exam with short answer questions. Topic: reproduction
LECTURE 8 – MECHANISMS FOR CELL DIFFERENTIATION IN DEVELOPMENT COMMON OVERALL PATTERNS IN DEVELOPMENT When comparing the mouse and human embryo at different stages, it is noted that they are very similar as both hatch from the blastocyst, developing in a similar manner to humans; it also develops much quicker, allowing for effective use as a model. Similarly, chicken develop in an analogous manner to humans, despite the obvious differences in the later phenotype. Gastrulation is the point in develop at which pluripotent stem cells become the multipotent germ layers, differentiating into the cell types that make up the body; for example, the mesoderm forms the muscle, blood, bone, and cartilage. However, if not done correctly, there are consequences for the rest of development and hence, it is often deemed the most important point in ones’ life. Across a variety of organisms, the process is similar with cells moving and invaginating, forming the multipotent layers; this similarity has given rise to the use of various animal models with the process similar, but since they develop quicker and can be done outside the body, there are many advantages. During gastrulation, a tube is formed at the back end which eventually goes to the mouth with this process causing cells to proliferate, differentiate, migrate, and communicate. COMMON CELLULAR AND MOLECULAR FEATURES OF DEVELOPMENT To understand this, many animal models are used. The four essential cellular processes across multicellular organisms are: 1. Cell Proliferation: This is done as the organism must get larger 2. Cell Specialisation: We begin as a single cell, but must go to 220 cell types. 3. Cell Interaction: These interactions can be physical, or through signalling with the appropriate signals at the right time triggering migration or differentiation. 4. Cell Movement: Without cell movement, development cannot proceed appropriately. Underpinning the cellular processes are various molecular mechanisms, with cells beginning with an identity that must either be renewed or changed. These include gene expression (selective expression change in proteins produced change in effector functions), autocrine and paracrine signalling, epigenetics (determine what genes are expressed by remodelling chromatin), and microRNA. Part of the genome codes for proteins that are responsible for multicellularity, where more genes dedicated to this in organisms with more cells. This is seen as there are more transmembrane molecules used for cell adhesion, signalling, and ion transport in worms then unicellular yeast, whilst there are more gene-regulatory proteins in humans when compared to yeast. The development of the cerebellum requires Engrailed-1, with the lack of this protein preventing cerebellum formation. However, it was noted that by using the Engrailed protein of the fruit fly (Drosophila) helped cerebellum growth, showing the similarity across organisms in the production of the same protein. COMMON MECHANISMS OF MAINTAINING CELL IDENTITY CELL MEMORY When a new cell is produced, it retains the memory of its cell type (i.e. a muscle cell will divide into two muscle cells), with neurons maintaining their identity to prevent them from becoming another cell type. One of the key methods of doing this is the use of positive feedback loops, with the expression of a protein turned on, with this protein then moving to the promoter region (transcription factor) to produce more of this protein with this loop ensuring that the cell identity is retained, even after cell division. However, there are other mechanisms of cell memory such as DNA methylation and chromatin remodelling. MASTER GENES AND GENE CIRCUITRY Generally, this is where there is a signal that causes the expression of a master gene, which is responsible for a circuit, changing the expression of other proteins that then cause the development of various organs. In the development of the eye in the Drosophila, it was found that there was a pocket of cells that expressed ey (the master gene) with this then placed nearby, forcing the expression of this gene in the wrong area and hence, caused the production of an eye. However, the lack of innervation in this area meant that this eye could not see. Another case is in muscle development, with the master gene being MyoD with forced expression in chicken skin fibroblasts then creating muscle cells; this was observed as it was both long and multinucleated, as well as contractile. MORE CELLS AND SPECIALISATION – INCREASING SIZE AND COMPLEXITY OF THE EMBRYO There are many mechanisms that control the specialisation including: - Combinatorial Gene/Protein Control: Since there are limited numbers of genes (20,000), combinations of these genes are needed to control development. In each cell division, the induction of a regulatory protein/s cause differences in the gene combinations of each cell, increasing the complexity of the organism. - Symmetric and Assymetric Division: In assymmetric division (as is observed in the case of the P granules in the C. elegans germ line), the cells are born differently with the granules moving to one side of the cell, with the cell cleaved to ensure all granules are on one side and hence, in one cell. Specifically in the case of the C. elegans, it is done until the 32 cell stage, with the cell that contains the P granules producing the germ cell lines. Symmetric division is seen when the sister cells produced are the same, but from here, the provision of signals means that it forms differently whilst the other is maintained due to the lack of a signal. MORPHOGENS – CRITICAL INDUCTIVE SIGNALS IN DEVELOPMENT Morphogen: A secreted molecule that goes into the environment, acting as a signal. These are integral in forming gradients that alter the development of the organism. In cases where a morphogen is released, the concentrations are higher at the source whilst the diffusion of the morphogen means that the levels decrease further away from the source, meaning it has less of an effect on development (less likely to bind to a cell further away). However, morphogen inhibitors can also act, with the source of the inhibitor at a point on the opposite side thereby having the opposite effect and if in conjunction with the morphogen itself, the final signal is uniform distribution of the inducer. Different morphogens lead to different cell types, as it provides short range instruction in the embryo regarding development in terms of proliferation, shape changes, migration, differentiation, and death. This information must be transduced from the morphogen through binding onto a specific receptor and hence, cells without this receptor will not be able to respond to the morphogen. Ligand classes can also have inhibitors that prevent ligand binding at the receptor. - Sonic Hedgehog Gradients: In the wing bud of the chick, there is a polarising region which is responsible for the development of the limb. If removed and placed in a different location on a new chick (meaning there are now two regions but in different locations), it is noted that the morphogen gradients cause a double-up of digits. A similar concept is observed with Sonic Hedgehog and FGF10 gradients in lung branching, as FGF10 binds onto its receptor but then Sonic Hedgehog is secreted at the bud, inhibiting it directly in front, creating two new centres on the sides, which then helps to keep the cycle perpetuating. CELL-CELL ADHESION There are a class of cadherin genes that code for cadherin proteins. The proteins are dimeric with calcium binding causing a conformational change, activating them. If two active dimers on each cell interact, they bind and lock in with respect to each other. E-Cadherin and N-Cadherin differ and only bind onto the same type of cadherin (i.e. E- Cadherin binds to E-Cadherin and so on). When sorted out (through calcium addition), it is noted that they bind based on their expression, as well as level of expression (cells expressing higher levels of E-Cadherin will bind to other cells expressing more E-Cadherin). These are critical in neural tube formation as is observed in the Xenopus with it forming it from the surface ectoderm. This is because the surface ectoderm has E-cadherin whilst N- Cadherin is only in the Neural Tube. As such there is a sorting out process as shown above. The neural plate invaginates into the neural tube whilst the neural crest is above it, separating it from the surface ectoderm. They are also seen in the Neural Crest movement, with Cadherin 7 found in the PNS. N- Cadherin is in the neural crest, with the downregulation of this coupled with the upregulation of Cadherin 7 causing the migration away from the cluster and at the right locations, Cadherin 7 is then downregulated with N-Cadherin upregulated again (once differentiated). CELL-ECM ADHESION To help stay put, Integrins bind to ECM proteins at the basement membrane where they adhere. These are seen in the linings of blood vessels, skin, etc. to prevent the cell from moving out. Integrins are large proteins that stick out of the membrane (and therefore cell), binding to ECM proteins like collagen, preventing the cell from moving excessively. LECTURE 9 – MODELLING DEVELOPMENT WITH EMBRYONIC STEM CELLS ICM TO PRIMITIVE ECTODERM – A CRITICAL STEP IN DEVELOPMENT The transition from the ICM to the Primitive Ectoderm happens between days 3.5 and 5.5 in mouse development, with it like humans despite the developmental period differences (20 days in mice when compared with the 9 months in humans). Further similarities include the process of hatching from the zona pellucida, and the subsequent binding to the uterine walls. The ICM is a small pocket of cells (15-20) which are pluripotent, forming all adult cells seen in the full organism. The primitive ectoderm forms all germ layers with it needing differentiation, as well as maintenance of its pluripotency. However, this process is very complex as it goes from 30 cells to 6500 cells within hours; this rate is much faster than cancer cells. For this process to occur, the key requirements are: - Maintenance of Pluripotence: This is because both the ICM and Primitive Ectoderm are pluripotent - Differentiation Signals: The ICM must convert to the Primitive ectoderm, which requires various signals for differentiation. - Proliferative Burst: As explained earlier