Problem Set #2 ? Induction in C. elegans embryos Zoology 470 ...

Problem Set #2 – Induction in C. elegans embryos
Zoology 470 – Spring 2013
20 Points Total
Problem Set Guidelines
1. Due date: This problem set is due by the end of class on Monday, April 1, 2013 (no fooling!)
2. Sources: You may use any sources at your disposal to answer the following questions.
Legitimate sources include classmates, knowledgeable friends and colleagues, written
documents, and any other scientific resources you find useful. If you work with other
classmates on this problem set, we ask that you list the other students with whom you
worked to answer these questions. Although you may discuss these questions as part of a
group, you are expected to answer the questions as an individual. If you believe that
published references will help you answer these questions, you may cite those references.
However, citation of additional references is not required, nor is it expected.
3. Answering the questions: This problem set is designed to be answered concisely. Brief but
complete answers should be written in the space provided. It is acceptable to type your
answers, but you must provide a hard copy of the document you generate. Where you are asked
for explanations, you must provide them to receive full credit. If you find it helpful, feel free to
include diagrams in your answers. You need only turn in your answers on pages 2 & 3.
Necessary information: All of the information and techniques needed to answer the questions on
p. 2-3 have been presented in class, or are to be found in Gilbert's Developmental Biology. This
problem set requires you to learn about a new signaling pathway, the Notch/Delta pathway, which
is described in Chapter 3 of Gilbert (p. 98). You may also find it helpful to review your reading on
C. elegans early development from Ch. 5.
Problem Statement
Several labs, beginning in the 1980s, began investigating inductive interactions that lead to
differentiation of the anterior-most cell from the four-cell embryo, AB.a, and its posterior sister
cell, AB.p. These two cells give rise to cells of the pharynx, a mesodermal structure. Normally,
AB.a generates cells that form the anterior pharynx, while AB.p normally gives rise to cells
that make the posterior pharynx. These two types of cells express different proteins and
differentiate quite differently. There are several papers that you are free to consult. However, it is
not necessary to read these papers to answer the questions on pages 2-3.Papers that may be
relevant include the following, which can be downloaded from Learn@UW:
Priess, J. and Thomson, N. (1987). Cellular interactions in early C. elegans embryos. Cell 48:241-50.
Mello, C.C., Draper, B.W., and Priess, J.R. (1994). The maternal genes apx-1 and glp-1 and
establishment of dorsal-ventral polarity in the early C. elegans embryo. Cell 77:95-106.
Mango, S.E., Thorpe, C.J., Martin, P.R., Chamberlain, S.H., and Bowerman, B. (1994). Two
maternal genes, apx-1 and pie-1, are required to distinguish the fates of equivalent blastomeres
in the early Caenorhabditis elegans embryo. Development 120:2305-15.
Evans, T.C., Crittenden, S.L., Kodoyianni, V., and Kimble, J. (1994). Translational control of
maternal glp-1 mRNA establishes an asymmetry in the C. elegans embryo. Cell 77:183-94.

Zoo 470 – 2013 – Problem Set #2 Page 2
Name:________________________________ Student Number:__________________
If you worked in a group, other collaborators: _____________________________________________
1. Testing how AB.a and AB.p differentiate. Recall that there
are four cells in the C. elegans embryo: AB.a, AB.p, EMS, and
P2, whose spatial relationships are shown correctly at the right
(anterior is to the left in the diagram).
a. Jim Priess, Craig Mello, and Bruce Bowerman developed ways to
manipulate blastomeres in the early embryo. They found they
could move cells around by pushing them with a blunt needle, so that AB.a could be moved to lie in
the position normally occupied by AB.p. They also developed ways to remove cells from the embryo
by sucking them out of the eggshell with a pipette.
a. (4 points) Jim believed that P2 normally sends an inductive signal to the AB daughter it is
touching that leads that AB daughter to form posterior pharynx. Using the simple blastomere
manipulations Jim developed, describe one experiment you could perform to show P2 contact is
sufficient to induce either AB daughter cell to form posterior pharynx.
Moving P2 so that it touches AB.a instead of AB.p should lead to the following: AB.a should now
make posterior pharynx. Since the question only asks for sufficiency. However, if such contact is
indeed necessary, then AB.p, if it no longer touching P2, would not be expected to make posterior
pharynx, and instead would make anterior pharynx.
b. (4 points) Craig Mello and Jim Priess created the embryo below on the right (b). A normal embryo is
shown for comparison (a).
If Jim’s hypothesis is correct, will this embryo make more or less anterior pharynx than normal?
Circle the correct answer:
Embryo b will make more the same less anterior pharynx than normal
Clearly state your reasoning: Contact with P2 is needed for posterior pharynx. Therefore, since
neither AB daughter can touch P2 in this case (see the picture), neither should make posterior
pharynx, and therefore an excess of anterior pharynx should result. THis is in fact what happened
with this embryo; Jim and Craig followed it until became a much more advanced embryo, and looked
posterior vs. anterior pharynx.
2. apx-1, glp-1, and pharynx differentiation
a. (2 points) Craig and Bruce (working independently) showed that a gene called apx-1 (for anterior
pharynx in excess) is required for posterior pharynx differentiation. apx-1 is a maternal effect gene. If a
mother (hermaphrodite) worm has the genotype apx-1(-)/+ [+ = wild-type copy of the gene], i.e., it is a
heterozygote, what percentage of its offspring will have defective pharynges [“pharynges” is the plural of
“pharynx”!]?
Percentage of offspring from apx-1(-)/+ with defective pharynges: _____0%________
Since the mother is a heterozygote, and it is her genotype that matters (apx-1 is materal effect), all
of the offspring will appear normal.

Zoo 470 – 2013 – Problem Set #2 Page 3
2. (cont.)
b. (4 points) Please refer to p. 98 of Gilbert for information on the Notch/Delta pathway. Craig
showed, using a special mutant in the glp-1 gene, that glp-1 is needed for correct differentiation of
AB.p. glp-1 encodes a Notch protein. Craig went on to show that apx-1 encodes a Delta family
protein. You are now working in Bruce Bowerman’s lab, where techniques were developed for
isolating blastomeres and recombining them, as we discussed for Wnt/Frizzled signaling in class.
You are eager to try blastomere recombinations using cells lacking either apx-1 or glp-1 function.
You assess anterior pharynx formation using an antibody that recognizes anterior pharynx.
Based on your study of Notch and Delta, what would happen if an AB.p cell lacking APX-1 protein
were combined with a P2 cell lacking GLP-1 protein? Clearly explain your answer.
APX-1 the Delta-like ligand; GLP-1 the Notch-like receptor. P2 normally needs to produce the ligand
(signal), and AB.p normally needs the receptor to sense it. Therefore in this case there would a
normal mount of anterior pharynx, since AB.p doesn’t need the ligand, and P2 doesn’t need the
receptor.
3. Translational control of glp-1
a. (1 point) Tom Evans, who was working in Judith Kimble’s lab here at UW at the time, showed that
glp-1 mRNA is found everywhere in the four-cell embryo, but that the protein is only found in two
cells in the four-cell embryo. What technique did he use to assess mRNA localization?
Technique: ___in situ hybridization__________________________
b. (3 points) Tom hypothesized that the glp-1 mRNA is subject to translational control. To see which
part of the mRNA was involved, he used molecular biology techniques to make a piece of DNA that
encoded a reporter protein he could detect (β-galactosidase) fused to DNA encoding parts of the
glp-1 mRNA. He then assessed whether the artificial mRNAs produced from this DNA, when
introduced into embryos, were only translated in two cells. What part of the glp-1 gene would you
predict would need to be attached to the β-galactosidase coding region to cause it to only be
expressed in two cells? Explain your answer.
Part of the glp-1 gene required for translation of the fusion protein only in two cells: 3’-UTR
Reasoning: Variants of the following rationale are acceptable:
The 3’-UTR is required for translational regulators (protein, miRNAs, etc.) to bind to an mRNA and
regulate its expression (the polyadenylation “signal” sequence is also there). Since this is the region
that controls where the protein is translated, it would have to be hooked up the coding region for βgalactosidase
to get the restricted protein expression pattern.
c. (2 points) Neither of the two cells in which GLP-1 protein is normally found is EMS. Based on this
information, what are the most likely cells that contain GLP-1?
Two cells at the four-cell stage that contain GLP-1: _____AB.a and AB.p______
AB.a and AB.p are both capable of responding to the P2 signal, so they each would be expected to
have the receptor to receive the signal. The signal is APX-1/Delta, so they would each be expected
to have GLP-1/Notch.