---

B. Müllerian Inhibitory Substance:

Hormone produced in male mammals that is responsible for regression of the Müllerian duct during secondary sexual development.

---

C. Silencer:

DNA regulatory region that can be some distance for the coding region of the gene whose expression it regulates. Silencers suppress expression of the gene of interest.

---

D. Genomic Imprinting:

Phenomenon whereby genes are expressed or fail to be expressed depending on whether the chromosome on which they reside is derived from the mother or the father. Methylation of DNA is thought to play a role in stable imprinting in mammals.

---

E. Cytoplasmic Determinant:

Substance which confers on cells that possess it the propensity to differentiate in a particular manner. Cytoplasmic determinants can be asymmetrically distributed based on cleavage patterns in the early embryo.

2. An invertebrate embryologist friend of yours in Hawaii is studying a new species of ascidian. Although the fertilized eggs of this species have regions of brightly colored cytoplasm, she claims that embryos in this species do not have myoplasm that functions in the same way as that in other, "classic" ascidians, because they have a "red crescent" instead of a yellow one. You don't believe her theory, so you set out to disprove it. Describe one way you could you show that the red crescent functions like the yellow crescent in other ascidians (2 points).

A few possibilities: (1) Redistribute the red crescent by compressing the embryo. All cells that receive red crescent cytoplasm should become muscle cells. (2) Isolate cells that have red crescent cytoplasm; all isolated cells with red cytoplasm should become muscle in isolation. (3) Use cytochalasin to suppress cytokinesis. All cells receiving red cytoplasm should make muscle.

   3. Circle true (T) or false (F) for each of the following (6 points)

4. Germ plasm is a common feature within animal embryos. Answer the following questions about the nature and formation of this material.

A. What is germ plasm? (1 point)

Germ plasm is a specialized cytoplasm containing germinal granules, "nuage" material, mitochondria with a special appearance, etc., that is inherited by those cells that will form the germ line.

B. Oskar is a maternal effect gene required for formation of germ plasm in Drosophila.

Describe one experiment that demonstrates this besides the mutant phenotype:

Injection of a RNA with the oskar coding region and bicoid 3’UTR that localizes to the anterior end of the Drosophila oocyte results in production of ectopic pole plasm in the anterior of the early embryo.

C. PIE-1 protein is thought to be important for the differentiation of the cells that form the germ line in C. elegans. What is PIE-1 thought to do? (1 point)

PIE-1 is thought to repress transcription in germ line precursors.

D. Recall that mex-1 gene function is important for regulating pie-1 gene function. You breed a hermaphrodite that is heterozygous for a complete loss-of-function mutation in the mex-1 gene with a male who is also heterozygous for the same mutation in mex-1. What would the phenotype of the embryos be who inherited two non-functional copies of the mex-1 gene (i.e., they're homozygous for mex-1), and why?

Mex-1 is a maternal effect gene, so the mother would have to be a homozygote for her offspring to be affected. Since she is a heterozygote, the offspring will be normal.

5. Neural crest cells differentiate based on their position of origin, and/or in response to cues within their local environment.

A. Provide two pieces of evidence that neural crest cells respond to such local environmental cues (2 points)

Transplant cervical crest to the thoracic region or vice versa, and the derivatives produce neurotransmitters appropriate to their new location

Isolate NC in vitro; depending on what combination of growth factors, etc. they are incubated with, various differentiated cells result

B. You are studying a knockout mouse in which homozygous embryos are missing rhombomeres 2-6. What non-brain structures would you expect to be missing? (1 point)

Pharyngeal Arches and Derivatives (e.g., facial cartilages, ear structures, etc.)
 
 

6. You work in Marc Tessier-Lavigne's lab, and you are studying axon guidance. Please answer the following questions about guidance of axons in embryos.

A. You are reviewing previous axon guidance experiments. Cite two pieces of evidence that indicate netrins are required for attractive guidance of axons that migrate ventrally in animal embryos (2 points).

KO Mice:
Commissural Axons have difficulty migrating to the ventral region of the spinal cord.

UNC-6 Mutant Worms:
Ventral Migrations are disrupted.

Floor Plate is an attractive cue in vitro; it also expresses high levels of netrin-1.
 

B. Your Ph.D. thesis work involves a set of cells in the ventral spinal cord that normally migrate dorsally. Design one experiment that would test whether netrin acts as a chemorepellant for your neurons (2 points)

Place in vitro near clump of recombinant cells expressing netrin-1; they should grow away from the netrin-1 expressing cells or In netrin-1 KO mouse, axons should stray towards ventral region of the spinal cord
 

 

7. Transcriptional regulators influence a wide variety of developmental processes in Drosophila.

A. Bicoid is a transcriptional regulator of zygotic hunchback expression in the early embryo. Describe what happens to levels of hunchback mRNA in the following situations. Use diagrams if you find these helpful (3 points)

i. When an embryo is derived from a mother that is homozygous for a loss-of-function mutation in bicoid:

hb is activated by bcd; in this case, hb RNA will be largely absent from the anterior

 ii. When an embryo is derived from a mother with 2 extra copies of the bicoid gene:

Now we should see an expansion of the region in which hb is transcribed too far towards the posterior

iii. When an embryo is pricked at its anterior end to allow cytoplasm to leak out, and then the same embryo receives an injection of posterior cytoplasm into its anterior end:

Now we should see something similar to (a)

 
B. Another well-studied example of transcriptional regulation in embryos is the way in which gap gene products regulate the expression of pair rule genes in Drosophila. In general terms, describe how this regulation occurs ( 2 points).

The overlap of domains of expression of gap proteins determines where stripes of pair rule expression will appear. The regulatory region of each pair rule gene is sensitive to local combinations of transcriptional activators and repressors.
 
 

 

8. You are studying hedgehog proteins, which you may recall are important in pattern formation events in many embryos.

A. Based on what you know about the molecular basis of segment formation in Drosophila, why would mutations in hedgehog affect the formation of segments? (2 points)

hh is sent by an engrailed expressing cell to the neighboring wingless expressing cell. The wg expressing cells maintains its expression in response to hh. Without hh, wg is lost, and this leads to loss of en in the adjacent cell.

 

B. Your friend is studying sonic hedgehog knockout mice. He wants to show that the only limb defects associated with the knockouts are due to failure of cells in the zone of polarizing activity (ZPA) to send the appropriate signals. Assuming that all experiments possible in chicks can be performed in mice, design an embryological experiment that would show this. You may not use engineered cells or introduce molecules into your experimental subjects (2 points)

The ZPA normally produces shh. If we transplant WT cells from the ZPA into the limb bud of a KO mouse, it should rescue the polarity defects.
 

 C. Your friend wants to be sure that the normal dorsal-ventral polarity of the limb bud ectoderm is not affected in the knockouts. What molecule’s expression might he examine? (1 point)

radical-fringe

 

9. You are assaying for mesoderm differentiation in Xenopus embryos under a variety of circumstances, using the loose lips gene as a marker. loose lips encodes a transcription factor normally expressed in the dorsal, anterior mesoderm in Xenopus. In each case, state whether you expect the levels of expression of loose lips to be higher than normal (high), normal, or lower than normal (low). (4 points).

Treatment	loose lips level
Inject large quantities of b-catenin mRNA into the one-cell zygote	High
Injection of high levels of chordin mRNA into ventral blastomeres at the four-cell stage	High
Injection of large quantities of BMP-4 mRNA into dorsal blastomeres of the early embryo	Low
Combine animal cap cells with ventral, vegetal cells at the 16-cell stage	Low (Or Normal)
Isolate dorsal, vegetal cells with no further treatment	Low (Or Normal)
Injection of a mutant form of Brachyury that acts as a repressor	Low

10. During limb development, the FGF family of proteins are thought to be crucial for the establishment of the limb bud.

A. Describe one experiment that demonstrates that FGF-10 is necessary for establishing the sites of limb bud formation in the flank of amniotes (2 points):

FGF-10 KO mouse: no limbs form.

B. Describe one experiment that indicates FGF family members are sufficient as a functional substitute for the apical ectodermal ridge (AER) (1 point)

Add FGF-2 (or —4 0r —8) soaked bead to a bud that has had its AER removed; limb bud grows out

11. In an actual episode of the X-files called Postmodern Prometheus, the evil Dr. Pollidari has been experimenting with the homeotic gene proboscopedia in Drosophila.

A. In a terse exchange with Scully and Mulder, Pollidari asks Scully to explain to Mulder what "homeotic genes" are. If you had been Scully, what would you say" (1 point)

Hometoic genes are genes which when mutated can cause transformation or conversion of whole body parts.

B. Pollidari goes on to say that humans have such genes, too. Name three expected similarities between human homeotic genes and Drosophila homeotic genes, and one expected difference. (4 points)

Similarity #1: Encode homeodomain transcription factors

Similarity #2: Sharp anterior onset of expression

Similarity #3: 3’ to 5’ order on chromosome correspond to anterior-posterior location of expression

Difference #1: Vertebrates have four sets of Hox genes compared with a single set in flies.

Difference #2: In flies, homeotics often have a sharp posterior boundary of expression.

C. Imagine that in a later episode, the writers realize that with the availability of human embryonic stem cells, it might be possible for Dr. Pollidari to make a knockout of a human homologue of proboscopedia. After Pollidari performs the unethical experiment, he is enraged to find little phenotypic effect. Being a bright young developmental biologist, you are not surprised by this. Why? State your reasons (1 point)

Functional redundancy of Hox genes means that it might be necessary to knock out more than one paralogue to see an effect.

12. It is possible to produce amphibian embryos that "exogastrulae", i.e., their marginal zone tissue does not involute, but instead elongates on the exterior of the embryo (see the figure). You reexamine neural induction in such embryos. What do you expect to see regarding expression of neural markers, and why (2 points)?

Planar neural induction should still occur, so, as in the Keller sandwiches, we should see neural markers expressed at the correct distance away from the mesoderm up to the level of the hindbrain.

RETURN TO THE PREVIOUS EXAMS SECTION