2011_Questions_Week_12_Answers

2011_Questions_Week_12_Answers - 1.The earliest study...

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Unformatted text preview: 1.The earliest study concerning liver induction was performed by Willer and Rawles (1931), who observed that chick explants with two hears (cardia bifida) often developed two livers, but never developed a second liver in the absence of cardia bifida. A) With the benefit of hindsight, what was the major inductive molecule secreted by the hearts? -FGF4 B) What embryonic tissue is stimulated by this molecule, and what is the earliest known marker of the liver? -FGF4 induces ventral foregut endoderm directly adjacent to the cardiac mesoderm to become hepatic endoderm. The transcription factor, Hhex, is the earliest known marker of the region. 2. Endoderm development contains examples of inductive signaling and permissive signaling. Give an example of both using pancreatic development as an example. -Inductive signaling: dorsal pancreatic endoderm is induced by notochord (without it, you do not get Pdx1 expression; this is because it suppresses Shh in the endoderm in this region). Another example: dorsal (and likely ventral) pancreas are induced by endothelial cells (the dorsal aorta in the case of dorsal; likely the vitelline veins in the case of ventral). -Permissive signaling: pancreatic buds require mesenchyme, as shown by the classic experiments of Golosow and Grobstein. A strong line of evidence that this is permissive is that pancreatic endoderm is specified well before the appearance of pancreatic mesoderm (before 14 somites vs. after 20 somites respectively in mice). 3. In 1968, Nicole Le Dourain showed that the septum transversum mesenchyme can be substituted for a variety of different mesenchymal tissues and still promotes migration A) What tissue was she referring to when she said 'migration'? -The liver bud, which delaminates and undergoes an epithelial to mesenchymal transformation into the mesenchyme B) In the context of more modern experiments, what is a possible molecular explanation for her observation? -All mesenchyme will be vascularized and thus have this important proliferative signal for the expanding liver. 4. Compare and contrast the origin initial induction of the hepatic vs. pancreatic endoderm. -The liver forms from three populations of ventral foregut: two lateral populations and one medial population that come together when the foregut tube forms. The ventral pancreas also has two medial populations, located caudally to the hepatic precursor regions, but no ventral region. Unlike the liver, two distinct pancreas are induced (dorsal and ventral) that will eventually fuse. The dorsal pancreas has a different origin, dorsal foregut endoderm. 5. Hhex mutant embryos have a proliferative defective in endoderm that retards their migration. As a result, the most caudal endoderm is directly adjacent to the cardiac mesoderm. Explain why this results in a lack of ventral pancreatic formation. -Pancreatic endoderm is specified caudal to the hepatic endoderm (it must migrate past the cardiac mesoderm, which specifies liver and inhibit pancreatic formation). In Hhex mutants, the tissue simply isn't any further caudal at the time of liver induction. This results in a complete loss of ventral pancreatic specification. 6. Lammert et al. (2001) showed that co-cultures of dorsal aorta + dorsal endoderm generated pancreatic gene expression, including insulin-positive cells, while dorsal endoderm cultured alone did not express pancreatic markers. Propose an experiment that would allow you to identify if the inductive signal coming from the endothelium is secreted or if it requires direct cell-cell contact (hint: think of the Grobstein experiments). -Using their cell culture method, insert a permeable membrane between the endoderm and dorsal aorta. If induction of pancreatic markers still occurs, it is a secreted signal. 7. What is the difference between a transgene expressing Cre, and one expressing Cre-ER? -A standard Cre will be active from the moment the protein is translated. Cre is normally nuclear and will go into the nucleus, and act on LoxP sites. Cre-ER proteins are fusions with a modified estrogen receptor that sequesters Cre in the cytoplasm. When the drug Tamoxifen is added, this triggers the translocation of the protein to the nucleus, where it will activate recombination. Tamoxifen is usually injected into the mom, and then diffuses into the embryo through the placenta. This is a way of adding temporal specificity to Cre (useful when timing is critical, and to look at what is the cell fate of a Cre-expressing population over time). 8. What do you think would happen if you expressed Gremlin specifically within the interdigit mesenchyme of mice? -You would inhibit BMPs, therefore no apoptosis, and there would be webbing between digits (syndactyly). 9. Why is a lungfish considered to have a fin that is somewhat similar to a tetrapod when compared with currently living fish? -Lungish have a single bone that connects the fin to the body. This is an ancestral stylopod. 10. What is the major difference between Tiktaalik and Eusthenopteron limbs? -Tiktaalik is the earliest fossil related to the tetrapod lineage that has digit-like appendages. Eusthenopteron has stylopod and zeugopodal-like bones, but lacks digit-like appendages. 11. Compare a bat wing with a bird wing. A) Is this considered to be homologous or convergent evolutionary trait? B) What about a bat wing and a horse's limb? -A) Convergent evolution (similar structures that developed independently during evolution); B) Homolgous (structures that evolved from a commmon ancestor). 12. The following image contains part of a figure discussed in lecture and part of a figure that we did not discuss. In this experiment, Carboxypeptidase A regulatory sequences were fused to CreER to generate CPA::CreER transgenic animals. These were crossed to Rosa::LacZ reporter mice, and Tamoxifen was administered to pregnant mice at E10.5 and E13.5. Mice were harvested Developmental Cell at E18.5, and pancreatic tissues were s tained as indicated below. Based on these mental Cell nt Progenitor Domain of the PancreasMultipotent Progenitor Domain of images, what do you conclude about the ability of CPA -positive cells to act as a representative the Pancreas progenitor population over time? Endo, Endocrine cells, Amy, Amylase, a mature exocrine marker; DBA, a pancreatic duct marker, DAPI, marks nuclei of all cells, Bgal, beta-galactosidase (product of LacZ). -At E11, CPA-positive cells give rise to all three populations of the pancreas (exocrine, endocrine, duct). At E14, CPA-positive cells do not give rise to endocrine or duct cells. They only give rise to exocrine cells (amylase positive). pa1+ Cells Are Multipotent before E14 Figure 5. Cpa1+ Cells Are Multipotent before E14 + Cpa1CreERT2;R26R stages. were labeled with one dose E18.5. different embryonic ;R26R embryos were labeled with one dose of TM at different embryonicembryosPancreata were harvested atof TM atCpa1+ cells pulsed stages. Pancreata were harvested at E18.5. Cpa1 cells pulsed at E13–14, respectively) gave rise to endocrine (A–C, arrows), exocrine (E–G, hollow .5, and E12.5 (labeling occurs at E11–12, E12–13, and E10.5, E11.5, and E12.5 (labeling occurs at E11–12, E12–13, and E13–14, respectively) gave rise to endocrine (A–C, arrows), exocrine (E–G, hollow + arrowheads), and duct (E–G, white arrowheads) progeny. In only exocrine (H, hollow , and duct (E–G, white arrowheads) progeny. In contrast, Cpa1+ cells pulsed at E13.5 (labeling E14–15) generate contrast, Cpa1 cells pulsed at E13.5 (labeling E14–15) generate only exocrine (H, hollow arrowheads), but not endocrine (D) or duct (H) cells. Endo, and pancreatic polypep, but not endocrine (D) or duct (H) cells. Endo, four major endocrine hormones (insulin, glucagon, somatostatin,four major endocrine hormones (insulin, glucagon, somatostatin, and pancreatic polypeptide) were simultaneously recognized with a mixture of antibodies; Amy, Amylase, multaneously recognized with a mixture of antibodies; Amy, Amylase, a mature exocrine marker; DBA, a pancreatic duct marker. (I–L)a mature exocrine marker; DBA, a pancreatic duct marker. (I–L) + Summary of the lineage exocrine; D, duct. the lineage of Cpa1+ cells at different embryonic stages. En, endocrine; Ex,of Cpa1 cells at different embryonic stages. En, endocrine; Ex, exocrine; D, duct. 2 three late endocrine progenitors, as well as endo+bgal+ mature essive days for analysis (Figure 6 and Figures successive days forprogenitors, as welland endo+bgal+ mature late endocrine analysis (Figure 6 as Figures S3B). day after progenies, the detected (Figures day after TM injection, the initial population of One endocrineTM injection,wereinitial population of 6I andendocrine progenies, were detected (Figures 6I and 6L). 6L). TM-responsive cells expresses that Cpa1+ cells appear to self-renew data suggest that Cpa1+ cells appear to self-renew These sive cells expresses both bgal and Cpa1 as exThese data suggest both bgal and Cpa1 as ex+ pected that ure 6A, arrows). Consistent with the labeling of (Figure 6A, arrows). Consistent with the labeling of (making more Cpa1+ cells) and lay down daughters(making more Cpa1 cells) and lay down daughters that go through a series of differentiation steps to generate an early multipotent progenitor cell differentiation steps to generate go through a series of type, these cells were ultipotent progenitor cell type, these cells were mature pancreatic cells in vivo (Figure 6M). mature pancreatic cells in vivo (Figure 6M). r the early endocrine marker, Ngn3 (Figurenegative for the early endocrine marker, Ngn3 (Figure 6D), 6D), the late docrine marker, Pax6 (Figure 6G), and all endo- endocrine marker, Pax6 (Figure 6G), and all endoDISCUSSION crine DISCUSSION ones (Figure 6J). Starting from 2 days after la- hormones (Figure 6J). Starting from 2 days after laÀ + beling, observed that Cpa1À bgal+ cells appear in the we observed that Cpa1 bgal cells appear in the trunk Formation of the mammalian pancreas as a complex n of the branches (Figures 6B and 6C, arrow- region of the branches (mammalian and 6C, arrow-a complex Formation of the Figures 6B pancreas as + + three-dimensional organ requires timely generation, mitips ddition to Cpa1+bgal+ cells residing in theheads) in addition to Cpa1 bgal cells residing in thegeneration, mithree-dimensional organ requires timely tips (Figures 6B and 6C, arrows). This observation, togethertypes gration, and differentiation of different cell types from B and 6C, arrows). This observation, together gration, and differentiation of different cell from + with multipotent progenitors. We performed genome-wide TF ta that most Cpa1+ cells are multipotent beforethe data that most progenitors.are multipotent before multipotent Cpa1 cells We performed genome-wide TF + E14 5 and Figures S4), of the developing Cpa1 e 5 and Figures S4), suggests that some Cpa1+(Figureexpression analysissuggests that somepancreas andexpression analysis of the developing pancreas and disdisare capable of limited self-renewal (i.e., producing more le of limited self-renewal (i.e., producing more covered multiple distinct gene expression domainscovered multiple distinct gene expression domains that that À Cpa1+ indicate the presence of specific pancreatic progenitor ltipotent tip cells as well as Cpa1À differentiated multipotent tip cells as well as Cpa1 differentiated progenitor indicate the presence of specific pancreatic progenies).domains. Using a combination of serial immunofluoresdomains. Using a combination of serial immunofluores). Starting from E14, some progeny of Cpa1+ cells labeled we cence and genetic lineage tracing experiments, we profrom E14, some progeny of Cpa1+ cells labeled cence and genetic lineage tracing experiments, proat E12 pose in to express Ngn3 (Figures 6E and 6F, arrow- begin to express Ngn3 (Figures 6E and pancreatic progenitor that one type of multipotent pancreatic progenitor pose that one type of multipotent 6F, arrowheads), indicating that they have become committed markers be recognized by a combination of markers can dicating that they have become committed can be recognized by a combination of + + + + High + ...
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