Assignment Task
1. A hypothesis has been proposed that Wnt growth factors secreted in a gradient from the caudal (tail, posterior) end of the gastrulating embryo are critical for determining the anterior/posterior (rostro-caudal) axis of the embryo, including differences in the parts of the nervous system that develop along the A-P axis . To simplify, let’s assume that there is only one Wnt growth factor (In reality, there are many mammals have 19 different Wnt genes!).
This question has three parts. Please completely answer each question within the page limits specified ( total = 1 page ). Please copy each part of the question and insert it before that part of your answer. Pasted-in questions do not count against the page limits. Be as specific as possible in choosing experimental approaches (but do not describe details of techniques). For each part of the question, state the results you expect if the hypothesis is correct.
(1) To test the hypothesis proposed above, you would first like to know if Wnt is expressed in a pattern consistent with this hypothesis. (a) If the hypothesis is correct, what would you expect the distribution of both Wnt mRNA and protein to look like in the gastrulating embryo? Remember that secreted protein can diffuse, but mRNA is intracellular and doesn’t diffuse. (b) Which techniques would you use to determine where in the embryo the Wnt mRNA is made and where the Wnt protein is localized ? You may propose to use any of the model organisms commonly used.
(2) Propose experiments to specifically test the hypothesis that Wnt is necessary for formation of the spinal cord. Include which model organism and techniques you would use and the results you expect if the hypothesis is correct. Wnt functions in the development of many embryonic structures, so eliminating its expression globally would cause abnormal development and wouldn’t specifically affect the nervous system.
(3) Different parts of the embryonic neural plate form different structures and are specified by different genes, such as homeobox genes, that can be used as markers.
Propose experiments that would allow you determine whether a Wnt gradient leads to the formation of different parts of the nervous system in a correct rostro-caudal sequence. Explain the experimental design, state which techniques you would use and the expected results if the Wnt gradient hypothesis is correct. If necessary, you may use embryos of a different model organism to answer this question (3).
Please cite references to show the source of ideas and results and explain how they support your hypothesis .
2. As a researcher interested in placental/fetal development, you believe that factors secreted by the maternal decidua (either decidua cells or immune cells in the decidua) impact cell signaling mechanisms important for (a) differentiation of the epiblast and (b) formation of villous and extra-villous trophoblast cells. You also think that (c) production of these factors by decidua/immune cells is regulated by estradiol from the maternal ovary.
Please provide experimental approaches (in vitro and/or in vivo) to test these hypotheses. State the specific aims/goals of the experiments and expected results. Also briefly provide relevant background on the role of the decidua (if any) on these two processes. Please provide broad experimental ideas/design and relevant experimental tests (e.g., CRISPR; RT-PCR; RNA-Seq; proteomics; pharmacologic interventions; protein neutralization, etc.) useful to address the specific aims.
Example: I will isolate “these” cells using “this procedure” from “this tissue” and do a gene array of factors of the “this gene family” because these factors have been proposed to be important for “cell communication”. I expect to see differences between “this” and “that” and then will do the following to confirm my ideas. Do not list “nitty gritty” experimental details such as volume of sample; number of cells; pH of media etc
3. Gastrulation
This past week we covered the work by Ciruna and Rossant (2001). This paper investigated the molecular mechanisms downstream of Fgfr1 signaling that regulate early embryogenesis, including gastrulation. In general, from this work, it is unclear whether the reported molecular changes in Fgfr1-/- embryos (for example, changes in mSnail expression) are directly regulated by the Fgfr1 signaling pathway.
(A) Please explain how Fgfr1, which is located on the cell’s plasma membrane, can elicit a transcriptional response in the nucleus ?
(B) Please design an experiment that would test whether Fgfr1 signaling directly causes any of the transcriptional changes reported Ciruna and Rossant (2001).
(C) Finally, using more modern techniques, please design an experiment to identify other downstream molecular targets of Fgfr1 in gastrulating embryos. Explain how you could then confirm whether any of your identified targets alter EMT in manner consistent with that seen with loss of Fgfr1 function. Please identify the model organism and the techniques that you would use in these experiments.
4. As we learned, FGF4 signaling in the inner cell mass (ICM) of the mouse blastocyst is essential for primitive endoderm (PE) specification and determination, and for the segregation of PE and epiblast (EPI) cells. But FGF4 is expressed throughout the ICM rather than being regionalized, and PE and EPI precursor cells are intermixed and distributed in a salt-and-pepper fashion. It is now known that an early stage in the ICM, EPI precursor cells express the gene encoding FGF4 but do not express the fgfr2 gene, whereas PE precursor cells express the gene for the receptor FGFR2, but not fgf4 . This leads to the question of the mechanism by which inner cell mass cells are specified to express these genes differentially at an early stage so that some later will become PE cells and others will become EPI cells .
(A) Based on results and conclusions of experiments in the literature, propose a hypothesis involving molecular and cellular mechanism(s) that could explain how some inner cell mass cells are specified to become PE cells and others to become EPI cells .
(1) By what mechanism could FGF4 and FGFR2 expression be specified in different cells?
(2) How could regulation of FGF4 secretion and signaling lead to formation of two different cell types that are intermixed and closely packed within the ICM?
(B) Propose experiments to test your hypothesis. Please cite references to show the source of ideas and results and explain how they support your hypothesis .