THE UNIVERSITY OF CALGARY

FACULTY OF SCIENCE

MIDTERM EXAMINATION II

CMMB 403

November 6, 1996

TIME: 120 minutes

TOTAL POINTS: (25)

1. Noted developmental biologist, Dr. Squiggly Squid, has just discovered a new kind of marine life having an enormous suction cup protruding from the vegetal side of the larva. Intrigued by the mechanism of suction cup formation, she took a closer look at the development of this organism. Of particular note is a green crescent located in the vegetal hemisphere of the fertilized egg. Early cleavages were as follows:

She performed a series of isolation and culture experiments and obtained the following results:

 Cell(s) cultured  Resulting pluteus
 AB, A or B alone  incomplete embryo, no suction cup
 C or D alone  incomplete embryo with suction cup
 A and B together  incomplete embryo, no suction cup
 CD alone or C and D together  incomplete embryo with two suction cups
 B and D together  complete embryo, no suction cup
 A and C together  complete embryo with suction cup


She also injected green crescent material from C or D into B and obtained an incomplete embryo without a suction cup. However, when A was injected, a suction cup formed on an incomplete embryo.

a) Which cell or cells give rise to the suction cup during normal development? (0.5 point)

C

b) Discuss the roles of each of these four cells in controlling development of this embryo. Support your answer with experimental evidence. (0.25 points for each cell).

A: Insufficient to produce a complete embryo. Must be combined with a vegetal hemisphere cell to produce a complete embryo. Permissive for suction cup formation.

B: Insufficient to produce a complete embryo. Must be combined with a vegetal hemisphere cell to produce a complete embryo. Represses suction cup formation.

C: Insufficient to produce a complete embryo. Must be combined with an animal hemisphere cell to produce a complete embryo. Contains suction cup determinants (possibly in the green crescent).

D: Insufficient to produce a complete embryo. Must be combined with an animal hemisphere cell to produce a complete embryo. Contains suction cup determinants (possibly in the green crescent).

2. Draw a cell lineage map from fertilization to the 8-cell stage of C. elegans development.

(1 point)

The derivation of Abal, Abar, Abpl, Abpr, E, MS, Pc and C should be indicated graphically.

3. Briefly define the following terms. (1 point each)

a. Balbiani ring

Giant puffs on the polytene salivary gland chromosomes of Chironomus tentans. These puffs result from unfolding of the chromomeres that constitute bands on the chromosomes and represent sites of intense transcription.

Evidence:

Puffs incorporate radioactive RNA precursors.

Puff regression (e.g. by alpha-amanitin) causes a commensurate reduction in incorporation of tritiated uridine.

b. glp-1

Found in C. elegans. Receptor for the signal from P2 to ABp and from MS to Abal and Abar that is necessary to specify pharyngeal fate. The transcript for glp-1 is maternal and is subject to translational regulation from elements in the 3' UTR. These elements regulate both the temporal and spatial translation of the messenger. The best answers will describe the experiment in which the 3' UTR was ligated to a reporter gene to demonstrate the role of the 3' UTR.

c. homeobox

A short sequence of nucleotides that encodes a DNA-binding domain in gene regulatory proteins. Homeoboxes were originally identified in the homeotic genes that control pattern formation in Drosophila. Example of a homeobox gene that we have discussed thus far in this course is bicoid.

d. XTC-MIF

Mesoderm-inducing factor secreted by Xenopus tissue culture cells (XTC cells). XTC-MIF was found to induce mesoderm in isolated animal caps of Xenopus blastulae. XTC-MIF has been purified and found to be homologous to activin, a growth factor in the TGF-ß family.

4. Describe the technique of nuclear transplantation in either Rana pipiens, Xenopus laevis or Mus musculus (the mouse). For the organism you choose, describe the results that are obtained by transplanting embryonic nuclei into enucleated eggs and compare the results to those obtained with the other two organisms. (4 points)5. Describe an experiment that demonstrates that cellular adhesive properties can influence gene expression. (4 points) 6. Describe the Spemann organizer, its determination and its roles in dorsalization of lateral mesoderm and in determination and patterning of neural ectoderm. Include in your answer any intrinsic or extrinsic factors that have been demonstrated to be involved in the processes you describe. (4 points)7. What are the requirements for nucleic acids in pre- and post-blastular development? How were they discovered? (3.5 points total)

Rana pipiens

Briggs and King devised the technique of nuclear transplantation using Rana pipiens. The donor cells are prepared by dissociating them in a Ca++ free medium and withdrawing the nucleus into a micropipet of slightly smaller diameter than the cell, which causes the cell to burst, releasing the nucleus. The host egg is activated with a clean glass needle. When the nucleus rises to the animal pole to form the second polar body, it is removed with a needle. The nucleus is then implanted using the micopipet.

Using endoderm nuclei, Briggs and King observed that the ability of nuclei to promote normal development decreased during the gastrula stage (a graph showing their data is useful here). Endodermal nuclei from post-gastrula embryonic stages promoted development of abnormal embryos with defective mesodermal and ectodermal development and good endodermal development. These results indicated that the developmental potency of nuclei becomes restricted during development and that nuclei become specialized to perform a limited repertoire.

The developmental potency of nuclei becomes extremely restricted after embryogenesis. Nuclei from most post-embryonic cells are incapable of promoting any development, although some stem cells have been shown to promote some development. For example, primordial germ cells of tadpoles had a high degree of potency. However, spermatogonia of frogs lacked developmental potency, even though they are genetically totipotent.

One source of developmental restriction is the inability of nuclei to adapt to the rapid mitotic requirements of early development, resulting in chromosomal abnormalities.

No adult nucleus has been shown to promote completely normal development of eggs. However, erythrocyte nuclei transplanted into oocytes could promote development to the abnormal tadplole stage.

Xenopus laevis

Host eggs are exposed to ultraviolet light, which destroys the egg nucleus and activates the egg. Donor nuclei of embryonic cells are prepared as for Rana pipiens. Results obtained with embryonic nuclei have been comparable to those obtained with Rana pipiens; i.e., developmental potential decreased progressively during development. (Graph of data would be useful.)

Donor tadpole gut epithelial cells are dissociated chemically, and their nuclei are removed using a micropipet of smaller diameter than the cell, which liberates the nucleus, allowing it to be injected into an activated, enucleated egg. Donor nuclei are from a strain having one nucleolus per cell, whereas the host eggs are wild-type. This allows the investigator to confirm that the nuclei in the nuclear transplant recipients are of donor origin.

To maximize the opportunities to demonstrate the developmental potential of donor nuclei, recipient embryos are dissociated at the blastula stage, and nuclei of blastula cells are used for serial nuclear transplantation. A low percentage (~1.5%) of intestinal cell nuclear transplants developed into normal feeding tadpoles, some of which developed into adult frogs.

Mus musculus (mouse)

McGrath and Solter developed a technique for nuclear transfer that did not involve penetration of eggs with a micropipet (a drawing illustrating the technique is helpful). This involved aspirating the pronuclei of a zygote or nuclei from embronic cells into a micropipet within a membrane-enclosed karyoplast. In the presence of cytochalasin B and colcemid, the karyoplast will separate from the embryo. Fusion of karyoplasts containing either pronuclei or embryonic nuclei with cytoplasts is promoted by aspirating inactivated Sendai virus into the micropipet before the karyoplast is pipetted into the perivitelline space via the enucleation site of the zona pellucida.

Developmental potency of donor nuclei decreased dramatically between the early and late two-cell stage. Four-cell and eight-cell nuclei are rarely able to promote even one cleavage division.

5. Describe an experiment that demonstrates that cellular adhesive properties can influence gene expression. (4 points)

Elimination of the genes encoding E-cadherin in either Drosophila or the mouse has profound effects on cellular adhesive properties and, hence, normal development. The role of ß-catenin in theWnt pathway implies that E-cadherin-mediated adhesion would also have profound effects on gene expression. This prediction has been upheld by studies on mouse embryonic stem cells (ES cells) that are null for the gene encoding E-cadherin. These cells are defective in cell aggregation (a defect that can be corrected by transfection with cDNA encoding either E-cadherin or N-cadherin driven by a constitutive promoter).

Expression of the gene encoding the transcription factor T-brachyury is regulated by the presence or absence of E-cadherin. Thus, the cadherins play a role in linking cell surface receptors to gene expression. Normal ES cells can form a number of organized tissues in vitro, a property that is lacking in cadherin null cells. However, constitutive expression of E-cadherin restores their ability to form epithelia. In contrast, expression of N-cadherin restores the ability to form neuroepithelium and cartilage, but not epithelia. Thus, specific cadherins appear to stimulate differentiation of particular types of tissues.

6. Describe the Spemann organizer, its determination and its roles in dorsalization of lateral mesoderm and in determination and patterning of neural ectoderm. Include in your answer any intrinsic or extrinsic factors that have been demonstrated to be involved in the processes you describe. (4 points)

The Spemann organizer forms opposite the sperm entry site in the dorsal marginal zone (dorsal lip of the blastopore). Determination of the Spemann organizer is the function of the Nieuwkoop center, which is located in the dorsal vegetal region of the embryo. The Nieuwkoop center signals the overlying marginal zone cells to become dorsal mesoderm (notochord and somites), which assumes the role of the organizer. The organizer neuralizes the dorsal ectoderm and dorsalizes lateral mesoderm. (A drawing illustrating the relationships among these regions is helpful.)

The native signal responsible for determination of the organizer is uncertain, although there is evidence that Vg1 may be involved. Two factors that are expressed in the organizer and may be involved in mediating its function are X-bra and Noggin. X-bra is a nuclear protein that is generally present in the marginal zone in response to signals from the vegetal hemisphere, whereas Noggin is a secreted factor produced initially within the organizer from zygotic transcripts and later from the notochord, which is derived from the organizer.

X-bra and Noggin work combinatorially to dorsalize lateral mesoderm (i.e., induce muscle), whereas Noggin causes cells that do not express X-bra to neuralize.

As the dorsal lip of the blastopore involutes and migrates anteriorly, it contributes to the patterning of the overlying ectoderm. The ectoderm overlying head mesoderm becomes brain, whereas that overlying trunk mesoderm becomes spinal cord. Noggin has been shown capable of inducing anterior neural markers, but not posterior markers. Hence, other factors are necessarily involved in determination of posterior neural ectoderm.

7. What are the requirements for nucleic acids in pre- and post-blastular development? How were they discovered? (3.5 points total)

This question was graded on 14 different criteria, each worth 0.25 points:

Pre-blastular development:

Post-blastular development:

Evidence: