midterm_1_04_key - Bio 120 Midterm 1 20 April 04 KEY Bio...

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Unformatted text preview: Bio 120 Midterm 1 20 April 04 KEY Bio 120 Development Midterm 1 April 20th 2004 KEY Question 1 Short definitions Define the following terms (as used in developmental biology). Use diagrams as necessary; if the term applies to a particular species or animal group, say which. 5 points each Accurate labeled diagrams are acceptable in lieu of verbal definitions. Anything in parentheses below is not essential but can be given credit. Compaction Process in early mouse/mammalian cleavage (starting at 8-cell stage) in which cells adhere more tightly to each other. Cells also become polarized such that only exterior surfaces have microvilli. Blastodisc Cleavage stage of chick (avian) embryo; flat disc of cells that lies on top of yolk. Primitive streak Region of thickening of the zona pellucida (in chicks) or epiblast (mice) that extends anteriorly from the posterior marginal zone. Dynamic zone in which future mesoderm and endoderm cells move into the interior of the blastodisc/epiblast. Corresponds to dorsal midline of embryo. Somite Transient (epithelial) segmented block of paraxial mesoderm in vertebrates; will give rise to muscles, axial skeleton, dermis. Chimera An organism made up of cells from 2 (or more) different zygotes. Blastomere Any cell of a cleavage stage embryo. Homeobox Region of DNA that encodes a homeodomain. Gene containing such a region is known as a homeobox gene. Homeodomain is a DNA-binding protein domain. (Originally found in homeotic genes, which are often involved in A-P patterning). Neurulation Stage of vertebrate development during which the dorsal ectoderm the neural plate, which then undergoes morphogenesis to form the neural tube. Visceral endoderm 1 Bio 120 Midterm 1 20 April 04 KEY Embryonic tissue in mammalian embryo, derived from primitive endoderm (or inner cell mass). Outer cellular layer of the egg cylinder. (Expresses genes such as Hex that are required for axis formation in the epiblast). Rhombomere Segmental unit of the vertebrate hindbrain. Question 2 In each group of statements, either three are false and one is true, or one is false and three are true. Indicate which is the ‘odd man out’ and whether it is true or false. 10 points each; no partial credit. (a) Regulation of gene expression Transcription factors are proteins that regulate mRNA export messenger RNAs are spliced in the cytoplasm Ribosomes bind DNA to promote translation Chromatin contains both DNA and proteins TRUE (b) Axis formation in amphibians Gastrulation begins at the same side as sperm entry Beta-catenin is a dorsalizing factor TRUE Ultraviolet irradiation causes dorsalization Lithium activates GSK-3 (c) Mouse development The first cleavage of a mouse egg is meridional The epiblast undergoes compaction The embryo forms from part of the inner cell mass TRUE The blastocoel will give rise to Hensen’s node (d) Germ layers in amphibians Mesoderm normally arises from the animal cap of the late blastula The axial mesoderm becomes the notochord TRUE The gut is predominantly derived from ectoderm Xnrs are transcription factors (e) Neural induction The neural tube is derived from lateral plate mesoderm Neural crest cells are migratory TRUE Inhibition of BMP signaling promotes epidermal fates in ectoderm Planar signals cannot induce neural fates in Xenopus 2 Bio 120 Midterm 1 20 April 04 KEY Question 3 (a) In 1924 Hans Spemann and Hilde Mangold reported results of an experiment that later became known as the ‘organizer’ experiment. Describe the experiment they did, in as much detail as you can. 30 points They grafted the dorsal blastopore lip from a gastrula of one species of newt (NOT Xenopus, although experiment works there too) into the ventral side of blastula of another species. They found that this graft induced formation of a second axis, i.e. neural tissue and dorsal mesodermal cells. They used two different species (‘heterospecific’ transplants) because one species (the donor) was not pigmented and the other (the host) was pigmented (don’t need to remember species names). This allowed them to determine if the cells in the second axis were from the graft or from the host tissue. They found that the second axis contained cells from both host and graft. Most of their experiments resulted in duplication of trunk and tail because they used late stage dorsal blastopore lips. No points for describing Nieuwkoop center experiments—sorry. (b) State one conclusion that you can draw from the Spemann/Mangold experiment, explaining why. 10 points Any one of the three following: 1. The dorsal blastopore lip tissue is determined (with respect to this experiment) but ventral mesoderm or ectoderm are not. 2. The dorsal blastopore lip can induce ventral mesoderm to make dorsal mesoderm (dorsalizing activity). 3. The dorsal blastopore lip can induce ventral ectoderm to make neural tissue instead of epidermis (neuralizing activity) (c) Name one molecule thought to be involved in organizer activity in Xenopus, and say what its molecular function is thought to be. 10 points OK: --noggin, chordin, follistatin: bind to BMP4 and prevent it from interacting with its cell surface receptor --Frzb: binds to Wnts and prevent it from interacting with its cell surface receptor --goosecoid, pintallavis, Xnot, Xlim-1, HNF-3b: transcription factors that specify organizer fate not OK: Brachyury, beta catenin, Wnts, BMPs, Xnrs. None of these are specifically made by the organizer. 3 Bio 120 Midterm 1 20 April 04 KEY Question 4 (answer this question OR question 5) (a) You are a grad student in a lab at UCSC studying the development of the anteroposterior axis in the rare bird species, Swale’s Warbler. You observe that, as in chick development, the anteroposterior (AP) body axis develops from the posterior marginal zone (PMZ) of the blastodisc. Your advisor suggests that you do some transplant experiments. You find that: --When you transplant a PMZ into a second blastodisc at 180° from the host PMZ, twinning (duplicate body axes) occurs, with each PMZ inducing an axis. --When you transplant a PMZ into a second blastodisc (of about the same age), at 90° from the host PMZ, a single body axis forms from the host PMZ only. What can you conclude about the role of the PMZ in the development of this bird? 20 points The first experiment suggests that the PMZ is sufficient to induce development of the body axis/primitive streak. The difference between the first two experiments suggests that the host PMZ can inhibit the development of a second primitive streak (lateral inhibition) if it is close enough, but not if it is further away. You can also conclude that the PMZ is partly determined, but that the rest of the blastodisc does not seem to be determined. (b) A TGF-b family protein, Wt1, has been cloned from Swale’s Warbler by a new research group at UC Merced. By analogy to Xenopus, you decide to test whether this gene has any function in PMZ development in Harrington’s Warbler. The UCM team has kindly sent you DNA clones encoding Wt1 as well as antibodies specific to the Wt1 protein. However, nobody has yet developed the technology to do gene knockouts in Swale’s Warbler, and you want to graduate soon. Describe TWO experiments that you could do with the reagents you have that would address the role of Wt1 in PMZ formation. State the rationale, method, possible outcomes, and what type of evidence your experiments would provide. 30 points Since you cannot knock out the gene by mutation, here are some alternative approaches: Examine whether Wt1 is expressed in or near the PMZ, using in situ hybridization or antibody staining or reporter genes. If Wt1 is in the right place at the right time then that would support the idea that it specifies PMZ formation. (Correlative evidence). You could try to inhibit Wt1 function by antisense mRNA/RNA interference/morpholinos. If you knew the nature of the Wt1 receptor you could also do dominant negative receptor experiments—note that the question does not say if the receptor is known. Not much credit for saying ‘dominant negative Wt1’ unless you can explain how this would work. (Loss of function evidence) Finally, to ask if Wt1 is sufficient for PMZ formation, you could try something like injecting Wt1 RNA or protein into a certain part of the blastodisc and seeing if it induced an extra PMZ. (Gain of function evidence) If all these experiments worked then Wt1 would be a good candidate for a protein involved in PMZ formation. 4 Bio 120 Midterm 1 20 April 04 KEY Question 5 (answer this question OR question 4) (a) You are studying the embryogenesis of the extremely rare Bonny Doon Mountain Hog. You find that, as in the mouse, the 16-cell stage hog embryo contains interior (‘inside’) and exterior (‘outside’) blastomeres. Fate mapping experiments show that the outside blastomeres give rise to trophectoderm (TE) at the blastocyst stage and that inside blastomeres give rise to the inner cell mass (ICM). You do the following experiment to assess whether hog blastomeres at the 16-cell stage are determined to become ICM or TE. You mark individual inside or outside blastomeres with a non-diffusing dye at the 16-cell stage and recombine them with fifteen unmarked blastomeres, placing the marked cell either on the inside or the outside of the aggregate. You let the aggregate develop (such aggregates can develop into normal embryos) and then see where the progeny of the marked blastomeres end up at the blastocyst stage. You find: Origin of cell and location in recombined aggregate Inside cell in inside position Inside cell in outside position Outside cell in outside position Outside cell in inside position Total number of recombinant blastocysts 19 13 14 18 Number with Marked cells in TE only 2 8 13 17 Number with marked cells in ICM only 9 2 0 0 Number with marked cells in TE + ICM 8 3 1 1 What can you conclude from these data, and why? 20 points Inside cells can generate TE or ICM depending on their new location, so they cannot be determined by this stage. Outside cells mostly make TE (their normal fate) whether they are placed inside or outside, so they are already ‘determined’ (with respect to this experiment). (b) You have isolated a gene from the hog, called HOG1, that encodes a member of the TGFb family of proteins. You look at where HOG1 is expressed in embryogenesis and find that it is turned on in posterior extra-embryonic endoderm adjacent to where the primitive streak will form. Embryonic cells do not express detectable levels of HOG1 mRNA or protein. You delete (knock out) the HOG1 gene using transgenic hog technology, and find that the resulting mutant hog embryos (those lacking HOG1 function) do not form a primitive streak. Suggest a model for the normal function of HOG1. Describe one experiment you could do to test your model, stating what reagents you would need, and possible outcomes. 20 points. HOG1 is required for embryonic axis formation yet it is not expressed in the embryo, but in extra-embryonic tissues. HOG1 thus has a non-autonomous role, consistent with its secreted nature. HOG1 could be made in the extra-embryonic cells and diffuse to the embryonic cells, where it somehow induces primitive streak formation. Experiments: Try to make an ectopic source of HOG1 in extra-embryonic tissues and see if this induces a streak (gain of function). 5 Bio 120 Midterm 1 20 April 04 KEY If the HOG1 receptor has been identified you could look at its expression. You would predict that if HOG1 is signaling directly from extra-embryonic endoderm to the epiblast/primitive streak, then the receptor should be expressed at or near the primitive streak (correlation). You could propose making a dominant negative HOG1 receptor if you explain how this works. (c) Hog embryos, like other pig embryos, begin gastrulation before implantation into the uterus. Speculate on what this means for the control of anteroposterior axis formation in this and other mammalian species. 10 points. Since gastrulation precedes implantation this suggests that environmental cues from the uterus do not bias the axis forming process. It also seems unlikely that gravity is important, as mammalian blastocysts are not known to undergo any kind of stereotyped rotation (unlike birds). An alternative possibility is that mammalian axis formation initiates at random and that no external cues are required to break symmetry. 6 ...
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