THE UNIVERSITY OF CALGARY


FACULTY OF SCIENCE

MIDTERM EXAMINATION II

CMMB 403

Answers in bold-face



November 3, 1997 TIME: 50 minutes

TOTAL POINTS: 25

1. Answer either question A or B (6 points).

A. Describe the experiment that demonstrated the distinct role of the dorsal vegetal cells in Xenopus development.

The roles of the dorsal vegetal cells in development were demonstrated by transplantation experiments (a drawing of the experiment outlined in Browder et al., 1991, Fig. 6.8, would be helpful). Fertilized eggs were irradiated in the vegetal hemisphere with ultraviolet light, which causes ventralization of the embryos. At the 64-cell stage, two vegetal blastomeres were removed and replaced by donor blastomeres from either the dorsal or ventral side of a non-irradiated donor embryo. Only the dorsal blastomeres were capable of rescuing the irradiated embryos, which formed a normal complement of dorsal structures.

The investigators utilized donor cells that had been injected with a fluorescent tracer. The fluorescent donor cells were confined to the gut and did not become dorsal cells themselves. The dorsal structures were derived from the marginal zone cells of the host. Thus, the dorsal vegetal cells have the capacity to release a signal to induce the overlying animal hemisphere blastomeres to develop as dorsal mesoderm. The dorsal vegetal cells form the so-called Nieuwkoop center.

B. Discuss the relationships among ß-catenin, Xtcf-3 and siamois.

ß-catenin, the vertebrate homolog of armadillo, accumulates opposite the sperm entry point by the end of the first cell cycle in Xenopus embryos. It continues to accumulate in dorsal vegetal cells (i.e., opposite the sperm entry point) through the early cleavage stages. This dorso-ventral asymmetry in ß-catenin is a consequence of the subcortical rotation that is triggered by fertilization. ß-catenin accumulates in the dorsal vegetal cells as a consequence of wingless signaling, which stabilizes the protein in the dorsal cells by decreasing the phosphorylation of ß-catenin by the serine/threonine kinase, zw-3/shaggy (Xgsk3). Greater dorsal ß-catenin then participates in establishing dorsal mesoderm, which leads to formation of the gastrula organizer.

The target of ß-catenin/plakoglobin may be a transcription factor called XTcf-3. XTcf-3 is a homolog of a group of vertebrate high mobility group (HMG) box transcription factor genes. Its mammalian counterparts are referred to as "architectural transcription factors" that affect spatial structure of enhancers of target genes, thus facilitating contacts between other factors that are bound to the enhancer.

In vitro assays demonstrated that XTcf-3 is capable of binding to ß-catenin. XTcf-3 and ß-catenin also interact in vivo, which causes the translocation of ß-catenin into the nucleus. XTcf-3 directly binds the siamois promoter. In the absence of ß-catenin, XTcf-3 inhibits gene expression. However, on the dorsal side of the embryo, ß-catenin binds the XTcf-3, and now activates the gene. siamois is a homeobox gene likely playing a major role in specifying formation of Spemann's Organizer. The N-terminal region of XTcf-3 is required for binding to ß-catenin.

So, a dorso-ventral difference in ß-catenin that forms within an hour or two of fertilization directly regulates expression of siamois in the blastula, thus contributing to forming Spemann's Organizer on the dorsal side of the gastrula.

2. Answer either question A or B (6 points).

A. What are the molecular and morphological effects of misexpression of Sonic hedgehog in the gastrula-stage chick embryo?

Initially, Shh is expressed throughout Hensen's node. Somewhat later, activin -ßB is expressed on the right side of Hensen's node. As a consequence, cAct-RIIa is induced on the right side, shutting off expression of Shh on that side. As a consequence, Shh expression becomes limited to the left side of the node and in the notochord. Asymmetric Shh expression induces a small domain of cells adjacent to the left side of the node to express nodal. A larger domain of nodal expression follows in the left lateral plate mesoderm.

The role of Shh in inducing nodal expression was shown by applying ectopic Shh to the right side, which bilateralizes nodal expression. This randomizes heart looping. Also, endogenous left-side Shh was removed by applying activin to the left side, which suppressed nodal expression.

B. What is a dominant-negative receptor, and how has this technology been used to identify native mesoderm inducers?

Growth factor receptors are typically dimers. Hence, excess expression of a non-functional, mutated monomer would render the dimers non-functional. The mutation is overexpressed by injection of its mRNA into embryos. The effects on embryonic development are then assessed. Thus, the ligand for the receptor would not have its normal effects. One or more of the following examples should be given to show how they have been used to identify native inducers: (Keep in mind that you are asked to present evidence regarding the identities of native inducers. Thus, experiments using exogenous inducers on animal caps must be complemented by experiments with intact embryos.)

Dominant-negative activin receptor mutations.

Expression of a dominant-negative BMP-4 receptor blocked mesoderm induction by BMP-4 in isolated animal caps and prevented ventralization of embryos by BMP-4. Overexpression of the receptor in embryos caused mesoderm to be dorsalized. These results suggest that BMP-4 may be involved in ventralization of the mesoderm by counteracting the dorsalizing signals.

Expression of dominant-negative FGF receptor inhibits mesoderm induction in animal cap explants and causes defects in trunk and posterior development in intact embryos, while not affecting anterior development. This result suggests that FGF is necessary, but not sufficient, for induction of trunk and posterior mesoderm

3. Answer either question A or B (8 points)

A. How have our ideas of nuclear potency, which were derived primarily from nuclear transplantation studies on frogs, changed as the result of recent advances in transplanting mammalian nuclei? In your answer, you should demonstrate that you understand the results of nuclear transplantion experiments in both frogs and mammals.

Previous experiments involving both frog and mammalian nuclear transplantation failed to demonstrate totipotency of nuclei from adult cells. No adult cell nucleus had ever been demonstrated to promote completely normal development when transplanted into an enucleated egg. Briggs and King's original experiments with Rana pipiens showed that nuclear potency diminished during the gastrula stage. A low percentage of normal larvaue resulted from transplantation of neural plate nuclei from neurulae. A similar trend of loss of potency was shown in Xenopus laevis. Gurdon showed that some Xenopus tadpole intestinal epithelial nuclei are totipotent. A small percentage of nuclear transplant recipients developed into adult frogs. Epidermal cell nuclei from Xenopus tadpoles have also been shown to be capable of promoting normal development.

Attempts to reprogram nuclei to increase their potency have improved the results of nuclear transplantation. For example, erythrocyte nuclei of Rana pipiens will show increased potency if they are injected into oocyte cytoplasm at the first meiotic metaphase.

Wilmut and his colleagues have now shown that a nucleus from a sheep udder cell that had been cultured under low serum conditions could promote normal development of an enucleated egg. The cultured cell became arrested in the diploid G0 phase of the cell cycle. By arresting donor nuclei in G0, it is thought there is better synchrony in the timing of DNA replication between the transplanted nucleus and the cytoplasm of the recipient egg once development is initiated, thus reducing the possibility of chromosomal abnormalities.

B. Discuss the putative roles of Noggin in embryonic induction in Xenopus. Present experimental evidence to support your answer.

Noggin mRNA is oogenic and is also express zygotically. The zygotic expression begins at the mid-blastula transition (MBT) in the organizer region and later in the dorsal mid-line of the archenteron roof. When ventral marginal zone tissue is exposed to noggin protein, muscle is induced. Thus, noggin is a possible candidate for the third signal in the three signal model. In combination with X-bra, Noggin induces dorsal mesoderm in animal caps.

Zygotic expression of Noggin can induce animal caps to form neural tissue and to express the anterior and general neural markers NCAM and XIF3 mRNA; XAG-1 and otx2 [formerly otxA] protein in the absence of detectable mesoderm. Noggin protein, however, has little or no ability to induce posterior neural markers (e.g., ß-tubulin mRNA, En-2, Krox20 and XlHbox6 protein). Thus, noggin can apparently induce anterior neural tissue, but it cannot induce posterior neural tissue.

Noggin is an inhibitor of the function of bone morphogenetic protein (BMP)-4, which is a TGF-ß family member that has strong mesoderm ventralizing and antineuralizing activity.

4. Provide clear definitions for five of the following terms (1 point each).

A. zw3/shaggy

zw3/shaggy is a component of the Wnt signal transduction pathway. It is downstream of dishevelled and upstream of armadillo. It is repressed by dishevelled and is a repressor of armadillo. It is a serine/threonine kinase. Through wingless signalling, zw-3/shaggy causes a decrease in the phosphorylation of the armadillo protein and an increase in its stablility. Thus, zw3/shaggy is thought to regulate armadillo by phosphorylation.

The zw-3/shaggy homolog in Xenopus is glycogen synthase kinase-3 (Xgsk-3), which directly phosphorylates ß-catenin in vitro at an amino terminal site. Deletion or mutation of this site greatly reduces phosphorylation of ß-catenin in embryos, and inhibition of endogenous Xgsk-3 with a dominant negative protein also reduces phosphorylation of ß-catenin. Phosphorylation of ß-catenin by Xgsk-3 targets it for rapid degradation in the embryo. When this phosphorylation by Xgsk-3 is inhibited in the embryo, ß-catenin accumulates in both the cytoplasm and in the nucleus. After fertilization of Xenopus eggs, dorsal-ventral differences in Xgsk-3 activity may arise, perhaps linked to cortical rotation. These differences in kinase activity lead to dorsal-ventral differences in ß-catenin phosphorylation and thus steady-state levels.

B. frizzled

frizzled is the wingless receptor. Thus, it is proposed to receive the signal from Wnt family members and pass the signal down the wingless signal transduction pathway via dishevelled.

C. Hemimethylated DNA.

After replication of DNA with methylated cytosines in CpG dinucleotides, the cytosines on the newly synthesized strand are initially unmethylated. This strand is said to be hemimethylated. The pattern of methylation is restored via the action of maintenance methylases, which use the replicated hemimethylated DNA as a template. Methylation imprints genes and renders them inactive in transcription.

D. GLP-1 protein.

This transmembrane protein, which is encoded by a maternal transcript in C. elegans, is the receptor of signals required for determination of pharynx. It is involved in two inductive interactions: P2 induces ABp and MS induces ABa to become pharynx in GLP-1-dependent interactions. Translation of the glp-1 mRNA is restricted to cells of the AB lineage of the embryo and is regulated by elements located in the 3' UTR of th glp-1 mRNA.

E. Nieuwkoop's animal cap assay.

Nieuwkoop demonstrated that if the animal cap of the Xenopus embryo is isolated during the blastula stage, it will not form mesoderm. However, if it is exposed to either vegetal base or mesoderm inducers, mesoderm will form. This experiment provided evidence that formation of mesoderm depends upon an inductive signal derived from the vegetal hemisphere. The most complete answers would include a drawing of an embryo, showing the location of the animal cap in the blastula. Induction of mesoderm is indicated by elongation of the explant and formation of an enlarged vesicle. Histological examination would show the presence of mesodermal tissue. Molecular assays would reveal the presence of mesoderm markers such as messenger for X-brachyury or muscle-specific (alpha-cardiac) actin.

F. Three signal model of mesoderm induction.

A general mesoderm-inducing signal originates in the vegetal hemisphere, causing the overlying marginal zone cells (equatorial region) to form mesoderm. The dorsal vegetal cells produce a specific signal, which causes dorsal marginal zone cells to become the Spemann Organizer (dorsal mesoderm; notochord and somite mesoderm; dorsal lip of the blastopore). Evidence for a third signal was shown by experiments in which ventral marginal zone was exposed to dorsal mesoderm. As a result of this interaction, the ventral marginal zone tissue became dorsalized and formed muscle, rather than ventral mesoderm derivatives, such as blood. Thus, the third signal is proposed to emenate from the Spemann Organizer and cause ventral mesoderm to become dorsalized.

G. Epitope tagging.

This is a technique in which genes are engineered such that an epitope (such as a short sequence of MYC) is attached to a protein to enable the protein to be recognized by an antibody to the epitope. The antibody can then be used to immunoprecipitate the protein bound to the epitope.

H. sog

sog is the Drosophila homologue of chordin. It is expressed ventrally and promotes ventral development, including specification of the nerve cord. It is an inhibitor of dpp. Results showed that mRNA injection of either sog or chordin can produce ventral development in Drosophila. Sog can produce dorsal development in Xenopus. This indicates molecular conservation of the dorsal-ventral patterning mechanism during evolution.

I. Follistatin.

Follistatin is an inhibitor of activin and bone morphogenetic protein-4 (BMP-4). Injection of follistatin mRNA in to Xenopus embryos at the two-cell stage resulted in exaggerated anterior dorsal development. Follistatin is expressed in the dorsal lip and axial mesoderm of neurulae. It is proposed to be one of the endogenous factors involved in dorsalization of the mesoderm and induction of anterior neural tissue.

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