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Developmental Biology BY1101 P. Murphy Lecture 10 Master Regulatory genes: The genes that control development. This lecture dealt with • Identifying developmental regulatory genes at the molecular level: What kind of molecules do the genes that guide Drosophila development encode? How might they carry out their functions? • Are there similar genes in other animals? • It opened the question of what the similarities and important differences are between development in fruitflies and humans? __________________ So what are developmental regulatory genes? We began by considering the following as a possible classification of all genes in the genome: (1) Structural genes (2) “Housekeeping genes” (3) Regulatory genes. The structural genes provide the building blocks for the organism (or house as analogy). The housekeeping genes look after the everyday running of the organism. The regulatory genes are the genes needed to govern the construction of the organism. These genes could be called “The genetic toolkit for development” _______________________________________________ In lecture 9 you heard about the classical genetics approach and the mutational screens that first identified the presence of developmental regulatory genes in the fruitfly Drosophila. They found a number of different Catagories of Drosophila developmental genes (with sequential activity): •Maternal effect genes ↓ •Segmentation genes 1. Gap genes 2. Pair rule genes 3. Segment polarity genes ↓ •Homeotic genes ↓ other Drosophila genes (e.g. structural genes) Hierarchical relationships often exist between genes across groups e.g. maternal effect genes control segmentation genes. Within segmentation genes, particular gap genes might control particular pair-rule genes etc. But the mutational screens that identified the genes above didn’t automatically place the genes in the hands of the researchers. Rather, they showed the existence of the genes and their approximate location on the genetic map. The next step was to actually isolate the genes. The burning question at the time was, what kind of proteins are produced by genes that have such a profound effect on the development of the body plan. Among the first genes to be cloned (physically isolated and reproduced in the laboratory) were 2 homeotic genes. This was achieved using biochemical techniques and the knowledge of their approximate location in the genome. Note: The first Drosophila developmental genes to be isolated (cloned) were cloned based on known position on the genetic map (their position within the genome). When these first homeotic genes were cloned and analysed, it was seen from their DNA sequence that they encoded transcription factors, proteins that can regulate the expression of other genes. Each gene contained within it, a similar DNA sequence of 180bp that encodes a 60 amino acid domain in the protein that can bind to and regulate the control regions of other (target) genes. This region of DNA was given the name the homeobox. The domain within the encoded protein is called the homeodomain. The homeodomain has a structure of three helices that bind directly to regulatory sequences of target genes influencing their expression Homeodomain Protein bound to DNA of target gene One alpha helix fits neatly into the major groove of the DNA double helix. Other more variable parts of the overall protein (outside the homeodomain) determine what other proteins it interacts with how it and will influence gene transcription. ___________________________________ Not all homeobox-containing genes are classified as homeotic genes: Other classes of Drosophila developmental genes also have homeobox sequences. • • • The maternal effect gene bicoid encodes a homeodomain transcription factor. The products of many segmentation genes are also transcription factors. BUT NOT ALL Other segmentation gene products (proteins) operate more indirectly- – Some are components of cell-signaling pathways and are involved in cellcell communication (lecture 8)- therefore instructing neighbouring cells to change the genes they express. This is particularly true of the later acting segmentation genes- segment polarity genes. It makes sense that the later acting segmentation genes could act through producing signaling molecules and receptors as well as transcription factors. Why? -At early stages of segmentation there are no cell membranes so no need for cell signaling. After cellularisation, systems are needed to signal between cells to ultimately define and refine the segmental units. So The genes that direct Drosophila development, when cloned, were found to encode 1. Transcription factors that regulate the expression of sets of target genes: Homeodomain transcription factors. Other types of transcription factors that use different protein motifs to bind DNA and regulate transcription. 2. Signalling molecules that could be responsible for cell to cell communication. 3. Other components of signalling pathways (e.g. receptors) also used to mediate cell communication. Once the genes were cloned, their expression patterns in the embryo could be investigated by in situ hybridisation. This revealed that segmentation genes are expressed where segments will form, appropriate to their mutant phenotype. A gap gene; the expression of Krupple A pair rule gene: the expression of hairy A segment polarity gene: the expression of engrailed Homeotic genes are like other developmental genes in encoding transcription factors that control the expression of specific target genes. In the case of homeotic genes they must control genes responsible for the generation of anatomical structures (either directly or indirectly). We can use this to explain homeotic mutations. For example, the fly segment T2 (thoracic segment 2), expresses a particular transcription factor which identifies or labels the cells as T2. This is involved in turning on the genes needed for the production of legs on this segment. In a mutant, the T2 identifier gene might be expressed incorrectly in a head segment, inappropriately labeling this segment as “thoracic” instead of “head” and causing the eventual production of legs in the place of antennae. ___________________________________________ When the expression of the cloned homeotic genes was investigated it was found that the genes are active in a sub set of segments where they contribute to segment identity (i.e. they don’t work alone but in combination). Many of the homeotic genes were found to be clustered together in the genome Segment identity is determined by the unique subset of homeotic genes expressed in each segment. They encode homeobox transcription factors of a particular type and these important genes (remember what happens if they are mutated) were given the name Hox genes The cluster of Hox genes also showed the phenomenon of Colinearity: Position of the gene in the cluster reflects position along the AP axis where the gene is expressed (and position where the gene functions). The reason for colinearity is not fully understood but is a consequence of the mechanism of regulating where in the embryo the genes are expressed. _________________________ Accessing developmental genes in other organisms Because the homeobox sequence was found in several Drosophila developmental genes, its importance during development was very quickly realised: ↓ Perhaps it is found in several different developmental regulatory genes because it is of key importance to developmental mechanisms i.e. it has been perfectly conserved through evolution in different genes. If so, then perhaps similar sequences exist in developmental genes in other organisms i.e. the sequence has been conserved in developmental genes in different species that arose from a common ancestor because it plays such an important role during development. ↓ Molecular techniques were used to search for similar genes in other organisms. However, nobody could have imagined in advance, the extent to which these genes have been conserved during evolution. Homeobox genes have been highly conserved during evolution – – – – Homeobox related sequences are found even in yeast and plants as well as all animals, so it is a very ancient type of regulatory gene. The homeotic genes of Drosophila that include the homeobox, fall into a particular category of homeobox genes- the Hox genes. Hox genes are a subset of all homeobox-containing genes particularly involved in positional information which are also clustered in the genome. Genes very similar to these homeotic genes (Hox genes) in Drosophila are found in all animals from the simple jelly fish to the human, and all levels of complexity in between. Hox genes are found in all animals with an organized body plan This means that the Hox genes appeared with the task of organising the body plan of an animal (Hox genes as a subclass of homeobox genes, are not present in yeast, only in animals with an organized body plan i.e. head end distinguished from tail end etc). They are so valuable that they have been conserved in animals for hundreds of millions of years. Hox genes are a particular subfamily of homeobox containing genes that evolved together with a complex multicellular body plan. They have been utilised to convey positional information and organise the body plan 3 Features of Hox genes. 1. They contain a sub-class of highly conserved homeobox sequences, so they encode transcription factors. 2. They are involved in organising the body plan of an animal. 3. They exist in clusters of similar genes in the genome. As shown above, Drosophila Hox genes are clustered in the genome. Vertebrate Hox genes are also clustered. See Fig. 21.18 Campbell and Reece In mammals there are 4 clusters. Mammalian clusters can be aligned with the fruit fly cluster based on most similar sequence. So: •DNA sequence is conserved •And chromosomal arrangement of the genes is conserved. •They have also conserved the order and relative position along the AP axis of the embryo where they are expressed and function (colinearity) The genes are in fact so closely similar that the mouse version of one gene has been transferred to the fly and could rescue the effect of a lethal mutation in the fly’s own gene. But when Hox genes are mutated in vertebrates like the mouse, the dramatic phenotypes seen in the fly are not produced (i.e. no mutant mice with legs on their head). But neither do mice nor humans have a fully segmented body plan; it is more complex. One place where we see segmentation in mammals is in the vertebral column: The identity (shape and size) of each vertebra depends on its position along the head to tail axis. This is where one can see more subtle transformations of identity in mouse Hox gene mutants, like changes in the shape of vertebrae: vertebrae being produced in one position that are appropriate to another position (see example below) An extra thoracic vertebra with ribs is formed when the Hoxc8 gene in the mouse is removed (on the right). So Hox genes in vertebrates also play major roles in determining the body plan during development ⇒ Hox genes are also conserved at the functional level, they carry out similar functions in mammals in determining the body plan as they do in the fruitfly. Not surprisingly the body plan is more complex in mammals; more genes are needed to organize it and so the same dramatic changes are not seen when a single gene is mutated in the mouse. If several of these genes are mutated the individual generally does not survive. How do they function? They appear to operate by conferring a positional code: The Hox code. There are 39 Hox genes in the mouse and human, arranged in 4 clusters. These are expressed in overlapping domains along the A/P axis of the embryo, ↓ so position along the A/P axis could be established and distinguished by the combination of Hox genes expressed at that particular point. This in turn determines what structures are formed there. Such homeodomain transcription factors probably regulate development by coordinating the transcription of batteries of developmental genes, as simplistically illustrated in the figure below Different combinations of homeobox genes are active in different parts of the embryo and at different times, giving a distinct molecular pattern to each cell type according to its position (positional information and pattern formation). Not only homeodomain proteins are similar, but many of the other molecules and mechanisms that regulate development in the Drosophila embryo, have close counterparts throughout the animal kingdom. These fall into a number of families of conserved genes and proteins: As for Hox genes, access to these other important regulatory genes in man was gained through knowledge of their counterparts in Drosophila _______________________________ There are clearly major differences between how a fly and a mouse develop. For example, maternal cytoplasmic determinants do not appear to map out the body plan in a mammal like the mouse. Therefore, the fact that these regulatory genes are so similar was a major surprise. What is particularly interesting is to understand how very similar genes guide the development of such different organisms. ___________________________ Key concepts in lecture 10 1. The existance and many features of developmental genes were revealed by Drosophila genetics. 2. More than 100 genes are responsible for laying down the Drosophila body plan. 3. The first developmental genes were cloned in Drosophila and shown to encode transcription factors- the first Hox genes 4. Drosophila genes allowed access to vertebrate developmental genes because of the extremely high level of conservation of these genes during evolution. 5. Developmental regulatory genes do not all encode transcription factors- in addition to transcription factors that control gene expression directly some encode cell signalling molecules that allow cells to communicate. 6. These genes perform such important functions that they have changed little through hundreds of millions of years of evolution (they are highly conserved). 7. Hox genes map out the body plan in all animals from jelly fish to humans. Lecture 10 Learning outcomes: you should be able to…. A) Describe how the discovery of developmental mutations in the fruitfly Drosophila led to the isolation of developmental regulatory genes in Drosophila and other animals including humans. B) Define a master control gene or developmental regulatory gene, list what kind of proteins they encode and how we might explain the effect of mutations in these genes on the body plan of the animal (relate this back to the concept of positional information (lecture 7). C) Describe what Hox genes are, listing their 3 defining features. D) Present the similarities and differences between Hox genes in the fly and Hox genes in a mammal such as the mouse or human. Include a comment on the kinds of effects caused by mutating Hox genes in the fruitfly and in the mouse. Key terms to be familiar with: master control gene, developmental regulatory gene, homeobox, homeodomain, Hox genes, (in common with earlier lectures: signalling molecule, signal receptor, transcription factor), gene cluster, colinearity, gene conservation during evolution, vertebral column/vertebrae, thoracic vertebrae, lumbar vertebrae, change of identity in a particular position, the Hox code, combinations of genes active