Molecular Basis of Gene Interactions PDF

MOLECULAR BASIS OF GENE INTERACTIONS Session 1, 18/11/2024 Polygenic Inheritance: Interaction Between Genes - Epistasis -- The interaction between two or more genes to control a single phenotype - Many genes produce proteins - Genes rarely act independently of each other - Usually...

MOLECULAR BASIS OF GENE INTERACTIONS Session 1, 18/11/2024 Polygenic Inheritance: Interaction Between Genes - Epistasis -- The interaction between two or more genes to control a single phenotype - Many genes produce proteins - Genes rarely act independently of each other - Usually cooperate in the production of a trait -- molecular complexes, molecular pathways - Function of one gene can mask or modify the function of another cooperating gene -- epistasis - Therefore, the protein of one gene may stimulate or repress another gene and its protein - Example -- transcription factors. They can not only stimulate the transcription of a gene, but can also stop/repress it - Absence of a functional gene (a gene, which has acquired a mutation) often affects the function of another gene and its protein - If multiple gene products are required to form a complex, then if one gene has a mutation, it will affect the formation of the whole complex A chart of different phenotypes Description automatically generated Case of Two Genes Acting on the Same Color Trait Are Tomatoes -- - Once the tomato has been fertilized, all the cells that are needed to make the fruit are present, it just expands up to 40 days. - At 40 days, it is green -- green fruit - 40 days -- green fruit, which produces a precursor to make a yellow fruit - A dominant gene R1 is needed to make the yellow fruit -- to make the yellow pigment - After the tomato is yellow, it needs to turn red - To turn red, a dominant gene R2 is needed - Red pigment requires BOTH R1 and R2 genes - Yellow fruit is dependent on a dominant gene R1\_, if we have the recessive r1r1, the tomato will stay green - Likewise, red tomato is dependent on dominant gene R2\_, if we have the recessive r2r2, the tomato will stay yellow - To make red pigment, we need both R1 and R2 products - Yellow pigment requires R1 -- lack of R1 will give green - Lack of R1, the tomato will stay green ALWAYS - If we do a double homozygous cross with a green mutant (has recessive r1r1 and dominant R2R2) and a yellow mutant (has recessive r2r2 and dominant R1R1), - If the green mutant has R2R2r1r1 and the yellow mutant has R1R1r2r2, - The yellow can become yellow from green, but will stay yellow -- it only has R1 - If we cross these mutants, the F1 will produce a red tomato -- it will have R1r1 R2r2 -- has dominant R1 and R2 genes, shows complementation - If we self-cross the F1, we'll get a modified F2 phenotypic ratio, not 9:3:3:1 - The altered ratio is 9:3:4 -- if there is r1r1, the tomato will ALWAYS stay GREEN, it can't evolve to produce other pigmentation - Genetic ratio remains the same, but the phenotypic ratio is different - Still obeys Mendel's laws on IA, still has independent assortment, but the phenotypic ratio doesn't have it - R1\_ still makes yellow, but needs R2\_ to make red - If there is r1r1, tomato will stay green - There is an epistatic interaction between genes -- one gene masks the effects of another gene - The gene that does the masking here is said to be epistatic to the other. The masked gene is hypostatic to the other - R1 is epistatic to R2 - R2 is hypostatic to R1 - Doesn't matter what you have on r2/R2, if you have r1r1, you stay green/yellow - Epistatic ratios may allow a geneticist to determine the other of genes in a particular pathway Another Example -- Coat Color in Mice - Look at a recessive gene c -- it can mask ALL other color-coding genes - Consider a trihybrid cross with a triple heterozygote - A and B give different coat colors, but the *agouti* gene C is dominant - A\_B\_C\_ - *agouti* - aaB\_C\_ - black - A\_bbC\_ - cinnamon - aabbC\_ - brown - When we have C, there is a complex relationship between A and B, which segregates as 9:3:3:1 - All depends on the C to get the segregation ratio - When the c is homozygous mutant -- cc, the coat color is white - A\_B\_cc - Aabbcc - c masks the effects of the other two genes, it could code for a precursor or makes a precursor that A and B act on to make coat color - Where there is a cc, it doesn't matter what the A and B are, the mouse is white  A white mouse with red eyes Description automatically generated Punnett Square for a Trihybrid F1 Cross - We get 27/64 of the agouti - 9/64 black - 9/64 cinnamon - 3/64 is white - ALL the rest are white -- 16/64 - We still have the 9:3:3:1, wherever there is a dominant C Other Complicated Relationships Can Occur Between Genes - Within Dihybrid F1 crosses, we can find epistatic relationships that give modified 9:3:3:1 ratios: - 12:3:1 -- Dominant epistasis - 9:7 -- Complementary gene action - 15:1 -- Duplicate gene action - 13:3 -- Dominant suppression epistasis Dominant Epistasis -- squashes, gourd (crop plants -- pumpkin, cucumber...) - Ratio of 12:3:1 - It can be white, green or yellow - Dominant allele of one gene can mask the effect of both alleles of another gene - If you are B\_, you're white - If you are bb, color is determined by A/a allele - A\_ is yellow - aa is green - 12:3:1 ratio - B is dominant to A/a  Two Genes Needed to Produce Flower Color -- Complementary Gene Action - 9:7 ratio - There is a pathway that leads to anthocyanin (an enzyme) production - In the context of snapdragons -- anthocyanin gives pinkish-purple color - Precursor molecule needs enzyme C to make the intermediate molecule - The intermediate needs enzyme P to make Anthocyanin - Perform cross between double homozygous: CCPP x ccpp - CCPP are pink, ccpp are white - It still segregates, as expected -- we've got Independent Assortment, just the phenotypic ratio is different - Needed at least one wild-type allele for BOTH genes, otherwise it's white - C\_P\_ is needed because it gives functional enzymes of both genes, BOTH enzymes present - This is complementary gene action -- we need the complementary action of both genes to get the phenotype - If you have C\_pp, you go through the first step, but then you stop -- no end product, no pink color - Either C\_P\_, otherwise it's white A diagram of a flower Description automatically generated  A table with text and images Description automatically generated with medium confidence Double Dominant Alleles, Two Genes Working on the Same Part of a Pathway - 15:1 ratio - Two dominant genes, acting on the same part of the pathway - A precursor molecule, taken by enzyme A to make the product - Also, the precursor can be taken by enzyme B to make the same product - Two genes that make 2 enzymes, which can be taken by the precursor to make the product - They are NOT competing against each other to produce the enzyme, they both work on the same precursor - Carry out the same function - Genes don't have to work in opposition for interaction to be epistatic - They both work -- they don't work against each other, aren't mutually exclusive - Example -- Kernel color in wheat - As the seed/kernel ripens, it reaches a golden color - Cross AABB (colored) x aabb (colorless) - Get mutants of these genes, where it stays intermediate between the two - Then cross the F1s (AaBb) - We get: 9 cases of both functional enzymes from both genes, 3 functional enzymes from the A gene pair, 3 functional enzymes from the B gene pair, 1 non-functional enzymes produced at both genes - aabb stays colorless -- no dominant gene for an enzyme - All other get colored - Still obeys Mendel's IA law - Duplicate gene action -- 2 genes carry out the same function  A diagram of different types of wheat Description automatically generated  Certain Genes Have the Ability to Suppress the Expression of a Gene at a second locus -- Dominant Suppression Epistasis - Ratio of 13:3 - Malvidin production in *Primula* plant - Certain genes have the ability to suppress the expression of a gene at a second locus - Malvidin produces purple/blue petunias, also in blackberries and red wine - Both the synthesis of malvidin (controlled by the K gene) and the suppression of synthesis (controlled by the D gene) are dominant traits - Both D and K are dominant - When there is expression of D, it represses K, which stops the enzyme to work on the precursor to make malvidin - The F1 plant with the genotype KkDd will not produce malvidin because of the presence of the D allele - You can't produce Malvidin with the dominant D allele - Everywhere, where there is a D present, we get suppression, no functional enzyme - Suppressor -- a genetic factor that prevents the expression of alleles at a second locus, an example of epistatic interaction - K\_dd -- Malvidin production because of the dominant K allele present with the recessive dd - kkdd produces no malvidin because we have both recessive genes - 13:3 ratio of phenotypic distribution - Genotypes are segregating as we'd expect, phenotypes are altered because of the action of the suppressor - 9+3+1=13 - 3 left - Dominant Suppression Epistasis A diagram of a gene Description automatically generated  Human Example of Epistasis - Ratio of 12:4 - Bombay phenotype -- involves the interaction between *ABO* alleles and a third antigen H, coded for by a different gene (H) - Individuals who are homozygous recessive at the H-gene (hh) will be blood type O regardless of the genotype of the I-gene, which has the alleles for AB blood types - H codes for an antigen H that is the precursor molecule for the formation of the A and B antigens - Genetically individuals may be A, B or AB, but appear as O, if they are hh - You can't make A or B without H - Question: - Identify genotypes first, then phenotypes, populate the Punnett square - Where there is recessive h, it doesn't matter what A or B are - h is epistatic to A and B - Ratio of 12:4 A table with red and white squares Description automatically generated - If there is a question on an exam, which doesn't mention the H locus, then: - Assume you are asked about the ABO blood system, don't consider the H locus - If you are specifically told about the H locus, then consider it - You can only work with the information you are given Summary of Pattern of Inheritance - The interaction between gene products that causes modification, often due to the presence of mutant alleles WORKSHOP 3:  A screenshot of a computer Description automatically generated - If you have ee, it doesn't matter what you have on the B locus, you can't produce melanin - Not you, dogs won't produce melanin - E\_B\_ - black - E\_bb -- brown - ee\_\_ - yellow - So far, we have looked at genes as individual entities in the genome -- but they are joined up on chromosomes - What implications does this have? - Genes aren't always on the same chromosomes - Around 1900 Mendel's work on the transmission of traits was rediscovered by 3 botanists -- Hugo DeVries, Carl Correns and Erich von Tschermak - 1903 Sutton and co-workers suggested that an organism has many unit factors, much more than the number of chromosomes, and that these factors are located on chromosomes - In today's words -- genes and alleles of those genes are located on the chromosomes - However, deviations were observed - Bateson, Saunders, Punnett made an unusual observation - Looked at flower color and pollen shape in pea plants - L -- color - P -- shape - Purple is dominant to red - Long granule pollen is dominant to round shape - Take homozygous parents PPLL x ppll, we get PpLl heterozygous plant in the F1 - Cross them together to produce the F2 - Get the typical 9:3:3:1 ratio for 2 factors and 3:1 ratio for one factor/trait - Single factor -- pollen shape/color, without the other - They collected 381 progeny and expected a 9:3:3:1 ratio with some expected numbers of progeny individuals - 9 P\_L\_ = 214.31 P\_L\_ - 3 P\_ll = 71.44 P\_ll - 3 ppL\_ = 71.44 ppL\_ - 1 ppll = 23.81 ppll - HOWEVER...  A diagram of flowers and plants Description automatically generated Chromosome Linkage -- Explanation of Results - If the two factors segregated independently and on separate chromosomes, you would expect a 9:3:3:1 ratio - Genes on separate chromosomes, independent assortment, we get 9:3:3:1 - If it was a linked gene -- a gene on the same chromosome -- there would be a ratio of 3:1, as seen for single factor - Single factor -- 2 genes are linked together, genes come together - This is NOT what is seen because of RECOMBINATION - We have parental classes, we cross them together - We have swapping of alleles, and now we have the P, but this time in a combination with l -- Pl, and pL - Because of recombination, we get a mixing of these parental alleles to change the ratios of what we would normally observe - We don't see the 3:1 segregation ratio - This doesn't happen a 100% of the time - The more individuals in the progeny, the bigger the chances of observing recombination Morgan also Investigated 2 Traits at a Time - Looked at eye color and wing shape in *drosophila* - Red eyes -- wild type - Purple color -- recessive - Red -- A/A - Purple -- a/a - Normal wings -- wild type - Vestigial wings -- recessive - Normal -- B/B - Vestigial -- b/b - Made crosses with the wild type cross with the recessive homozygous (recessive = test cross) - AABB x aabb - Resulted in a AaBb, which again crossed with aabb - AaBb x aabb - The expected results are 1:1:1:1 because it's a test cross - 25% of one parental type, 25% of the other, and 25% of each recombinant type - A test cross uncovers hidden recessive alleles - Morgan's dilemma was that there are more of the parental types than the recombinant types. He expected an even distribution  A diagram of a test result Description automatically generated  A diagram of a test Description automatically generated Morgan's First Prediction - Genes are on the same chromosome - Once alleles are together, they tend to stay linked - F1 would be red eyed normal winged because of dominance - Genes close together on the same chromosome are linked - Genes on separate chromosomes are not linked - If the genes are on the opposite ends on a chromosome, MAYBE they are linked  Morgan Observed Cytogenetics - Chiasma -- a place on a homologous pair of chromosomes, where exchange is occurring during recombination. A place for homologous pairing - Cross-over -- the process of reciprocal exchange leading to recombination - In prophase 1, - Chiasma marks the site of recombination - Crossing over/recombination is RANDOM - The end product is a mix of 2 parental chromosomes and 2 recombinant chromosomes - 2 recombinant and 2 parental A diagram of a structure Description automatically generated  Morgan's Second Prediction, Based on the Observations They Made - "Chromosomes sometimes exchange segments, allowing their passengers to change vessels" - We get changing of parental alleles, switching pieces of DNA - One of the dominant drivers of genetic diversity - Took account on why he was seeing core of the parental types and less of the recombinant types - Consequences of recombination - If there was no crossing over, the meiotic chromosomes that we acquire would be identically the same as the parental ones - Crossing over allows for the generation of two parental products and 2 recombinant products - Allowing for 4 different types going through - Meiosis with no crossover between the genes will result in ALL parental meiotic products - One crossover between the genes, we generate a parental product, 2 recombinant products and another parental -- 4 different chromosomes A diagram of different types of genetic modification Description automatically generated What Morgan Predicted... - Genetic recombination: mixing of genes during gametogenesis produces gametes with combinations of genes that are different from the combinations received from parents - Genes on non-homologous chromosomes (unlinked) assort independently - Genes on the same chromosome co-segregate - Crossovers result in recombination between linked genes - Genes on the same chromosome would just co-segregate together, if there is no recombination The Basis of Those Ratios... - Crossover frequency is a function of the distance between two loci -- a physical constraint on the chromosome - Locus -- where a gene resides on a chromosome - Physical distance influences the number of crossovers that can occur between genes - More crossovers can occur in a larger distance - The closer two loci are, the less the chance of a crossover event between them - Effects of multiple crossovers on the detection of recombinant chromosomes - Having different numbers of crossovers between 2 genes can affect what you observe - Odd number of crossovers between 2 loci = a recombinant chromosome can be detected - Even number of crossovers between 2 loci = no recombination is detected - One crossover -- brings b, second crossover brings B back, third crossover, brings b to the end of the chromosome, indicating a recombinant - Switching of alleles (odd numbers) and switching back (even numbers)  A diagram of a number of crosses between two chromosomes Description automatically generated Mapping Genes - Recombination frequency of two genes tells us how far apart thy are on a chromosome - By comparing multiple pairs of genes, we should be able to tell the order of genes along the chromosome - We use genetic maps to map regions that affect traits, and we can use to do comparisons between different genomes of different related species - More recombination events -- further apart are the two loci - Smaller the value, closer they are together, less recombination - When looking at the positions of markers on maps, we can think of the loci as being train stations with different distances between these stations - What's the smallest recombination frequency? -- which two genes/train stations are the closest together? Linkage and Recombination - The closer the genes are the less likely they are to be separated by recombination - If this is true, then recombination frequency should be proportional to the distance between genes - Centimorgan or map unit -- cM/m.u. is a unit of measure for genes along a chromosome - A scale we use, a map unit distance - 1cM=1m.u=1% recombination - In Humans, 1cM=1million base pairs, in plasmodium sp. 1cM = 15 base pairs - Genome size and position on the chromosome influence the number of recombination events and therefore, calculation of map units Genome Size and Position on the Chromosome - The centromere has a region all around it, which influences recombination frequences for the chromosome - Centromeres are very important -- every crossover is dependent on a physical aspect of how close it is to the centromere. The closer, the more difficult it gets - If we were to plot it, - Each dot on the graph is a genetic marker - Physical distance -- Mega-base-pairs, genetic distance -- centiMorgan - We get a characteristic chair-like plot - When we are around the telomeric ends, we get more recombination because of the physical aspect - We get suppressed recombination, where we have breakdown of the linear relationship between genetic and physical distance - QTL1, QTL2, QTL3 - QTL1 has a linear relationship with the distances -- small physical distance and small genetic distance - QTL2 is in the centromeric region - Recombination frequency changes along the genome and changes in different genomes  - Start with the smallest frequence and work your way all the way up 2\) Question A diagram of a test result Description automatically generated - This is Morgan's data - How close are pr and vg? - Recombination frequency can be used as a measure of distance - Genetic distance is measured in units called mu (map unit), for example cM (CentiMorgan) - RECOMBINATION FREQUENCY:  - Based on this equation: - We can identify the parental types - Parental ALWAYS have the highest proportion -- it' easier to find a parental type - The others are recombinants A diagram of a tester Description automatically generated - We've already used Morgan's data to see how close in the *drosophila* genome pr and vg are along the chromosome - Using this method, if you have lots of different traits and crosses, you can work out the positions of different genes along the chromosome, based on their recombination frequencies - Know the parental alleles, identify them, have the highest proportion, know the recombinants, then calculate the recombination frequency and the distance between them 3\) Question  A diagram of a number of numbers Description automatically generated with medium confidence - Each recombinant chromosome will be present in the progeny 20% of the time - There are two, forming together 40% we need to split the percentage  - RF max = 50% - When RF = 50, genes are said to be unlinked - We know recombination occurs at random and distance between genes determines how many recombinations can occur A diagram of a dna sequence Description automatically generated - In the pair of genes further apart, more recombination events can occur, so the Parents=Recombinants, RF = 50%  So... How Do We Work Out Whether the Genes Are on Separate Chromosomes or Far Apart on the Same Chromosome? - We can identify more genes in the linkage group -- even better, add in genetic markers and build a relationship between markers and genes at different ends of the chromosome - For example, if you only have 2 genetic markers, and they are really far apart - It may be that when you build the linkage group, it appears as the genes are on 2 separate chromosomes - If you add more genetic markers, you get the relationship between the ones at the top or around the middle of the linkage group with the ones in the bottom part of the linkage group - Then bring together these parts to form one group A screenshot of a test Description automatically generated - d and e appear unlinked, but they are still on the same chromosome - d and e appear unlinked, but when you add f in, to see the relationship between them, you see that they are on the same group, it's just that they are too far apart to form links - If you added more, you'd be able to further cement that relationship SUMMARY:  NON-MENDELIAN INHERITANCE 20/11/2024 Session 2 - Inheritance patterns not inherited in the usual Mendelian fashion - Not inherited in the usual manner predicted by Mendel's pea experiments Examples of NM-inheritance - Poky mutants in Neurospora - Mirabilis jalapa (chloroplasts) mtDNA - Mitochondria, function: - Power-plants of the cell, generate ATP from chemical energy - Mitochondrial genome: - Circular dsDNA structures, unmethylated - Encode approx. 37 genes, this depends on the species - Vary in size -- approx. 20 kb -- 2Mb, again depends on species - Encode tRNA, rRNA, electron transport chain genes - Multiple mitochondria per cell - Multiple genomes per mitochondria - Majority of the genetic material is in the nucleus *Neurospora* - Filamented fungi - Poky mutant -- characterized by slow growth, excess of cytochrome c, lack of cytochrome a and b - No male and female parts, but crosses can be defined paternal and maternal in the cytoplasm - Depending on which one produces the trichogyne structure - Females produce trichogyne - Males produce the conidium structure - Genetic material is either black or red - Conidium fuses with the trichogyne, producing a structure, where: - It produces a 2n structure and a sac, where: - There is a 1:1 segregation ratio of genetic material, 4 ad+ and 4 ad- - All the spores have the cytoplasm from the "female" and they are all poky mutants - The poky mutant produces the trichogyne - If we reverse the cross, - We'll have normal cytoplasm, normal individual - Poky mutant produces the conidium - The 2n structure has all spores with normal cytoplasm - Inheritance of poky mutations is dependent on which one of the parents is "male/female" and brings the cytoplasm -- cytoplasmic inheritance - Poky mutation is inherited through cytoplasmic inheritance Diagram of a diagram showing a normal and a normal molecule Description automatically generated with medium confidence Pedigree Analysis  - Mating -- firstborn on the left, secondborn on the right... - Demonstration of maternal mitochondrial inheritance Mitochondrial Diseases - Leigh's disease, Alpers disease, Barth syndrome - Maternal inheritance only! - Faulty mitochondria is linked to other diseases -- diabetes, deafness, Parkinson's... - Multiple mitochondria are found in the cell - Each mitochondria has multiple copies of DNA - Around 13 genes for oxidative phosphorylation system protein subunits -- the remainder proteins for this system come from the nucleus - Depends on the organism - Subunit V drives ATPase to create ATP - All the protons produced create a hostile environment because of the free radicals - The circular DNA lacks a repair mechanism and has very few introns - If anything happens to those units, the energy-producing system is disrupted - Bad things happen in the mitochondria - We typically see single mutations in mitochondrial DNA - Pathology of mtDNA mutants can alter in different tissues - Tissues may have different thresholds for disease -- some may tolerate certain mutations, some can't, therefore, a disease is seen - Distribution of mutant and WT mtDNA will influence complementation - \% of mutant mtDNA differs between tissue types - Different cellular origins for particular parts of\_\_\_ A diagram of a family Description automatically generated  A screenshot of a computer Description automatically generated mtDNA Transmission Pedigree  Mitochondrial Bottleneck - Variety of mtDNA molecules in the maternal pool, represented by the bottle - The two genotypes in this maternal pool are represented by blue and yellow - Blue -- healthy - Yellow - faulty - When created, each oocyte receives a small subsampling of mtDNA molecules in different proportions - This is represented by the conveyor belt with oocytes as they are produced - It's totally random - Each oocyte will receive a random amount of faulty mitochondria - Difficult to predict which cells will become faulty in a population Cytoplasmic Segregation and Recombination - Mitosis can result in a disproportionate number of mutant or wild type organelles in a daughter cell due to random sampling errors ONLY - Once this occurs, there is a higher chance of the more populous organelle replacing the less populous one - Since organelles are essentially haploid, there is no complication of diploidy and its associated dominance/recessiveness - Non-Mendelian - Not only we have the initial bottleneck source of differentiation, but now we have a random distribution of organelles through cytoplasmic segregation - This makes it difficult to predict the inheritance of faulty organelles -- mitochondria in this case A diagram of a cell structure Description automatically generated mtDNA Transmission Pedigree - Heteroplasmy define a mixed population of Mito in a single cell - These can be inherited with uneven, unpredictable separation - Makes it difficult for clinicians to predict within families, who is going to be affected - Males are affected, but when they meet a mother that's unaffected, their children are fine - The carrier mother (third child of the parents) is unaffected, but is a carrier -- there is a chance her children would be affected - Because its cytoplasmic inheritance, mothers don't know what amount of faulty mitochondria they will give to their offspring - Inheritance of faulty mitochondria is uneven and unpredictable - Three-parent babies is an accepted method for women, who are affected with mitochondrial diseases, but want to have healthy babies - Donors give DNA for healthy mitochondria, NOT for other traits, which influence appearance  A diagram of a mother\'s egg Description automatically generated  - Method 2 -- repair is done after fertilization - An avenue of hope for families, which are affected by mitochondrial diseases Different Disease, Affecting Humans: A screenshot of a white text Description automatically generated Heritability of Organelles -- Chloroplasts - Some similarities with mitochondria - Both have large surface areas for energy production - [Both have inner and outer membranes] - Endosymbiotic theory -- basis of difference - Chloroplasts use sunlight to generate ATP, whereas mitochondria use chemical energy - Very complicated system of photosynthesis -- if one part gets disrupted, the whole process shuts down Chloroplast Genome - Very similar to the mitochondrial genome - Circular piece of DNA, unmethylated - Encodes around 100 genes, depending of species and size - Vary in size from 120kb -- 160kb - Encode genes for photosynthesis and the protein production pipeline to produce those proteins - Multiple chloroplasts per cell and multiple genomes per chloroplast Inheritance of Chloroplast DNA - A classic experiment -- How is stem and leaf color inherited in the four-o'clock plant - Cross flowers from white, green and variegated plants in all combinations - Variegated segregates into all 3 types of leaf, all segregate into the phenotype of the seed, it is dominant - Chloroplasts are inherited from the cytoplasm cytoplasmic inheritance - Depends on the seed - Conclusion: The phenotype of the progeny is determined by the phenotype of the branch the seed originated from, not from the branch the pollen originated. Stem and leaf color exhibits cytoplasmic inheritance  Lethality - Mice -- recessive lethal genes - First identified while studying the inheritance of coat color in mice - Yellow x Brown = 1:1 segregation of progeny - Yellow coats are heterozygous and dominant - AyA - However, when making the cross, - AyAy is a lethal allele - So, the segregation ratio is 2:1 not 1:2:1 - AyAy mice die as embryos - Effects of the AyAy mutation: - Yellow coat color - Tumors - Obesity - Insulin resistant type II diabetes - Increased propensity to develop spontaneous induced tumors - AyAy preimplantation lethality due to abnormalities in both trophoblast and inner cell mass of the blastocyst -- we usually don't see them, it doesn't implant - The mutation deletes the whole section of the 170kb Raly gene, except the promoter, which is essential for pre-implantation - Recessive lethal gene - AyAy -- no Raly gene expressed no pre-implantation - There are Dominant lethal genes: - Dominant lethal genes are expressed in both homozygotes and heterozygotes - How can this be inherited? - Late onset, Huntington's disease or variable penetrance - Conditional lethal genes: - Conditional mutants -- we can engineer microorganisms, which are functional in a specific temperature and then become non-functional at another temperature - Can be sex limited -- X-linked recessive - Favism -- deficiency in Glucose6PhosphateDehydrogenase -- linked to hemolytic anemia A yellow mouse with text and images Description automatically generated with medium confidence  X-Linked Recessive InheritanceA diagram of a family Description automatically generated Complications to simple Mendelian Genetics - If you look a nucleus under a microscope, see nuclear pore complexes -- a group of proteins, which enable all kinds of molecules (carbohydrates, polymerase, DNA, lipids, ribosomes, proteins...) to go in the nucleus - There is a conservation of these groups of proteins because they have the same role, it fulfills the function -- there has never been a need to change it in evolution - Into nucleus: DNA, carbohydrates, lipids, signaling molecules - Out of nucleus: RNA, ribosomal proteins Segregation of NUP88 Mutation - Nucleoporin 88 -- a protein - Present in higher plants and in vertebrates - Involved in binding microtubules to the pore complex - They bind to NUP88, during segregation in gametogenesis, it helps pull the nucleus in the cell - NN -- all normal, segregate normally - Nn -- normal, segregate normally - nn -- can't be found, no correct segregation, mutated protein - From Nn plants, all male gametes are fine, but 50% of female gametes die - Female n gametes don't make enough protein and fail to segregate during meiosis - Non-Mendelian segregation pattern due to faulty binding of microtubules to the NUP88 EPIGENETICS - Epigenetics is defined as inheritable genetic changes above and beyond modifications to DNA - Developed by C.H Waddington in the 1940s - "Epigenetic signals..." - Epigenetic markers are temporary modifications to DNA - Stem cells to differentiated tissue - From one generation to the next -- we can inherit epigenetic changes Epigenetic Marks - DNA methylation - Histone modification -- acetylation, methylation, phosphorylation - Gene switched on -- uncondensed, open chromatin due to acetylated histones, DNA is relaxed and open - Gene switched off -- condensed, closed chromatin, methylated cytosines, deacetylated histones, inaccessible for transcription DNA Methylation - Amount and type of methylation differs between organisms from 14% in Arabidopsis -- 0.3% in Drosophila - Typically, methylation is on cytosine - M5C is the most common methylation - In mammals these tend to be at CpGs - In plants, mostly on CpGs, but can also be on CHH and CHG - H = A, C or U - Effects: - Cytosine methylation within gene promoter regions is thought to inhibit binding of regulatory proteins and repress transcription - Methylation within introns and exons is correlated with highly expressed genes - However, methylation in the first exon is correlated with inhibition - In cells, we have transposons -- a short sequence of DNA that can replicated and move in the genome and insert itself - If you have pieces of DNA that can replicate and move in the genome, it that was left unchecked, the genome would become massive and unstable - Typically, transposons are repetitive, so you'd get these repetitive bits of DNA added to the genome - IN PLANTS - If the transposons moves and lands onto an essential defense mechanism (tumor suppressor gene), it would go very bad - So, to protect themselves, plants have developed a mechanism to recognize these small, repeated sections - As soon as they are detected, plants methylate them, which silences that region - If the transposon wants to jump, it can't because it can't transcribe itself - It's isolated and can't move around in the genome - Effect on transposons in plants: Methylation stops transposition - Methylation -- silences a region  Genomic Imprinting -- Example in Mice - The Igf-2 gene encodes an insulin-like growth factor - Functional allele required for normal size - Igf-2m allele encodes a non-functional protein - Epigenetic regulation results in the expression of the paternal allele ONLY - Paternal, allele is transcribed - Maternal allele is transcriptionally silent, epigenetically silenced - If we cross homozygous mutant with homozygous functional (wild type) - Offspring is normal, if the father has the normal version of the gene - Offspring is mutated (dwarf) if father has the mutated version -- it doesn't matter that the female has the normal version - ALL depends on the father's allele - The imprint of the Igf-2 gene is erased during gametogenesis - A new imprint is then imparted - Oocytes possess an imprinted gene that is silenced - Sperm posses a gene that is NOT silenced - The phenotypes of the offspring are dependent on the paternally derived allele A screenshot of a white background Description automatically generated  A diagram of a mouse Description automatically generated - Maternal allele is silenced Genomic Imprinting in Humans - Prader-Willi Syndrome - Many symptoms including insatiable appetite, leading to obesity - PWS is caused by a genetic lesion on chromosome 15 - Region silenced on maternal chromosome - Therefore, any disruption to paternal chromosomes, can develop the disease  WORKSHOP 3: A screenshot of a cell phone Description automatically generated  A screenshot of a cell phone Description automatically generated  A screenshot of a cell division Description automatically generated  WORKSHOP 4: A screenshot of a paper with text Description automatically generated - Less than 50 mu genes are linked  - Test cross -- cross with the double recessive - Allows us to uncover hidden recessive genotypes A screenshot of a paper Description automatically generated  A screenshot of a cell phone Description automatically generated  - Start with the smallest frequencies A screenshot of a paper with text Description automatically generated  A screenshot of a computer screen Description automatically generated - Identify where we've got the combination dP and Dp from the parents -- we find the parental classes, others are recombinants  A screenshot of a cell phone Description automatically generated  A paper with text and words Description automatically generated  COMPLEXITY AND DEVELOPMENT Session 6 22/11/2024 - How do we get from a single cell, a single fertilized egg to grown humans? - What are the mechanisms? - Mechanisms how hox genes affect development in animals How Did We Think the Embryo Formed? - Aristotle (4^th^ century BC): two possibilities - Everything in the embryo was already present - New structures arise progressively -- epigenesis - 1600s: preformation was the dominant mechanism explanation - All embryos existed since the beginning of time -- everything in the embryo is present right from the beginning. Inside the embryos are all the embryos for the next generation and so on... - Malpighi "observed" experimentally a tiny fully formed chick in the egg from the very beginning. Sometimes you can observe things, which aren't actually there, your mind makes them and creates false knowledge - Homunculi (a small human, which just needs to grow) exist in sperm A drawing of a wine glass Description automatically generated - Now we know embryos form progressively, the egg undergoes through a lot of developmental processes to become the adult organism DNA Does NOT Provide a Blueprint for Embryonic Development - Genes -- instructions to make development work - Blueprint -- describes final form - Does DNA describe a fully formed human, or a fly, or whatever? - NO - Series of iterative instructions - DNA provides these - Example -- origami - If you have a series of instructions how to fold the paper to become a full origami... - It's much simpler way of working out how to form the adult form - If a picture of the full origami is shown, we have no idea how to create it, unless we already know how - A series of folds to form the final structure - In development, you have a limited number of developmental processes, which go into creating the adult organism What Does a newly Fertilized Egg Need to go Through to Become a Fully Formed Organism? - Cell division -- a single cell within the fertilized egg, we have many cells in the grown adult - Growth -- the single cell is very tiny, compared to the adult organism - Cell death -- sculpting the final shape of the organism - Cell specialization -- some cells will perform one function, others may perform another - Patterning -- where all the bits of the embryo go -- the developing embryo needs to know where the head is going to be, the tail, the limbs... - Cell movement - Changing of shape - Communication between cells  - The processes are all going on at the same time during development Part 1 - Divide, Differentiate, Die A diagram of a human embryo Description automatically generated - Patterns of division is different across the species - In humans, we start with a fertilized egg, which needs to divide - The first few divisions are called cleavage divisions -- No growth, the egg simply divides in 2,4,8,16 - The embryo remains the same size - In this stage, the cells (1-16) are called totipotent -- can become absolutely any type of cell in the embryo - Evidence -- identical twins - When identical twins form, the embryo splits during early development and forms 2 complete fully formed humans - Evidence -- mice - Making chimeras experimentally -- fuse two 8-celled embryos from mice, they go on and form one mouse, but that mouse contains cells from both of the embryos -- any one of the cells could form any part of the mouse  - Driesch experiments -- sea urchins - He took a sea urchin embryo and split it during the 2-cell stage - Discovered that either of these cells could form a fully formed sea urchin, although it was half the size of the normal sea urchin - Often one of the two cells died A diagram of a cell life cycle Description automatically generated - Early embryo cells are totipotent - Once the cells start to form a blastocyst, they start to differentiate - In humans, the cells within the blastocyst form two lineages - Inner cell mass -- forms the embryo proper - They are pluripotent -- can form any cell of the embryo, but NOT the placenta - Outer cell mass -- form the placenta and so on... - Evidence for this is in mice - If you take inner mass cells (IMCs) and culture them, now termed embryonic stem cells, - You can genetically alter them, and then microinject them back into a recipient blastocyst -- from another mass - Implant these into a foster mother, and they will grow up to form mosaic mice, just like the once we formed, when fusing the two 8-cell embryos  - Inner Cell Mass: Embryonic stem cells - PLURIPOTENT, NOT TOTIPOTENT - However, you can't take the embryonic stem cells, reconstitute an embryo, inject that and it having grow up because they would not have a placenta - You have to inject them into a recipient blastocyst because the pluripotent cells, which will form the placenta - Embryonic stem cells in humans may have potential use in regenerative medicine - You have to destroy the embryo to get the stem cells -- ethical considerations - Main point -- these cells are PLURIPOTENT CELL SPECIALIZATION/DIFFERENTIATION - By the time we've got a blastocyst with embryonic stem cells, we are beginning to have specialization - Pluripotent stem cells -- embryonic stem cells - Multipotent stem cells -- can form several types of cells, but not the placenta - haematotrophic stem cells -- all the cell types in blood - Unipotent stem cells -- Form only 1 type of differentiated stem cells - epidermal stem cells, which form the skin - Terminally differentiated cells -- blood cells, skin cells, reproductive cells - Changes in gene expression as a key example: - To become a blood cell, hemoglobin needs to be expressed - To become a skin cell, keratin needs to be expressed - Gene expression is activated or repressed by transcription factors -- proteins, which bind to regulatory regions within the gene (the promoter, or enhancer/repressor elements) - This binding to these regions determines whether the gene is expressed, or not - Cell differentiation is considered to be a one-way process - Cells acquire their fate over a number of generations - Fate gradually becomes restricted: 1. Specification -- cells "know" what they will become, but can still change fate under the right conditions (cell signals, changes in gene expression...) 2. Commitment -- there is no way back! - Once differentiated, there is no way back - But what about cloning.... Cloning - Taking the nucleus from an adult cell, introducing it into an unfertilized egg, then creating a full organism from it - This suggests that we can reprogram a fully differentiated cell - Some of the earliest cloning experiments were done using frogs -- *Xenorphus* - Adult skin cells were cultured, or tadpole gut epithelial cells - The nucleus from one of the adult cells was transferred into an unfertilized egg, which had its nucleus removed - They were able to grow up to at least a tadpole most of the time - HOWEVER, gene expression is not 100% normal and the organisms that result do have abnormalities - Success rate is very low from these nuclear transfers into embryos, a very small amount of these grow up into an adult organism - It can be done at least to an extent - Programmed Cell Death is also very important for differentiation - Example -- digit separation -- in different species, there is a combination of different outgrowth and cell death - The chick limb initially forms as a kind of a paddle with webbing between the digits - The darker regions in pic. 1 represent where cell death is occurring between the digits - All the black regions will die, leaving the chick with separated digits - This is the same in humans, but sometimes it can go wrong MORPHOGENESIS -- Creating Form - Division, differentiation and cell death already have started to create three-dimensional form - Other things required to create form are: - Cell movement - Changes in cell shape - Changes in cell adhesion (sticking/attaching) - Some molecules are very important in these processes: - Cytoskeleton /microtubules within the cells change shape of cells A diagram of a curved line Description automatically generated - Changes in cell adhesion -- cells lose contact with each other and gain contact with other - Gastrulation -- the process, which starts to occur after the blastula - Blastula -- where we have inner cell mass and a layer of cells surrounding that - Gastrulation is the process of turning the one-dimensional layer of cells into a three-dimensional structure of the embryo, called the gastrula - In the majority of animals, which have 3 cell layers - Ectoderm -- outer layer - Mesoderm -- middle layer - Endoderm -- inner layer - Diploblastic organisms form only 2 layers  - First picture is the one-dimensional layer of cells - The second picture is the gut begins to form - Cell shape changes - Meanwhile, the mesoderm cells (red), begin to migrate away from the gut, to form the mesoderm - During this process, we have changes in cell adhesion - Filopodia (yellow) are really important in making contact with the opposite end of the sea urchin, and pulling the gut towards the outer surface of the embryo, where it's going to form the mouth - Examples of changes in cell shape, cell adhesion - Gastrulation is absolutely VITAL PATTERNING -- Putting all the bits in the right place - How is positional information conveyed to all the cells within the embryo? - How does the embryo know where it's head will be, the tail, etc.? - Cell-cell communication is key in this process - Organisers and induction - Morphogens - The hox code -- how does it specify identity or pattern within the embryo? The Spemann-Mangold organizer - Carried experiments in frog, where regions of the frog embryo were put into another embryo - They used embryos with different pigmentation patterns -- it is easier to tell which tissue came from which parent - Took the dorsal region of one embryo in the gastrula stage - Transplanted it into a ventral region of the second embryo - Transplanted the dorsal limb - Dorsal=back - Ventral=belly - Let the embryos grow up - Couple of days later, the tadpoles had two axes -- a second head and a whole extra body axis, containing tissue from both the donor tissue AND the recipient embryo. Only the extra axis had the tissue - Shows that the dorsal region can signal to the regions around it and induce them to become something that they wouldn't normally be - That's the definition of an organizer - In the past, the organizer was known as just the Spemann organizer - Mangold did the experiments in Spemann's lab A diagram of an embryo Description automatically generated - Induction -- the process when one group of cells is signaling to another group of cells, affecting how they develop - Organiser -- a signaling center that directs development of the whole embryo or a part of it (limb patterning...) - Second example -- the developing limb of an embryo of chicks about the anterior posterior axis - The chick limb has 3 digits -- digits 2,3,4 - How is the axis patterned? - Polarizing activity is very important in the role of an organiser - Polarizing activity is found in the posterior of the developing limb - Limb develops from a limb bud, which doesn't have any digits - If you transplant a second posterior region in the anterior part of the limp bud, you duplicate the digits - We still have the original posterior part, but we've transplanted a second one in the anterior - We get a complete duplication of the digits -- extra digits 4,3,2 - Zone of polarizing activity releases morphogen, which patterns the anteroposterior axis of the limb - Morphogen is diffusing across the limb, so that around the ZPA there is a high concentration, which specifies digit 4 - At a slightly lower concentration is digit 3, and at an even lower concentration, is digit 2 - When transplanting a second ZPA, you create a second region of high polarizing activity, which creates high morphogen concentration at the anterior, creating digit 4, then below that is digit 3 and then, digit 2 - Morphogen, released by the organizing center, which is patterning the limb bud  A diagram of a human leg Description automatically generated Morphogens - We'd come across again and again in developmental biology - Morphogen -- "form giving" substances, whose concentration varies across a gradient - Can directly activate cells at a distance - Produce concentration-dependent responses in receptive cells (Different outcomes at different concentrations) - Morphogenesis -- the time of embryogenesis, when 3D form of embryo forms - Morphogen can either diffuse through the cell signaling center, or there could be a relay of signals - However...  - The result will be a partial duplication of the limb bud - Small amount of morphogen is released, which is just enough to specify an extra limb 2 because limb 2 is specified at the lowest morphogen concentration - If we grafted a bit more, we'd expect to see an extra limb 3, but not 4 A diagram of a foot Description automatically generated with medium confidence Zone of Polarizing Activity = ZPA = ORGANISER - *Shh*, produced by the ZPA is likely to be the primary morphogen patterning in the limb's AP axis (Anterior Posterior) - *Shh* (Sonic Hedgehog, morphogen) is expressed highly in the ZPA - A gradient of Shh protein can be detected across the limb bud - A *Shh* soaked bead can mimic the action of the ZPA, transplanted in the anterior -- evidence that the morphogen is *Shh* - When done, we get the same duplication of the digits, just like when transplanting the ZPA - Mutations in *Shh* can lead to digit patterning defects - Mutation in a regulatory region of Shh leads to extra Shh being expressed/activated in the anterior - Shh is expressed in the anterior of the limb bud, as well as the posterior  THE HOX CODE - Roles in anteroposterior patterning of the embryo from flies to mammals - Mechanisms -- how do they work in development *Drosophila* -- Fruit flies example -- homeotic selector genes - Fly anterior/head to posterior/tail - Fly embryos look a bit different from human embryos - In the example, in the very early stage of the egg, cytoplasmic determinants (maternal effect genes) are really important in setting up the anterior-posterior axis - Then, from that, genes, such as hunchback, are expressed in the anterior, embryonic gene, further sets of genes are activated, which give polarity to the segments of the fly, but also set up a pattern of homeotic genes about the anterior-posterior axis - Homeotic selector genes -- genes located on the chromosome in order of expression - Not only expressed differently about the anterior-posterior axis, but they are also in the order, in which they are on the chromosomes - Two clusters, expressed in a nested pattern - Nested -- the most anterior genes (ultrabithorax) is expressed posteriorly from its initial expression, all the way to the posterior of the embryo A close-up of a bug Description automatically generated Mutations in homeotic selector genes can cause homeotic transformations - Mutations in them can cause homeotic transformations  - antp mutant has legs on its head, instead of antennae - A normal fly has one pair of wings on its 2^nd^ thoracic segment and a pair of halteres on its 3^rd^ segment - These mutations led us to an understanding of how the homeotic selector genes work Homeotic genes are conserved from flies to vertebrates - In vertebrates, homeotic genes are called hox genes - There's been a couple of events of genome duplication -- now there are 4 clusters within vertebrates - Instead of the clusters in drosophila, we have them 4 times -- hox a,b,c,d - They are still expressed in the same way and have a similar role - They give identity to individual segments along the anterior-posterior axis/ head-tail axis - Again expressed in the order, in which they are on the chromosome - First genes in the cluster are expressed the most anteriorly -- in the head - Final genes are expressed the most posteriorly -- in the tail - Nested pattern -- expressed all the way to the posterior, from where they are first expressed - Gene I is expressed from the head all the way to the tail of the embryo - Gene II doesn't start from the beginning, but goes all the way A diagram of a diagram of a person\'s body Description automatically generated with medium confidence Segments in Vertebrates - Vertebrates sort of have segments - Somites -- a precursor structure, which is found in the embryo along the head-tail axis of the embryo - Precursor of skeletal muscle, skin, cartilage, tendons, spine, ribs, skeletal elements -- the dermis -- along the embryo - Form from the mid layer of the embryo (mesoderm) in pairs - Somites form in pairs along the head-tail axis - What they give rise to is slightly different between the head end and the tail end - In the neck region, they give rise to elements in one place, but in the thoracic region, they give rise to elements in another place - Which part of the embryo the somites are going to become, is specified by the hox code, just like the segments in the fruit fly are controlled by the homeotic genes - Neck region expressed hox 5, thoracic region expressed hox 6 -- hox 9 - Nested pattern -- hox 5 is expressed from the cervical backwards  - Experimental evidence: - If you knock out a hox gene, it can lead to changes in segment identity -- what this segment will become - If you knock out hox 10, the lumbar region disappears, looks just like the posterior part of the thoracic region because it expresses hox 9, but not hox 10 - Ribs form on the supposed lumbar region A diagram of different types of cell division Description automatically generated  Learning outcomes of developmental mechanisms Know what the developmental concepts are, be able to interpret information and experimental evidence similar to that, discussed in the lecture, understand how they work A screenshot of a white paper Description automatically generated - Cleavage -- earliest stage of embryonic development. Initial cell divisions, but no growth of the embryo. Cells are totipotent (can become absolutely any part of the embryo). - Early development (blastocyst) -- here there are 2 lineages of cells. Inner Cell Mass -- pluripotent cells (can become any type of cell, except the placenta), which will form the rest of the embryo. The outer cell mass is responsible to form the placenta. - Apoptosis -- done to shape individual body parts of the embryo, digit separation - Gastrulation and morphogenesis -- when the embryo transitions from a one-dimensional structure to a three-dimensional one. Morphogenesis is the specific part of embryogenesis, where the 3D form starts to develop. Morphogen is induced from the ZPA (the organiser) and in varying concentrations. Morphogens give form to different digits in different parts of the embryo. - Homeotic selector genes in Drosophila are genes, which are expressed the way they are located on the chromosome. The regions, starting from the anterior and going all the way to the posterior express different hox genes. But once the hox genes have started expressing, they go all the way to the posterior axis. That way, different combination of genes tell the different parts of the embryo to differentiate. EVOLUTION OF DEVELOPMENT - How have changes in development facilitated evolution of adaptive novelty - How has evolution shaped development? Conservation of developmental mechanisms - Developmental mechanisms and individual genes that affect development are highly conserved amongst species - If that was not the case, we wouldn't be able to study model organisms to study development in humans - Example: Piebald trait - Mutation in the KIT gene leads to changes in pigmentation - Has the same effect between the mouse and the baby - Example: Anterior Shh gene causes digit duplication in chicks and humans - Same patterning mechanisms of limbs between chicks and humans - Shh being in the anterior, not only in the posterior leads to gene duplications double limbs  - Genes for developing wings in *drosophila* and fruit flies are strikingly similar, even though they are very distinct species - Hox genes are conserved from flies to vertebrates How do developmental mechanisms evolve? - Changes in the protein coding region of genes will affect everywhere that that gene is active -- more chances for a catastrophic change - Changes within the regulatory regions are much more likely to affect a subset of the gene functions -- more chances for a useful adaptation - If you completely knock out the gene function, the chances of that being catastrophic, and not adaptive, are quite high - That's because developmental pathways are re-used for multiple purposes, not just one thing - Example -- Shh gene - We came across Shh in terms of limb development and patterning the digits within the limb - It also does many other things in different parts of the embryo - One of its main roles is in the midline -- brain development, ear development, nervous system development, muscle development - Absence of Shh leads to incompatible with life embryos A close-up of a fetus Description automatically generated - Any changes within the coding region usually affect many structures, which will often lead to a decrease in fitness However, evolution via changes in the protein coding regions can occur, it's just less likely to lead to adaptive changes in evolution: - Evolution from changes in the protein coding region can happen, it's just less likely to lead to an adaptive changes in evolution - Example: Changes in ubx gene, which lead to leg loss from the abdominal segments in insects - If the insect is the ancestral form, it would have legs in all segments of the body (millipede/centipede) - At some point in evolution, the gene has picked up the polyalanine repeat - This mutation now represses leg formation, leading to an insect with 6 limbs  Gene duplication can allow diversification of gene function - If you duplicate a gene, one copy of the gene can continue to do its old role, and the new copy can evolve to do something different, or the functions and expression patterns of the original gene can be split between 2 copies - Example: duplication of the hox cluster between insects and vertebrates, genome duplication, involved in chordate evolution CHANGES IN REGULATION OF GENE EXPRESSION - Changes in pattern of gene expression - Changes in timing of gene expression - Changes in levels of gene expression Changes in pattern - Evolution of pelvic films in three-spine sticklebacks (fish) - Marine species, they mostly have fishy predators, spines were for protection to avoid being eaten - The pelvic spine forms as an action of the *pitx1* gene - 12 000 YA, this species got trapped in the great lakes in the USA and became freshwater - Predation changed; they were now mostly predated on by invertebrates - Some of the invertebrates could get a hold on them by the spines - Some of them lost the spines, including the pelvic spine - Had an adaptive advantage, they were eaten less - The way the adaptation happened, was by a single enhancer, which changed expression in the pelvic region - You can't knock out the whole gene because it has important functions and knocking it out would lead to the fish dying, you can change just a region A close-up of a fish skeleton Description automatically generated - Changes within just one region of the embryo, occurring because of changes in a single enhancer Changes in Hox gene expression - Chick and mouse have very different neck lengths, caused by changes in hox gene expression - Neck/cervical region of embryos expresses hox 5, thoracic/ribs region starts to express hox 6 - Changes in the boundary between hox 5 and hox 6 leads to changes in the position of the boundary between the neck and the thorax (ribs) - In chicks, the region, which express hox 5 and not hox 6 is much longer than in the mouse, leasing to a longer neck - This also changes where limb buds form - Limb buds form at the boundary between hox 5 and hox 6 in chick, it's also further back Changes in pattern: Loss of snake forelimbs - Ancestrally, snakes would've had limbs - When hox 6 is expanded along the whole flank of the snake, the forelimb development is repressed entirely - Changes in hox genes can have massive effects on the anteroposterior axis of the embryo - Beak shape changes in Darwin's finches - Some finches on some of the islands have short beaks, some longer, depending on what they eat - In terms of development, this relies on Bmp4 levels - Increase in Bmp 4 leads to wider beaks, - Such changes are vital for species to inhibit new niches -- vital evolutionary changes Modularity underpins regulatory change - Modularity of regulatory regions allows expression to be altered in just one region -- allows changes in evolution - Pitx1 is under the control of variety of enhancers, which control its expression in different parts of the embryo - =\> knocking out the function of just 1 enhancer, leads to changes in expression in just one region - If control of the expression was NOT modular, if there was a single regulatory region, controlling expression everywhere, - =\> knocking out function in that regulatory region, would kill the organisms, rather than making it adaptive - Animals have modules too, which can evolve independently - In kiwi, the hind limb is much more developed than the forelimb - In bats, the forelimb is much larger than the hindlimb because they need their forelimbs to fly - Segments are great examples of developmental modules - Insects -- ubx, repressing leg development in the abdominal segments Summary: - Developmental mechanisms are conserved - Changes in regulatory regions are more likely to lead to adaptive changes in the embryo and therefore, the organism, rather than changes in the protein coding region - Both modularity of regulatory regions and duplication and divergence are really important processes in facilitating adaptive change