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BIOC 212
Maxime Bouchard

Model Organisms Introduction • There are about 10 million species on Earth. • Researchers have selected model organisms to study all species (i.e.: zebra fish, mouse). These organisms are used to study our cells. Basic Process of multicellular organism Development. • Broken down into four basic components. • All occur in parallel and without global supervision (occurs due to genetic programming). • Understanding how the genomic information encodes something so complicated. Proliferation Human figure details visible in human embryo after just 8 weeks. Toes, fingers, most organs and the • brain are visible. Until week 38 (around birth), proliferation is mostly what takes place. • 8 weeks: ~35 mm (Advanced organogenesis). At 38 weeks ~50 cm. • Adults have 6x10 13 cells which originated from a single zygotic cell. Lining up these cells in order would wrap 75 times around the earth. • There are ~250 different cell types (i.e.: neurons, muscle cells, adipocytes). Thus, cell specialization must occur. Cell Specialization • Formation of germ layers. Extremely important step to generate 3 types of cell: 1. Endoderm: Precursor of gut, liver, lungs, pancreas. 2. Ectoderm: Forms the outer, protective layer called epidermis and nervous tissue. 3. Mesoderm: Precursor of muscles, skeleton, kidneys and connective tissue. • Endoderm forms the inner tube that connects the two ends. Organs grow out of it. • Forming the three cell types from the first ball of cells is the first step of diversification. • Gastrulation (formation of mesoderm). - Involves epithelial-mesenchymal transition. This also occurs in cancer (cancer cells undergo this transition to migrate around the body). - Cells become more pyramidal in shape and move within the embryo. Cells lose their lesion to become mesodermal. - Snail induces epithelial cells to become mesoderm by repressing E-cadherin (which links cells together in adheren (tight) junctions). Cells then lose lesion and change fate. • Second way to create cellular diversity: Sequential Induction - Two different tissues (A and B) adjacent to one another will interact. Within tissue A, tissue C will form because of this interaction. Tissue C can then interact with tissues A and B to form tissue D. The more tissues there are, the more possibilities for interactions. - This occurs in the eye (and a few others places too). Two individual, adjacent cell layers have distinct identities until retinal layer interacts with the pigmented epithelium (outer layer). These cell types do not form individual layers but are rather, combinations of molecules that are expressed. - Interaction between cell layers allows for differentiation into more layers. • Changing the fate of your neighbors - Group of cells sends signals to neighboring cells to change their fate. - Cells send an inductive signal. This ‘induces’ a fate. Cells receiving the signal, will interpret it, and turn on a new cellular program which will change their identity. - An example: Drosophila wing starts off as a pouch of cells that unfolds to form the wing. The midline (separates ventral and dorsal sides) of the pouch will form the edge of the wing. Wg is a morphogen and will travel to neighboring cells to turn on dll. Cells that are surrounding the midline will begin to turn on dll. Vg is expressed in a wider region between cells are more sensitive to Wg signal in this case. Same molecule secreted from midline and neighboring cells will turn on both dll and Vg but then those cells further away will not active dll, only Vg. This creates 3 cell types (those that express all 3, those that express only dll and Vg and those that expressed only Vg). Morphogen - Principle of a morphogen: one inductive signal creates multiple (in this case, 3) cell fates. - Acts at different concentrations. At high concentration, signal will drive a certain information. At medium concentration, it will drive another kind of information etc. - Morphogen itself does not determine fate, it is the way in which the cells receive it. - Molecule is secreted from a source. - It is time dependent. The longer the cell is exposed to a morphogen, the more it has a chance to move on in its interpretation of the signal. - Usually morphogens are sticky. - Can be modified and regulated by different mechanisms (i.e.: ECM or proteins surrounding the neighboring cells can exert an influence). - Induction over a short range may take place first. Proliferation can then occur so that the range of action of a single morphogen can be very large. - French flag model explains how morphogen acts (a range of concentrations where the cells act). - Different systems can receive the same morphogen signal. It is not the morphogen that determines the fate of the cell. It is the cells’ method of interpretation. This allows for a single morphogen to be used in many systems. • Two ways to create a morphogen gradient: 1. Classical way to create a morphogen gradient: source inducer diffuses. A gradient moves away from the source. 2. Inducer can be expressed everywhere and an inhibitor (in the form of a gradient) acts. Close to the inhibitor, the inducer is unable to act. There exists a gradient of inducer activity. This occurs in flies. • The result is the same for the both methods. • Some important signaling pathways for embryonic development: - Wingless (in flies) and Int (integration of a virus in vertebrates) together become Wnt. - Some were discovered in the embryo. Segmenting Genes • Much information we know from the pathways comes from a single screen where mutations affecting the patterns of the fly larvae (from Drosophila) is studied. Mutations are induced, and the patterns of results are studied. • The pathways being studied know are for Cancer and other diseases. • Posterior end has naked cuticle while anterior end is covered in bristles. They have a specific pattern and an identity for each segment of the larvae. • The mutations being generated would generate only 3 kinds of phenotypes: 1. GAP gene: a whole series of segments were missing 2. Pair-rule gene: either even numbered segments or odd numbered segments were missing. 3. Segment polarity gene: Correct number of segments but polarity is wrong. • About 15 patterning genes in 3 categories were found. • A mutation was then induced in the mother. Additional 8 genes were found that are involved in tail and anterior head formation. These gens are referred to as the maternal contribution. • The reason why these three categories exist is because there is a sequential event that occurs. Steps: 1. The polarity of the embryo was defined. Morphogen creates a gradient to do so. At high concentration, the hunchback gene is turned on. 2. Cells respond to a gradient to express three cell types. 3. The three types interact to act in sequential induction. 4. Response to primary segmentation. Two genes being activated (one is even skipped the other is fujitso). Alternative segments express one of the either. The Canonical Wnt Signaling Pathway • Often related to hair phenotypes because they study the cuticles. • Without this pathway, there is no signaling at the membrane. This leads to formation of a complex that is controlling beta-catenin that is the key molecule in the pathway. • CKI alpha and GSK phosphorylate beta-catenin. • They then enter ubiquitination and degradation pathway. • TCF then binds Groucho which recruits a repressive complex that is essentially shutting down genes. Wnt binds receptor frizzled and co receptor LRP. This decomposes the built complex. Beta-catenin • cannot be phosphorylated anymore and it accumulates in the cytoplasm, translocates to the nucleus where it binds TCF and displaces Groucho. This turns on transcription of a series of genes needed in proliferation. • In vertebrates, many Wnt have been found (1-5). They are linked to different diseases such as Schizophrenia. • In Wnt 4, the genes are for pseudo males. Thus, females is not the ground state. Instead, a single drives the gender towards female. • Wingless and Int are the same thing. Wingless is the molecule that was identified in the fly. In vertebrates, there were other groups of people that identified Int gene. They later realized these two molecules were the same in both species. So the term ‘Wnt’ was created to encompass both. In flies, some people perform the term wingless. • Canonical pathway: refers to the dominant pathway, the most well known / best characterized pathway. Once a second pathway downstream was identified, the original one adopts the term ‘canonical’. Sonic Hedgehog and BMP • Act as morphogens in the neural tube. • The spinal cord / neural tube is visible in the early embryo in green. When cross sectioned, the image to the right is obtained. The cells are proliferating and will eventually give rise to different kinds of neurons. In order for the neurons to obtain their distinct identity types, they rely on the morphogen sonic hedgehog. • Hedgehog was identified as a mutant in the fly first. Later, three were found in vertebrates (and labelled with respective to the video game); sonic hedgehog, indian hedgehog, desert hedgehog. • Sonic hedgehog is the most well known and best characterized of the three. It is also the most active of the three. It is essential in the patterning of the spinal cord. • During development, a cord of tissue runs all the way down below the spinal cord, the noto cord. This tissue expresses sonic hedgehog. It first signals to the floor plate (lower part of the spinal cord) and turns itself on in this tissue. It is a transfer of expression. Sonic hedgehog then starts to signal from the dorsal side, in the ventral direction. It then provides the identity to different cells. • Closer to the floor plate, there will be a higher concentration of sonic hedgehog, inducing one type of fate. Lower down, different fates emerge according to concentration levels. This is seen by the various colors in the diagram below. These domains have different combinations of molecules. Sonic Hedgehog Signaling Pathway • Double system: activation and repression occur simultaneously. This allows precision. • The challenge is to generate clear borders between different cell types. The double system allows for this to be possible. • Ground state: refers to non activated state. Absence of ligand. • Pathway: 1. Patch I represses the Smo receptor. 2. PKA phosphorylates Gli3. 3. Gli3 is phosphorylated, cleaved by proteolysis. This generates an inactive repressor molecule. 4. Gli3 translocates to the nucleus and binds target genes to actively repress them. • Repressor function allows for repression of genes. • Sonic hedgehog represses its receptor and then becomes inactive to represses Smo. This is a double negative that turns positive. • Gli3 becomes an activator from being a repressor. Once target genes are activated, the amount of sonic hedgehog will determine how much activation • occurs. It is therefore a gradient response. The ligand does not reach far distances to the tissue there remains in ground state. • Cyclopamine: Smo repressor. System remains inactive, despite the presence or absence of sonic hedgehog. Acts like sonic hedgehog mutant. The whole pathway is shut down by this molecule. Cyclopia was born as a cyclone because its mother ingested the plant that contains a cyclopamine • molecule. This molecule is the inhibitor of Smo. • Sonic Hedgehog in the embryo, acts to specify the midline to differentiate this line in the body. • A cyclone is the fusion between in the eyes because the midline does not form. In a sonic hedgehog mutant, the midline does not form properly and different organs will fuse together (i.e.: one kidney). • Mutants of sonic hedgehog can lead to loss of Shh signaling which causes holopresencephaly (variable penetrance). There is a range of severity that can occur and the most dramatic cases are lethal. BMP • Acts as sonic hedgehog’s partner. Expresses from the dorsal side towards the ventral side. It is the inverted signaling pathway. Less well characterized than sonic hedgehog’s pathway. • • Roof plate expresses BMP and signals downwards to turn on combinations of transcription factors in a concentration dependent manner to provide different identities to different cells along the ventral axis. • Is involved in spinal cord patterning. Re used in different systems. • Canonical Pathway: 1. BMP binds its receptor (formed of a dimer of receptors 1 and 2). These molecules dimerize upon BMP binding. 2. Receptor 2 will phosphorylate receptor 1. This is the activation trigger. 3. Phosphorylation of receptor 1 recruits Receptor-regulated Smads (R-Smads): Smads 1, 5 and 8. There are three different proteins that carry out the same function. ** Phosphorylation can be repressed by inhibitory Smads: Smads 6 and 7. This allows a break in the pathway. 4. Smads 1, 5 and 8 translocate down to recruit co-Smad: Smad 4 which binds to anyone one of the three forming a complex. 5. This complex translocates to the nucleus, binds target genes and turns them on in a graded manner. The more BMP, the more activation of the pathway. • Non canonical pathway: 1. Binding of XIAP activates tyrosine kinase TAK1. 2. Different pathways are activated. TAK1 activates the MAPK pathway (MKK), JKN pathway and NF-kB pathway. 3. Those pathways are involved in different target genes. The response of these genes varies. Controlling Cell Fate From the Nucleus • Combination of gene regulatory proteins (transcription factors), which are often activated by inductive signals, will determine the fate of a given cell. • Smads are transcription factors. • Gene regulatory proteins bind enhancers (DNA sequences), which can be located upstream or downstream of the gene. They bind specifically based on their identity. They act as on the on switch to turn on transcription of the gene. Hox Genes • Type of transcription factors, first identified in drosophila. • The genes are all found in a cluster, lined up one after another. At one extremity, they are expressed in the anterior part of the body and the opposite on the other. There is a correlation between the position along the chromosome and the expression domain in the body. Well conserved in bilateria. • • Important for organizing the pattern of body tissue. Provide identity to the tissue they are expressed in. They tell the cells where they are in the body. • Antp and Ubx are two types of Hox genes often seen mutated. • In a wild type fly, there are 3 body segments. In the second segment, the wings are present and in there are haleteres in the third segment. Haleteres are used by flies to maintain balanced. • Homeotic transformation: change cellular identity towards a different fate. - Ubx mutant develops a second set of wings in the third segment as opposed to haleters. - Ubx over expression in the second segment causes wings not to be developed. Instead, haleters develop in their place. - Antenapedia (Ant) cause flies to develop legs in the place of antennas. • Hox genes are well conserved among species. In the mouse, there are 39 Hox genes found in four clusters (on 4 different chromosomes). In vertebrates, the clusters were duplicated twice such that four clusters developed. • Colinearity: the alignment of genes along the chromosome correspond with their expression pattern along the embryo. Unique pattern provides identity. • HoxB2: Expressed in the posterior level but does not have a sharp boundary. • HoxB4: Expressed in the posterior level but has abrupt stop and sharp boundary. • Removing HoxA10 (normally expressed in the lumbar region) and expressing it all along the vertebral column: the whole spinal cord becomes lumbar. The vertebrates do not form ribs. Thus, each segment is a read out of its gene. The reverse experiment (inactivation of HoxA10/C10/D10 which all perform the similar function - triple knockout): lumbar and sacral vertebrate becomes thoracic like. Cell Interactions • There are two ways to create diversity: 1. Before cell division - Asymmetric cell division. The determinant (something that will influence the fate of the cell) is only exposed on one side of the cell. Upon division, only one of the two daughter cells will express the determinant. 2. After cell division - Post division, one of the two daughter cells is influenced by its environment. • One example: Specification of sensory mother cell (one cell that is specified to become the precursor to the whole sensory organ). - Performed by interaction of two cell post division. - After several divisions, the precursor cell develops a socket cell (holds the bristle) and a shaft cell (the bristle). Upon the second cell division, one cell normally dies. The other divides into a sheath cell (protects the neuron) and a neuron (links organ to CNS). - This occurs by way of lateral inhibition. All of the cells of the ectoderm compete against one another. Each cell does not want its neighbor to become a sensory mother cell. They all repress each other. - At some point, equilibrium is broken. One cells wins over its neighbors. Once the neighbors become weaker, they shift their balance so its other neighbor can win. This generates a pattern of nicely spaced out, separated cells that lock their fate in the ectoderm. - The winning cells will leave the ectoderm and become the sensory cells. The losing cells do not die - they simply accept the second fate. - Two cells are equivalent bus a transient bias creates a small asymmetry where one cell becomes better than the other. A system comes along to amplify this asymmetry. By one cell being repressed, it creates a positive feedback loop. - The Notch pathway is what is responsible for shifting the balance at the molecular level. - Delta does not diffuse to multiple tissues. It remains in place. Only cells that are adjacent interact with one another. Delta activates Notch in one cell and this creates a domino effect of negative regulation. The cells compete to a point where one delta is stronger than another cell which will prevent the activity of the other cell. - Diagram above is a more detailed image of the pathway. - Cis inhibition of Notch (delta prevents Notch activity) is an all or nothing response. When Delta is in a cell, it binds Notch in the same cell (thus, cis) to prevent its function. This occurs in both cells. - In trans (between two cells), Delta acts as an activator. Delta goes to the membrane and binds Notch on the other cell. This ligand-receptor binding turns on transcription as a graded response (as opposed to cis which is an absolute response). The more delta, the more Notch response. Notch Signaling Pathway • Notch signaling occurs by interactions between neighboring cells. • Notch and Delta exist on two neighboring cells. • Signaling Pathway: 1.Notch is glycosylated and processed by protein cleavage so that it may be re attached to the cell surface membrane. 2.Notch binds Delta and a conformational change in the protein occurs that induces cleavage of the transmembrane domain. TACE Proteases cut at one position and gamma-secretase cuts at a different position in order to release the NICD (Notch Intracellular Domain) into the cytoplasm. 3.NICD is a transcription factor that translocates to the nucleus. NICD transcriptional factor displaces the co-repressors (CoR), binds CSL transcription factor and brings in co-activators (CoA). 4.Turns on transcription of target genes (typically Hes and Hey gene families) • The signal from membrane to the nucleus usually goes through several phosphorylation steps. However, in this case, it is a piece of the cell receptor’s intracellular domain that directly enters the nucleus and performs the required function. • The extra cellular domain of Notch is released by protein cleavage and remains bound to Delta. This can be problematic because the system is blocked by the presence of this interaction. The degradation of this complex is important. • Mindbomb is a fly mutant that was characterized by researchers. It was first discovered and then only later understood. • This system is involved in a number of pathways. • The specification of the sensory mother cell occurs by lateral inhibition where a cell ‘wins’ over its neighbors, generating a repetitive pattern of equally spaced cell types.
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