Question Set # 4 Answer Key 7.22 Nov. 2002 1) A variety of reagents and approaches are frequently used by developmental biologists to understand the tissue interactions and molecular signaling pathways involved in particular aspects of development. Several of these approaches or reagents are listed below. To determine how well you understand how we know what we know in systems where the forward genetics used in fruit flys and worms is difficult (at best) find one example from your reading and lecture notes where this type of approach was used to demonstrate and important developmental principle. Give the example and the developmental principle illustrated. Over-expression of a mrna in the Xenopus zygote. Over expression of BMP4 ventralizes the embryo; overexpression of goosecoid dorsalizes the embryo; overexpression of a mutant FGF receptor subunit anteriorizes the embryo etc. Trans-species injection of an orthologous gene from Drosophila into a developing vertebrate embryo. Injection of Sog into the belly region of of Xenopus early gastrula stage embryos induces a second embryonic axis. Sog and Chordin are orthologous protein that have also retained a similar function. Could also use the Dickkopf example that is stated in guestion 4 below. A transgenic mouse carrying the entire regulatory region of Hoxb1 in front of the reporter gene LacZ. LacZ product is obvious in an anterior rhobdomere and then in the last 2 rhombomeres with a slowly decreasing expression down the spinal cord. A knockout mouse for a known patterning gene. Lim1 knockout mouse blocks head formation. Pups are born headless and then die. Hoxc8 knockout shows a homeotic transformation of the first lumbar vertebrae into a rib-bearing chest vertebrae. (etc) An incapacitated retrovirus with a strong promotor driving LacZ or green flourescent protein genes. A non-diluting lineage tracer. Could label cells in the posterior marginal zone of early chick embryos and show that the label winds up in the notochord and paraxial mesoderm. Could label a cell in the expanding progenitor 1
population of the cerebral lobes in a mouse embryo and find that a large wedge of cortex is labeled when the animals are examined after birth, Exposure of a specified but undetermined tissue to different inducing tissues hypothesized to change the prospective fate of cells. Xenopus animal cap exposed to ventral/ventral endoderm blastomeres. Get ventral to ventral lateral mesoderm labeled. 2) Pieces of axial mesoderm and neural tube from the same presumptive hindbrain region of mouse early neural tube stage embryos can be placed into culture medium for short periods of time along with either 0M, 10-5 M or 10-7 M concentrations of retinoic acid (RA). The tissue incubated in each concentration was then washed with fresh culture medium and cultured for either 2, 4 or 8 hours before being analyzed for Hox proteins. The following observations were made: In 0M RA the tissue expressed the same Hox proteins it would have in the intact embryo; namely Hox a 1,2 and 3 Hox b 1, 2 and 3 No Hox c proteins Hox d 1& 3 In 10-5 M ret6inoic acid the following Hox proteins were expressed Hox a 10, 11, 12 and 13 No Hox b proteins Hox c 10 & 13 Hox d 10-13 In 10-7 retinoic acid the following where expressed Hox a 6 and 7 Hox b 6 and 7 Hox c 6 No Hox d Name two techniques you might use to detect these proteins. Could use immunohistochemistry or quantitiative western blotting. Currently could take a proteonomics approach and use nuclear magnetic resonance on a protein prep to survey a large number of proteins that may have changed. Note the latter was not covered in class or in your reading and you wouldn t be expected to know it. 2
Which Hox proteins would you expect to appear in the tissue incubated for the shortest time in 0M retinoic acid? Hox a 1; Hox b 1; Hox d 1. in 10-5 M retinoic acid? Hox a 10;Hox c 10; Hox d 10 in 10-7 M retinoic acid? Hox a 6, Hox b 7, Hox c 6 Why do you think some Hox genes are not expressed at certain concentrations? There are RA response elements in regulatory regions of many patterning genes that suppress rather than facilitate transcription. By assaying protein could you determine if retinoic acid was acting directly on the regulatory region of the corressponding Hox genes? No Why? The method for determining if a transcription factor acts directly on the regulatory region of a gene rather than induce transcription of an intermediary protein that then acts as a trnascription factor to induce expression of the assayed gene produce uses a protein translation blocker like cycloheximide. However if you are assaying gene activity at the protein level this approach will not work because the protein translation blocker will also block translation of the Hox proteins themselves. What general effect is RA having on this presumptive hindbrain tissue? It is transforming it to more posterior levels. If you applied RA in these concentrations to late gastrula/early neurula Xenopus embryos what morphological changes might you detect in embryos exposed to the increasing concentrations of RA? You would see embryos in progressively higher concentration of RA lose head and midbrain structures in successively higher RA concentrations. These 4 paralogous groups of Hox genes are located on 4 different mouse chromosomes 6, 11, 15 and 2 respectively. Which gene of each Hox group are likely to lie nearest the 5 end of their respective clusters. Hox a 13, Hox b 13, Hox d 10 What principles of homeobox gene expression are apparent in this experiment? Colinearity. Expression of lowest number Hox in a paralogous group at the most anterior region of that paralogous groups range in the embryo and at the earliest time. Also the lowest numbered Hox genes in each group are located closest to the 3 end of the chromosomelocated nearest the 3 end of the chromosome.. 3
3)Exposure of Xenopus embryos to retinoic acid (RA) results in expansion of the Xhox 6 gene into presumptive midbrain regions of the animal and a posteriorization of the embryos. You obtain the upstream flanking region of the Xhox 6 gene and use restriction enzymes R, N K and B to produce the constructs illustrated below that you introduce into Xenopus neural tube stage embryos bilaterally using electroporation. You next take the groups of these embryos transfected with these constructs and divide each construct group into 2 experimental groups: those treated with exogenous RA (w/ra) and those not treated with exogenous RA (w/o RA). You obtain the following data on LacZ expression in the midbrain and forebrain MB = midbrain r = rhombdomere R N 3 5 K B K B M B - r 2 r3 r4 - r8 N K w /RA w /o RA w /RA w/ o RA w /RA w /o RA - - + + + + R N + - + + + + R B + - + - + + a) What region of this 5 sequence appears to confer restriction of Xhox6 to r4-r8 in the normal embryos (the embryos untreated with RA)? Explain your reasoning. K-B because expression in animals without RA treatment is restricted to r4-r6 only when the fragment R-K is present not when either R-N or N-K is present. b) Where in this regulatory region is the retinoic acid responsive element (RARE)? Explain your reasoning. RN because given that there is negative regulatory element in KB R-K should not show expression in either MB-r2 or r3 with RA unless their was a positive RARE in one of the other regulatory fragment. The only regulatory fragment that shows Xhox 6 expression in anterior segments in the absence of the inhibitory fragment KB is RN 4) Dickkopf1 (Dkk1) is believed to be a secreted protein that acts as a Wnt inhibitor. Evidence for this has been generated by overexpressing mouse Dkk1 in Xenopus embryos where it has been found to inhibits XWnt8 axis forming ability in Xenopus 4
embryos. Note that a Dickkopf orthologue has been found in the early dorsal lip (the early organizer in Xenopus. What phenotype do you expect to find in a Xenopus embryos overexpressing DKK1 during gastrulation and early neuralation in Xenopus. A big head because the first invaginated dorsal lip induces head structure in organizer transplant experiments. You have a mouse developmental genetics laboratory and are interested in determining whether Dkk1 serves a similar function as a wnt inhibitor in the mouse. Suggest two experiments that you might carry out to support the hypothesis? You could inhibit wnt signaling by expressing a protein that inhibits the wnt receptor (frizzeled) under the control of a gene selectively expressed in anterior structures early or you could use this same promoter to over-express GSK-3 in the same region. This would inhibit wnt signaling further downstream by phosphorylating B- catenin and causing it to be degraded. You could also use homologous recombination to produce a line of Dkk-/- KO embryos. All of these manipulations should produce a similar phenotype.. embryos with deficient or absent anterior structures. Where would you expect to see Dkk1 expressed in the early gastrulating mouse embryo? In the early (anterior)node this is the structure homolgous to the early dorsal lip of the blastopore. In Dkk1 -/- embryos the expression of several genes involved in anterior differentiation have been studied. Hesx1 expression is absent in these embryos and the late expression of Six3 is disrupted. However Otx2 expression is normal in the Dkk1-/- mutants. All three of these genes are transcription factors. Based on these observations alone where in the transcription factor cascade responsible for differentiation of anterior structures would you place Dikk1 signaling? You could say that Otx2 is upstream of wnt signaling and Six3 as well as Hesx1 are downstream. What would you not be able to deduce from these data? You could not determine whether Six3 is up or downstream of Hesx1 5) You take an incapacitated retrovirus that can only infect dividing cells, incorporate within it a strong and continuously active promoter in front of the cdna for the green flourescent protein GFP. You inject the construct into the spinal cord region of a neurulating chick embryo in low concentrations. You find that neurons and glia across the entire dorsoventral axis of the embryo are labeled with GFP when the chick spinal cord is examined just before hatching. You perform the same experiment on a chick embryo 2 days later and you find that only neurons in the dorsal spinal cord are labeled. What does this tell you about when cells in the dorsal versus ventral regions of the spinal 5
cord are generated? That the dorsal spinal cord progenitors are still dividing after the ventral spinal cord neurons have withdrawn from the mitotic cycle. Next you perform a variation of this experiment in the mouse embryo. You inject a female mouse 7 days pregnant with a pulse of bromodeoxyuridine and then a second mouse at 9 days pregnant with a similar dose of bromodeoxyuridine. These 2 times have been shown to be equivalent to the two stages at which you introduce the retrovirus in the chick. You examine the spinal cords of the 2 litters of mice after allowing the pups to survive until 2 days postnatal. You find that the early pulse has produced a few labeled cells in the ventral spinal cord and no cells labeled in the dorsal spinal cord but the later pulse labes some sensory neurons in the dorsal spinal cord. Is this data consistent with your retroviral data? Yes because the retrovirus data suggest that cells in the dorsal spinal cord are still dividing and will still produce some pulse labeled cells assuming some of these cells leave the mitotic cycle to differentiate soon after the pulse is given. Their labeled DNA will identify them for the rest of their life. Ventral cells will not be labeled by the later pulse because they have ceased to divide when the late pulse is given. During the early pulse some ventral cells are still dividing to give off post-mitotic neurons that will maintain the label in the DNA. However many cells in the ventral proliferative zone may have withdrawn from the cell cycle at the time of this pulse and others may go on to divide thus diluting out their label. All the cells in the dorsal proliferative zone will divide many times after the early pulse is given and thus dilute out the label by incorporating normal untagged thymidine in their DNA. Now suppose instead of using a continuously active promoter in your retrovirus you use the promoter for the Islet 1 gene and inject it into the same neurulation stage chick embryo spinal cord. You examine the spinal cords of these animals at the same time (2 days before hatching) What cells would you expect to be labeled? Why? Islet 1 is a transcription factor selective to most motoneurons therefore in the spinal cord you would expect to see motoneurons labeled. If you removed the notochord from the chick embryo and then injected the animal with the Islet 1 promoter GFP what would you expect to find in the spinal cord of theses embryos examined at the same time? Why? You would not see any cells labeled because the notochord is the source of sonic hedgehog protein which is a critical inducer of ventral spinal cord neuron types. 6) You have learned how the dorsal/ventral axis is determined in Xenopus. In zebrafish, the embryonic shield (a thickening of cells on the future dorsal side of the embryo) is critical in establishing this d/v axis. 6
a) If the shield is transplanted to the ventral side of the embryo, you get two embryonic axes (2 heads and spinal columns, one forming on the side of the normal shield, and one in the location where it is transplanted.) What tissue in Xenopus seems to have a similar function? Dorsal blastopore lip, or Spemann s organizer b) How can you tell experimentally if the shield is inducing a secondary axis or is differentiating into a secondary axis? Label donor cells (perhaps with dye, or with some kind of marker) to differentiate them from host cells. If you do this, you will find that the secondary axis is induced- tissue from the host becomes the secondary axis. c) What might happen if you remove the shield from the developing embryo? No dorsal tissue, namely no neural tube forms In zebrafish, BMP2B is a ventralizing factor, while chordino is a molecule that can inactivate BMP2B. It is hypothesized that the ratio between chordino and BMP2B may specify position along the dorsal/ventral axis. d) If this hypothesis were correct, where would BMP2B and chordino be localized? BMP2B would form a concentration gradient, strongest in the ventral side; whereas chordino would form a concentration gradient, strongest on the dorsal side (emanating from the shield) e) What might happen if you mutated chordino? Mutating chordino would show a similar phenotype as removing the shield. The embryo would be partially ventralized. f) Propose an experiment that would test the hypothesis that the ratio between chordino and BMP2B may specify position along the dorsal/ventral axis. (The genes for chordino and BMP2B have been cloned and that the reagents that have been available for Xenopus are now available for zebrafish.) express chordino or BMP2B ectopically and observe phenotype (other answers would be accepted: loss of function, gain of function, expression analysis, transplantation, etc). 7