its transformation into a fully formed leg (above middle). - pb is expressed in the segments that give rise to the proboscis (the mouth parts of the adult fly). The loss of pb leads to the transformation of the proboscis into a set of legs. A fly that lacks pb and simultaneously has Antp expressed within the antenna will have ten instead of six legs (above right). - Thom Kaufman was a graduate student with Burke Judd who himself was a graduate student with Ed Lewis. Walter Gehring Thom Kaufman
The Homeodomain and Hox Genes - The genes that comprise the Bithorax and Antennapedia clusters are called Hox genes and each encodes a protein that can bind to DNA and either activate or repress transcription. Earlier in the semester we discussed several different types of DNA binding domains. All eight Hox proteins contain a homeodomain. These domains are 60 amino acids in length and are folded into three alpha helix structures. The homeobox was discovered by Bill McGinnis while he was in Walter Gehring’s lab and by Matthew Scott while he as in the lab of Thom Kaufman. - The third helix contacts the major groove of the DNA double helix (and is therefore called the recognition helix). Amino acids 47, 50, 51 and 57, which all fall within the third helix, make physical contact with the nucleotides within the major groove and provide binding specificity. Additional specificity is achieved by interactions between amino acids 3 and 5 (located within the N-terminal of the homeodomain) and nucleotides within the minor groove. Mutations that change the identity of these six amino acids can completely alter the binding specificity of the homeodomain. Of particular importance is the position 50 amino acid. - It is important to note that the homodomain is a very common DNA binding motif. All Hox genes contain a homeodomain but all homeodomain containing proteins are not Hox proteins. For instance the Pax6/Ey protein has two DNA binding motifs – a paired domain and a homeodomain. However, it is not a Hox protein despite the presence of the homeodomain. In order for a gene to be considered a Hox gene, the loss of that gene must be accompanied by a change in identity of a cell population or an entire tissue. The loss of Pax6/Ey results in the loss of the eye but not a change in tissue identity. Bill McGinnis Matthew Scott
Co-linearity of Hox Gene Position and Function - In the diagram to the left the position of each Hox gene on the chromosome is correlated with its expression pattern within the embryo and the identity of the adult structure that it controls. All eight genes are located on the third chromosome. One interesting fact is that the relative position of each Hox gene on the third chromosome is co-linear with its expression pattern in the embryo. For example, the lab gene is positioned more anteriorly than the Dfd gene on the third chromosome. Similarly, within the embryo the lab gene is expressed in cells that lie more anterior to the those that express the Dfd gene. This relationship is true for seven out of the eight Hox genes. The only exception to this rule is the pb gene (which is not diagrammed in this picture). - A comparison of the eight Hox genes indicates that there are significant similarities between them. This is not only true of the homeodomain but also in the other sections of the proteins as well. One model that has been proposed to account for this high level of sequence conservation is that the ancient Hox cluster actually consisted of a single gene and that over the course of evolution a series of duplication events has led to the birth of the remaining Hox genes. One additional idea is that the positions of the Hox genes on the chromosome (from anterior to posterior) actually reflect the order in which the genes were duplicated. For example, it has been proposed that the lab gene was duplicated to give us the pb gene which was further duplicated to give us the Dfd gene (and so on). - A prediction of this model is that organisms with a relatively few Hox genes have a simpler body plan than those with many Hox genes. This is generally true – insects such as segmented worms have very few Hox genes and they also have only a limited set of unique body segments.
Hox Genes in Flies and Mice - The eight Hox genes that are found within the Drosophila genome are also present within the mammalian genome (including mice and humans). Much like the Drosophila clusters, the mammalian organization of the Hox clusters is co-linear with their expression pattern within the embryo. Since the Antp and Bx complexes are found in both flies and mammals it indicates that these genes evolved prior to the split between the two organisms. - Each Hox gene is found in a single copy within the Drosophila genome. However in the mammalian genome each gene is found in at least four copies thereby suggesting that additional duplication events have occurred after the split in the lineages that gave rise to flies and vertebrates (including mammals). In addition the Abd-B gene has undergone a unique set of duplications – there are fifteen copies of that genes. In contrast some Hox genes have less then four copies (the Abd-A gene is one example). It is possible that chromosomal deletions have occurred.
Mammalian Homeotic Transformations - Mutations that disrupt the mammalian Hox genes also lead to homeotic transformations. In mice, the best studied Hox genes are the ones that affect vertebrae development. As you can see from the diagram above, mutations in different Hox genes lead to transformation of one vertebrae type into another. As you can see in the top right panel these transformation can be relatively mild. That is because there are several copies of each Hox gene thus there is functional redundancy amongst the genes. More severe defects can be observed if multiple Hox genes are simultaneously removed. - Another type of homeotic mutation that is seen (this time in human patients) is the ovarian teratoma. In the two examples to the right, a portion of the ovary has been transformed into epidermal and cranial tissue. As you can see there is a set of teeth in the far right ovary.
Molecular Biology Study Questions - What structural feature is found in all Hox genes? - What phenotypic characteristic accompanies all Hox genes? - What does it mean for Hox gene expression and organization to be co-linear? - What evolutionary mechanism underlies the current eight Hox genes? - How do the numbers of Hox genes in flies compare with that in mammals? - How does the number of Hox genes within the mammalian genome affect mutant phenotypes? - What would you expect to happen if the pb gene is forcibly expressed within the leg segments? - What would you expect to happen if the Ubx gene is forcibly expressed within the wing segment? - How do different homeodomain containing transcription factors bind to different target sequences? - The Pax6/Ey gene contains a homeodomain – is it a Hox protein?
Preview of Upcoming Lecture Topics to be Covered Next Time RNA interference (RNAi) The microRNA (miRNA) Pathway microRNAs and Development Textbook Chapter Chapter 20 pg. 712-729 Weekly Article(s) “The Evolution of Color Vision” “Ingenious” “Gene Therapy”