Exam 3 Flashcards
- Describe the “Life Cycle” of a typical animal. Tips: Focus on the most important steps, those that are shared by most animals, and how does each stage influence the subsequent stages and development in general.
The steps of the life cycle includes fertilization, cleavage, gastrulation, organogenesis, lrval stages and metamorohposis for some species, maturty and gametogenesis.
Immeditatly following fertilization rapid cleavage begins to form a blastula.
During gastrulation mitotic division slows down and gastrulation begins where the three germ layers are formed, the ectoderm, endoderm and mesoderm. The three germ layers are formed through invagination of the blastula. It’s during gastrulation the bilateral symetry is formed which seperates our motuh from our anus by the GI track.
Organogenesis is when the organs begin to develop from the three germ layers. Different organs generally develop from different germ layers such as ectoderm develops into skin and nerves, the endoderm the digestive track and the mesoderm muscles and bones etc.
Gametogenesis often doesn’t occur until species are matured and it’s when germ cells are seperated from the smatic cells often in gonads.
- Define what “stem cells” are. How can you prove that a cell is a “stem cell” experimentally? What their relevance as a medical tool?
Stem cells is any cell in the body that can regenerate itself by continuant proliferation and that can give rise to more differentiated daughter cells with specialized functions. Stem cells can differ in their potency, the more powerful a stem cell is, the more potent it is. More potent stem cells produce less potent stem cells. The different types of potent stem cells are:
Totipotent – Give rise to any other cell type in the organism
Pluripotent – Give rise to the entire organism except the placenta
Multipotent – Organ/tissue-specific cells which can give rise to all cells in that organ/tissue
Oligopotent – Give rise to for example different lymphoid cells
Bipotent – Can only form 2 types of cells
Unipotent – Regenerating cells that only form one type of cell
Since stem cells are able to regenerate damaged tissues it can be used for various diseases. For example induced-pluripotent stem cells have been used to treat macular degeneration of the retina. Using stem cells as treatments they are patient specific
- Select a model organism and describe its advantages and the disadvantages, and at least one key discovery that was made that to it.
Pros: Drosophila are easy to breed, prolific, tolerant of diverse conditions, its genome is fully sequenced and annotated, cheap, no ethical restrictions, 60% of genome is homologous to humans.
Cons: The brain, cardiovascular, respiration anatomy of Drosophila Melanogaster is different from humans, less complex and adaptive immune system as in vertebrates, effects of drugs on the organism might differ strongly.
Drosophila melanogaster was used by Thomas Hunt Morgan for his discovery of genes and their placement on chromosomes (polytene chromosomes).
- Describe the concept of differential gene expression. Why is it important, and how can it be studied?
- Every cell nucleus contains the complete genome established in the fertilized egg. In molecular
terms, the DNAs of all differentiated cells are identical. - The unused genes in differentiated cells are not destroyed or mutated, and they retain the
potential for being expressed. - Only a small percentage of the genome is expressed in each cell, and a portion of the RNA
synthesized in the cell is specific for that cell type.
Gene expression measurement is usually achieved by quantifying levels of the gene product, which is often a protein. Two common techniques used for protein quantification include Western blotting and enzyme-linked immunosorbent assay or ELISA.
- The protein β-catenin can act as transcriptional activation downstream of WNT signals. Design an experimental research project to study β-catenin’s function.
You can use ChIP (chromatin immunoprecipitation) to find new enhancers. ChIP identifies where proteins bind to DNA sequences. We start by cross-linking (covalently bound) DNA which allows us to “freeze” where a certain protein sits at a specific moment because it is covalently bound there. Now we can lyse the cell and extract the complex DNA proteins with for example restriction enzymes. Now we can now use an antibody that recognizes the specific protein and binds to it. These antibodies are bound to something that is “heavy” such as small balls of agarose or magnets, since it is heavy it will precipitate (by for example centrifugation) to the bottom of a tube and since the antibody is bound to the protein, which is bound to the DNA, the DNA will precipitate to the bottom as well. Now we can wash the sample which discards the supernatant leaving only the immunoprecipitation. Now we remove the protein by for example proteinases, and to reverse the crosslinking we can use high temperature to purify the DNA. Once the DNA is purified we can perform quantitative PCR and then DNA sequencing, we can map the position of the genome where this piece of DNA is along the genome. In this way we can find new regulatory DNA sequences (enhancers) which transcription factors induce.
Define “Cell Lineage Tracing”, How could it be achieved?
Lineage tracing is the identification of all progeny of a single cell. Lineage tracing provides a powerful means of understanding tissue development, homeostasis, and disease, especially when it is combined with experimental manipulation of signals regulating cell-fate decisions. In lineage tracing, a single cell is marked in such a way that the mark is transmitted to the cell’s progeny, resulting in a set of labeled clones. Lineage tracing provides information about the number of progeny of the founder cell, their location, and their differentiation status.
There are different techniques to lineage tracing such as direct observation, vital dyes and genetic markers. Direct observation is the oldest technique of lineage tracing, it was done in 1905. They studied early cleavages by light microscopy. A more modern technique is the use genetic markers. A very common genetic marker is fluorescent proteins such as GFP, these are introduced by transfection or viral transduction. Genetic markers in comparison with vital dyes do not “spill over” to neighbouring cells.
- What is Pax6? What does pax6 teach us about development and evolution? 5 points
PAX6 protein function is highly conserved across bilaterian species. For instance, mouse PAX6 can trigger eye development in Drosophila melanogaster. Additionally, mouse and human PAX6 have identical amino acid sequences.
The tissues of the vertebrate eye arises from different embryonic origins, the lens and cornea are derived from the surface ectoderm, the retina and the epithelial layers of the iris and ciliary body comes from the anterior neural plate. The activation by transcription factors and other inductive signals ensure correct development of the eye. The lens placode development is controlled by Pax6. The single eye field is separated into two, forming the optic vesicle and later (under influence of the lens placode) the optic cup. The lens develops from the lens placode (surface ectoderm) under influence of the underlying optic vesicle. Pax6 acts in this phase as master control gene, and genes encoding cytoskeletal proteins, structural proteins, or membrane proteins become activated. The cornea forms from the surface ectoderm, and cells from the periocular mesenchyme migrate into the cornea giving rise for the future cornea stroma. Similarly, the iris and ciliary body form from the optic cup. The outer layer of the optic cup becomes the retinal pigmented epithelium, and the main part of the inner layer of the optic cup forms later the neural retina with six different types of cells including the photoreceptors. The retinal ganglion cells grow toward the optic stalk forming the optic nerve.
Claudio lecture: page 546 (518) book
The development of the eye starts at about day 8.5 (in mice), the optic vesicle (OV) (an ectoderm derived protrusion of the encephalon) and presumptive lens ectoderm (PLE or surface head ectoderm), when these come in contact they start to exchange signals which leads to a thickening of PLE that forms what is known as a placode, the placode then invaginates to form a lens pit which follows the inward cell movements of the optic cap, on day 10-11 the lens pit pinches off from the surface head ectoderm and forms a small hollow ball of cells called the lens vesicle.
The development of the eye undergoes reciprocal induction.
The optical vessels is the inducer of the lens, only the surface head ectoderm is competent to respond. The optical vessel which is the inducer secretes paracrine factors BMP4 and FGF8, these molecules reach the surface head ectoderm/PLE which have receptors for them, these molecules activate a distinct transduction pathway within PLE which culminates in the activation of transcription factors in the lens cells, these transcription factors are Sox2 and L-Maf cooperate to turn on all genes that are required to make a lens. Pax6 is the master control gene controlling eye development. The Notch pathway is also important for this and becomes activated when cells come in contact when delta proteins are integral membrane proteins and cannot travel across the extracellular space. At this point the tissue layers (optic vesicle and PLE) are close and once the lens starts to form it starts secreting factors causes the optic vesicle to become the optic cup which differentiates into two layers the pigmented retina and the neural retina.
- Describe the mechanisms of sex determination and how they differ in different groups of animals. Why does sex exist?
In mammals the Y-chromosome contain the master-switch gene for sex determination, called the sex-determining region Y or SRY in humans. In most normal cases, if a fertilized egg (zygote) has the SRY gene, the zygote develops into an embryo that has male sex traits.
In drosophila the Y-chromosome doesn’t determine sex, but it is important for forming sperm in adults.
Sex determination depends from species to species. In mammals the presence of a chromosomes XX and XY determines sex. The Y chromosome determines male sex because the Y chromosome contains the SRY gene which produces a protein called sex-determining region Y protein that activates a testis-forming pathway which leads to the production of anti-mullerian hormone, testosterone and dyhyrdrotestosterone further leading to male sex characteristics.
Birds have a different sex determination, theirs is dependent on chromosomes ZZ and ZW, there is no counterpart to the SRY gene on the Z or W chromosome, but instead it is dependant on DMRT1 gene, we also have this gene in mammals and they are a member of the testis-forming pathway, but in order for testis development 2 DRMT1 alleles are needed. DMRT1 gene is present on the Z chromosome, making ZZ the male and ZW the female in birds.
Drosophila also have XX and XY as we humans do, but in this case the Y chromosome doesn’t determine sex but rather the sperm production capabilities in Drosophila. The sex determination in drosophila is determined primarily by the ratio of the number of X chromosomes to the number of autosomes. There is a balance between female-determining factors encoded on the X chromosome and male-determining factors on the autosomes that determines the sex-specific pattern of transcription. XX, XXY, XXYY are females, XY and XO are males.
Can Homo Sapiens be considered a “model” organism? Can you think of research questions that involve humans that discover new knowledge in the fields of developmental biology and genetics?
An organism suitable for studying a specific trait, disease, or phenomenon, due to its short generation time, characterized genome, or similarity to humans. Homo sapeins wouldn’t make good model animals since ethical issues, long life span, expesnive etc. However using humans for example drug testing is being done in late satages as well as certain disease treamtents for terminally ill patients etc.
Describe gastrulation. Why is this developmental stage so important?
During gastrulation the formation of the primitive streak occurs, cells will migrate to the midline of the gastrula and cause a thickening, this is what the primitive streak is. At the cranial end of the primitive streak the primitive pit is formed by epiblast cells forming a circular cavity, then migrating epiblast cells will join the primitive streak at the cranial end forming the primitive node, this is what becomes the primary tissue organizer where transcription factors (TGFB, Nodal, WNT, BMPs) further induce tissue formation in later stages.
Epiblast cells in the lateral edge of the epiblast layer will undergo epithelial to mesenchymal transition (EMT) to be able to migrate into the primitive streak. The first cells of epiblastic EMT to move into the primitive streak will transform into the endoderm. The second cells that do this will transform into the mesoderm. Multiple mesodermal structures such as the notochord (cells that pass through the primitive pit become notochord) will develop. The formation of the notochord is very important during embryonic development as it provides structural support, defining the midline of the embryo as well as providing chemical and physical interactions with the dorsal lying ectoderm to differentiate. It is the notochord that defines the anterior-posterior axis.
Important because: It primes the embryo for organogenesis by creating the three germ layers that are able to differentiate into tissues, it forms the body axis due to morphogenic gradients which allow for the correct position of the organs
Explain the concept of genomic equivalence how was it discovered, and what are the evidence for it? Are there any exceptions?
The concept of genomic equivalence is that each cell in the body has the same genetic material and therefore all the information necessary to create a complete organism. Animal cloning from a somatic nucleus “proves” this idea.
Evidence of genomic equivalence:
First evidence came from regeneration. A lens was surgically removed from a salamander eye which was then reformed, the new lens was formed from the cells of the dorsal iris. The cells that formed the lens was a very big difference structurally from the cells of the iris still maintained the potency to make a new lens, which gave the idea of genomic equivalence.
Second early evidence comes from polytene chromosomes, these are big chromosomes that you can dye and then analyse to see if any sections are lost after replication. When these chromosomes underwent numerous rounds of DNA replication scientist could see that the daughter cells didn’t loose any of their regions, indicating that all daughter cells have the same DNA. This occurred even in cells that underwent differentiation.
The ultimate evidence of genomic equivalence is experiments of nuclear transfer. The nucleus of an oocyte was removed (can be removed through UV-radiation, microsurgery) and was replaced with the nucleus of another cell, in this first experiment they took the nucleus of a blastula when they did this they noticed that an adult frog originated that had the phenotype of the donor nucleus. The most famous of this type of experiment is the one done by John Gurdon (SCNT – Somatic Cell Nuclear Transfer - 1962).
This is now known as organism cloning. He took an unfertilized egg of a frog but instead of using an embryonic nucleus he used the nucleus of an adult individual, he took the oocyte of a xenopus laevis and the nucleus of an albino xenopus laevis and when he inserted the exogenous nucleus into the oocyte some blastula would develop to a tadpole which would develop into a fertile living frog, and this frog would be looking like the donor of the nucleus (albino coloured), it would be a genetic clone of the organism that donated the nucleus.
Unnucleated cells such as red blood cells.
- Imagine there exists a gene called GeneX which is poorly studies, and no one knows what its functions are. How can you study it? 4 points
First find expression then we can knock out the gene and see how the phenotype turns out.
We can start by measuring the gene expression, this can be done
smFISH (single molecule fluorescence in situ hybridization) is when you use a fluorescent DNA probe which is a piece of DNA which has been covalently attached to something that is fluorescent or something that creates a colorimetric product (in smFISH it is a dye). You need to design a probe that has the sequence complementary of the targeted gene with the addition of a dye, this allows us to visualize the structure where mRNA is present. This can however produce colored mRNA at places where the targeted gene isn’t expressed because of the likelihood that this same sequence exists somewhere else in the genome. To test whether this is case or not you can have a knockout mouse where the targeted gene has been removed, meaning that if you use this probe you should see a signal if done on a normal embryo and you shouldn’t see a signal on the knockout, if there is a signal on the knockout then the sequence is present on other places in the genome. This shows that the probe ONLY binds to the targeted gene. This also has the limitation that it can take a long time to find the correct DNA sequence for the mRNA. This can be overcome by DNA sequencing by RNA-seq.
You can isolate the full content of mRNA from a cell is recognizable because they have a PolyA tail. Due to the PolyA tail you can produce a complementary DNA molecule of that mRNA. You do this by adding a oligonucleotide primer and then you add a reverse transcriptase which a polymerase that produces a DNA strand by using mRNA as a template, doing this yields a single strand of DNA by the addition of another polymerase you get double stranded DNA. If you do this then you would end up with the proportional amount of DNA in the sample as there is on the genes producing that mRNA. Now you end up with a sequencing library which allows us to map the sequence onto a reference genome and understand where the sequences came from, what genes they came from.
We can combine these two technologies.
- What is epithelial-to-mesenchymal transition? 4 points
The epithelial–mesenchymal transition (EMT) is a process by which epithelial cells lose their cell polarity and cell–cell adhesion, and gain migratory and invasive properties to become mesenchymal stem cells; these are multipotent stromal cells that can differentiate into a variety of cell types. EMT is essential for numerous developmental processes including mesoderm formation and neural tube formation. EMT has also been shown to occur in wound healing, in organ fibrosis and in the initiation of metastasis in cancer progression.
- What are transcriptional enhancers, and what model do you think best describes their mechanism of function? 4 points
In genetics, an enhancer is a short (50–1500 bp) region of DNA that can be bound by proteins (activators) to increase the likelihood that transcription of a particular gene will occur. These proteins are usually referred to as transcription factors. Enhancers are cis-acting. Translation enhancers upstream of the SD sequences of mRNAs, which likely contribute to a direct interaction with ribosome protein S1, enhance the yields of protein biosynthesis. Nevertheless, the mechanism of the effect of translation enhancers to initiate the translation is still unknown.
- What is the difference between the “knock out” and the “conditional knock out” technologies? 4 points
In knock-out technologies a particular gene is removed or mutated to assess its function in development.
In conditional-knock-out technologies a particular gene is removed or mutated in a specific tissue and/or in a specific developmental stage.
