THE UNIVERSITY OF CALGARY

FACULTY OF SCIENCE

MIDTERM EXAMINATION II (alt.)

CMMB 403

November 6, 1996 TIME: 120 minutes

TOTAL POINTS: 25

1. Draw an 8-cell embryo of C. elegans, and identify each cell. (0.5 point)

The names and locations of Abal, Abar, Abpl, Abpr, E, MS, Pc and C should be indicated on an accurate drawing.

2. Describe the inside-outside hypothesis of mammalian development and discuss an experiment to test it. (4 points)

This hypothesis proposes that the determination of mammalian blastomeres to become inner cell mass or trophectoderm is dependent upon whether they are located on the inside or the outside of the embryo. To test this hypothesis, chimeras were produced with outer blastomeres derived from 4-cell stage embryos of one strain of mice and inner blastomeres from another strain. The outer blastomeres always formed trophectoderm. In another experiment, blastomeres of an albino strain of mice were separated from one-another at the 4-cell stage and allowed to divide once before being placed in the middle of a cluster of 4 cells derived from an embryo of a black strain of mice. The strains also had different electrophoretic variants of the enzyme glucose phophate isomerase, so tissue samples of embryos could be examined to determine the blastomere of origin. The inner cells developed into the embryo, whereas the outer cells formed the trophectoderm.

3. You are trying to find a way to make human neurotransmitters in the lab. To try to induce expression of human neurotransmitters, you have made a heterokaryon between a mouse neuron and a human fibroblast. You make the following observations:

a) The heterokaryon makes certain human-specific neuronal cell surface markers.

b) The heterokaryon makes no neurotransmitters when cultured in isolation.

c) You know from previous experience that interspecies synapse formation does not occur.

d) When cultured with mouse neurons, the heterokaryon forms synapses with these mouse neurons and makes only murine-specific neurotransmitters.

e) Similarly, when cultured with human neurons, the heterokaryon forms synapses with human neurons and now makes only human-specific neurotransmitters.

Explain these results. (5 points)

Some human neural-specific genes were activated in the fibroblast-derived nucleus by interaction with the mouse neuron cytoplasm, including genes encoding proteins necessary for synapsis with human neurons. However, the expression of neurotransmitters depended upon synapsis with neurons, and the neurotransmitters produced by the heterokaryons were dependent upon species-specific feedback caused by the synapsis. Thus, synapsis with a mouse neuron caused production of mouse-specific neurotransmitters, whereas synapsis with a human neuron caused production of human-specific neurotransmitters. Cell fusion is sufficient to cause expression of human neurotransmitter genes, but there is insufficient information given to know whether synapsis causes expression of neurotransmitter genes or whether it triggers release of stored neurotransmitters.

4. Identify integrins and fibronectins and discuss their functional relationship. (4 points)

The following material could be included in your answer. Answers were graded based upon accuracy and comprehensiveness.

The main molecules that mediate cell anchorage to the substratum are members of a family of transmembrane linker proteins known as integrins. Integrins traverse the cell membrane, anchoring the actin microfilaments on the inside and binding to the fibronectin of the extracellular matrix. Linkage of integrin to the microfilaments is indirect and involves a number of attachment proteins. The cytoplasmic domain of integrin binds to talin, which binds to vinculin, which binds to alpha-actinin, which binds to actin filaments. Each integrin molecule is a dimer of alpha and beta subunits. There is a diversity of alpha and beta integrins (over 24 members of the integrin family are known); different combinations of alpha and beta subunits provide different cell types with different affinities for the extracellular matrix.

The significance of integrins during development is demonstrated by mutations of genes encoding integrins in Drosophila, which either cause developmental abnormalities or death. A number of correlations have also been drawn between changes in the expression of integrins and developmental processes.

One of the most prevalent components of the extracellular matrix is fibronectin. All known cellular receptors for fibronectin belong to the integrin family. The significance of the fibronectin/integrin interaction is exemplified by knockouts of the genes encoding either fibronectin or two fibronectin receptors (integrins alpha4 and alpha5) in mice, which cause developmental defects that are severe enough to cause embryonic lethality. Fibronectin molecules are dimers having individual domains that bind to collagen, heparin and cell surfaces. The cell surface binding domain is characterized by the presence of an essential Arg-Gly-Asp (RGD) tripeptide. The RGD sequence is so critical for binding that short peptides having the RGD sequence will compete with fibronectin for binding and inhibit the attachment of cells to a fibronectin matrix.

In addition to their roles in fibroblasts, integrins are expressed in epithelial cells. In fact, virtually every cell type of multicellular organisms expresses integrins, mediating their interactions with the extracellular matrix. Integrins not only play a structural role; they are also involved in communicating signals from the extracellular matrix into the cell. The clustering of integrins at the sites of contact with the matrix can activate several intracellular signaling pathways. The signals that the integrins initiate appear to influence such such cell properties as differentiation, proliferation, survival, and gene expression.

The cytoplasmic domains of the integrin subunits have no intrinsic enzymatic activity. They function by initiating the assembly of a functional signaling complex that contains catalytic signaling proteins, such as protein kinases.

5. Describe the Spemann organizer, its determination and its role in dorsalization of lateral mesoderm and in determination and patterning of neural ectoderm. Include in your answer any 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.

6. Describe Illmensee and Mahowald's experiment demonstrating the role of pole plasm in germ cell determination in Drosophila. (4 points)

Pole plasm was removed from the posterior pole of a donor embryo and injected into the anterior pole of a genetically-marked preblastoderm host. Cells resembling pole cells that formed in the anterior region were transplanted into the posterior region of genetically distinct hosts to test their potency in development. After reaching sexual maturity, the hosts were mated. 10-20% of the progeny had genotypes that were consistent with their derivation from the transplanted pole cells. Drawings to illustrate the experiment are helpful.

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

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

Pre-blastular development:

Post-blastular development:

Evidence: