Natural Selection
Updated October 2019
www.BioInteractive.org
Page 1 of 11
Activity
Educator Materials
Modeling the Regulatory Switches of the
P
itx1 Gene in Stickleback Fish
OVERVIEW
This hands-on activity supports the short film The Making of the Fittest: Evolving Switches, Evolving Bodies and
aims to help students understand eukaryotic gene regulation and its role in body development using the example
of a well-studied gene called Pitx1. Students will interpret molecular diagrams of eukaryotic gene transcription to
familiarize themselves with the molecular components and mechanisms responsible for regulating gene
transcription. They will then create models showing how Pitx1 gene transcription is regulated in two
morphologically different populations of stickleback fish.
KEY CONCEPTS
• Virtually all cells in the body have the same genetic makeup*, but not all genes are expressed in all tissues at
all times.
• Whether a gene is expressed is dependent on the regulatory switches that the gene possesses, and the
activators or repressor proteins present in that cell.
• Activators bind to regulatory switches in a sequence-specific manner. The binding of the activators to the
switches activates transcription.
• Some genes, especially genes involved in body development, have multiple switches. Each switch
independently regulates the expression of the gene in different parts of the body at different times in the
animal’s life cycle.
• The presence of multiple switches enables the same gene to be used many times in different contexts and
thus greatly expand the functional versatility of individual genes.
• Mutations in regulatory switches affect expression of a particular gene in a particular tissue without affecting
the gene’s protein product (shape and function).
• Changing the expression of a gene involved in body development can have profound effects in shaping the
body.
(* There are some notable exceptions:
(1) Mature red blood cells have no DNA.
(2) Germ line cells (sperm and egg) have half the genetic material.
(3) B cells, which are part of the immune system, have DNA that has been rearranged to make antibodies.
(4) Cells acquire somatic mutations. In some cases, these mutations confer a selective advantage for that cell and that
mutation will be fixed in a set population of cells, like in the case of cancer cells.)
STUDENT LEARNING TARGETS
• Interpret visual representations of eukaryotic gene transcription.
• Explain how mutations in regulatory regions of genes differ from mutations in coding regions.
• Construct models to represent the regulation of gene transcription.
CURRICULUM CONNECTIONS
Standards
Curriculum Connections
NGSS (2013)
HS-LS1-1, HS-LS3-1, HS-LS3-2; SEP2
AP Bio (2015)
3.A.1, 3.B.1, 4.A.3; SP1
IB Bio (2016)
2.7, 7.1, 7.2
Common Core (2010)
ELA.RST.9–12.3, ELA.RST.9-12.7
Vision and Change (2009)
CC2, CC3; DP3
Modeling the Regulatory Switches of the
Pitx1
Gene in Stickleback Fish
Natural Selection
Updated October 2019
www.BioInteractive.org
Page 2 of 11
KEY TERMS
activator, cis-regulatory region, coding region, enhancer, eukaryotic gene regulation, mutation, promoter,
regulatory switch, repressor, RNA polymerase, transcription factor
TIME REQUIREMENTS
• Two 50-minute class periods.
SUGGESTED AUDIENCE
• High School: Biology (General, AP/IB)
• College: Introductory Biology
PRIOR KNOWLEDGE
Students should be familiar with:
• basic concepts related to genetics and the central dogma
• how lac and trp operons work
• the role of genetic variation in a species in natural selection and evolution
MATERIALS
For Part 3, students will need:
• 4 white pipe cleaners (30–46 cm) or pieces of white twine (46 cm)
• magic markers (blue, green, red, yellow, purple)
• Scotch tape
• poster board or 4 sheets of letter-size paper
• any items that can represent proteins (any types of materials that come in different colors can be used; see
“Teaching Tips” for ideas)
• ruler
• scissors (optional)
TEACHING TIPS
Key Points to Emphasize:
• Students may wonder why Pitx1 is expressed in such diverse tissues. This is because the Pitx1 gene contains
multiple regulatory switches that allow for transcription of that gene in multiple tissues. The expression of
Pitx1 is important in various tissues because the Pitx1 protein is itself a regulatory protein that serves many
roles in the development of the fish. Pitx1 controls the expression of multiple genes, not just those
responsible for forming the hind limb.
• In addition, students should recognize that some activators and repressors are present in multiple tissues.
Approximately 5–10% of the human genome encodes regulatory proteins, which act in different
combinations to produce a great diversity of gene expression patterns.
• When students think of cell-type-specific proteins, they may automatically think of structural proteins like
insulin in pancreatic cells, or myosin proteins in muscle cells. Remind students that cells also contain
regulatory proteins, like Pitx1. The combination of both structural and regulatory proteins defines the cell
type.
• Lead students to understand that the expression of activators that activate Pitx1 transcription (for example,
the activator that binds the pelvic switch) is also under regulatory control. Thinking about development raises
chicken-or-egg questions. Although the development of a complex animal from a single cell is not fully
understood, great progress has been made in recent decades to understand how different sets of genes
move the development of an embryo through different stages to maturity. These processes involve
Modeling the Regulatory Switches of the
Pitx1
Gene in Stickleback Fish
Natural Selection
Updated October 2019
www.BioInteractive.org
Page 3 of 11
messenger RNA and proteins packed into the egg, and dividing cells having positional information about
where they are in the developing embryo. Students are encouraged to read more about embryology,
development, and differential gene expression.
• The film uses the term “regulatory switches.” Some textbooks will refer to them as enhancers.
Procedural Tips:
• Have students work in groups of two or three.
• The Regulation of Eukaryotic DNA Transcription
animation can be used as a supplemental resource. Note that
the diagram in the animation looks different from the ones used in the film. Make sure that students are
comfortable with looking at a variety of schematic illustrations used to represent similar molecular processes.
• You may want to show students the film the day before or assign it as homework to provide enough time to
complete Parts 1 and 2.
• In Part 3, have your students tape the models onto a poster board or on four sheets of 8.5” x 11” paper.
Alternatively, your students can take digital photographs of their models and submit them for review. Be sure
to instruct your students on how they should submit the work they do in Part 3.
• Students can use different types of materials (i.e., colored stickers, Play-Doh, Legos, beads, etc.) to model the
proteins in Part 3. The proteins needed for this activity are listed below. Suggested colors are in parentheses.
Provide students with four sets of each of the proteins, although not every protein will be used:
o RNA polymerase (purple)
o pelvic switch binding activator (green)
o jaw switch binding activator (red)
o pituitary switch binding activator (yellow). This piece will not be used in the activity, but you may want to
provide it for students as a “red herring.” You may discuss with your students where this activator would
bind and in which tissues you might find it.
o general transcription factors (optional, blue)
o mediators (optional, orange)
ANSWER KEY
PART 1: REVIEWING THE REGULATION OF EUKARYOTIC GENE TRANSCRIPTION
1. Figure 1 is a diagram, similar to the one shown in the film (8:00–8:24), showing key components of gene
transcription. Label the
boxes in Figure 1 with the
letters a–e, which
correspond to the terms
listed below. For example,
write letter “a” in the box
pointing at the coding region
of a gene.
a. protein-coding region
b. regulatory switches (or
enhancers)
c. promoter
d. mRNA
e. RNA polymerase
b
d
c
e
a
Modeling the Regulatory Switches of the
Pitx1
Gene in Stickleback Fish
Natural Selection
Updated October 2019
www.BioInteractive.org
Page 4 of 11
2. Describe the function of regulatory switches. (Note: Some textbooks refer to
regulatory switches as enhancers.)
Regulatory switches are regions of DNA that can be bound by a particular activator or repressor in a sequence-
specific manner. A switch can either be near the coding region or many megabases away. The switch controls
the transcription of genes in different tissues and at different times in development.
3. Gene transcription is a complex process that involves the interactions of proteins and regulatory regions of
DNA. The animation in the film (8:00–8:24) and Figure 1 show some of the factors involved but not all. In the
cell, a number of proteins bind to different regions on the DNA to regulate gene transcription. Use your
textbook to learn about the proteins involved in eukaryotic gene transcription. Mark the proteins that are
involved in eukaryotic gene transcription and regulation.
☐proteasomes
☒general transcription factors
☐lactase
☒activators
☐operons
☐ribosomes
☐DNA polymerase
☒mediators
☒RNA polymerase
4. Which protein(s) from the list above bind(s) to regulatory switches in a sequence-specific manner?
Activators
5. Which protein(s) from the list above bring(s) bound activators in contact with proteins bound to the
promoter?
Mediators
6. This drawing is missing all the protein components of eukaryotic gene transcription. Draw in the proteins
identified in question 5 to show active eukaryotic gene transcription. Be sure to label the proteins and DNA
regions in the figure. You can use any shape to represent these proteins.
Students should label all the proteins and DNA regions. Students may choose to draw mediators and general
transcription factors as a single circle, which is acceptable as long as they are bound to the correct proteins
and DNA.
Activator
General transcription factors
Mediators
RNA polymerase
Regulatory switches
Protein-coding region
Promoter
Modeling the Regulatory Switches of the
Pitx1
Gene in Stickleback Fish
Natural Selection
Updated October 2019
www.BioInteractive.org
Page 5 of 11
PART 2: GENE REGULATION IN DIFFERENT TISSUES
Activator
1
Activator
2
Activator
3
Activator
4
F
igure 2 illustrates how Pitx1 transcription is regulated in different tissues. The center image is that of a
stickleback embryo. The drawings in the surrounding boxes show the Pitx1 gene region and activator proteins
present in the jaw, pelvis, eye, or pituitary tissues. While the diagram only shows one activator in one tissue,
many activators are present in a particular tissue at any one time. For simplicity, we are only showing one
activator molecule present in a particular tissue. Activator molecules with specific shading can bind to switches
with the same shading
.
1. List all the tissues shown in Figure 2 that express the Pitx1 gene.
The jaw, pelvis, and pituitary
2. If a fish does not produce activator 1 proteins because of a mutation in the gene that encodes those proteins,
Pitx1 will be expressed in which of the following tissues?
☐jaw
☒pelvis
☐eye
☒pituitary
3. If a fish does not produce activator 3 proteins, Pitx1 will be expressed in which of the following tissues?
☒jaw
☒pelvis
☐eye
☒pituitary
Modeling the Regulatory Switches of the
Pitx1
Gene in Stickleback Fish
Natural Selection
Updated October 2019
www.BioInteractive.org
Page 6 of 11
4. Assume that a fish inherits a
deletion mutation in the pituitary
switch that inactivates the switch.
You isolate DNA from jaw, pelvic,
eye, and pituitary tissues. In the
DNA of which tissue(s) would you
expect to see the pituitary switch
mutation? Draw an “X” over the
mutated switch in the
appropriate tissue(s).
Make sure that students have
placed an “X” over the pituitary
switch in all four tissues. The
mutation will be the same in all
tissues because it is an inherited
mutation.
5. When a mutation in the pituitary switch prevents activator 4 from binding, where would you expect Pitx1 to
be expressed?
☒jaw
☒pelvis
☐eye
☐pituitary
6. A fish inherits a mutation that results in a new regulatory
switch (“eye switch”) that regulates Pitx1 expression in the
eye. This new switch binds a particular activator found in
the tissues of the eye (activator 3). See Figure 3.
W
here would you expect Pitx1 to be expressed?
☒jaw
☒pelvis
☒eye
☒pituitary
7. A fish inherits a mutation in the Pitx1 coding region. This is a nonsense mutation that introduces a premature
stop codon, resulting in a
nonfunctional truncated protein.
You isolate DNA from jaw,
pelvic, eye, and pituitary tissues.
In which tissue(s) would you
expect to see this Pitx1 coding
region mutation? Draw an “X”
over the Pitx1 coding region in
the tissues where you would
expect to see the mutation.
Modeling the Regulatory Switches of the
Pitx1
Gene in Stickleback Fish
Natural Selection
Updated October 2019
www.BioInteractive.org
Page 7 of 11
Make sure that students have placed an “X” over the Pitx1 coding region in all
four tissues. The mutation will be the same in all tissues because it is an inherited mutation.
8. Where would you expect Pitx1 to be expressed in the above scenario?
Pitx1
is not expressed in any tissue.
9. The Pitx1 protein has important functions in various tissues during stickleback development. The complete
absence of the Pitx1 protein from all tissues is lethal to the organism. However, as shown in the film, the Pitx1
protein can be absent in the pelvis alone, and the fish survives. The absence of Pitx1 in the pelvis confers a
unique phenotype. Mark the fish below that lacks Pitx1 expression in the pelvis.
a☐ b☒
10. A
quarry in Nevada contains fossil stickleback fish that once lived in an ancient freshwater lake at this site
about 10 million years ago. By examining many stickleback fossils in each rock layer, Michael Bell has
determined that over many generations the skeletons of stickleback living in the lake changed. In some rock
layers, most of the stickleback fossils lack pelvic spines, as pictured below.
Based on what you know about the molecular mechanisms that control the development of
stickleback pelvic spines, identify which figure below most likely represents what the Pitx1 gene
region looked like in these stickleback fish that lacked pelvic spines. The X represents a mutation that
inactivates that particular gene region. _
C
____
Fossil stickleback fish
lacking pelvic spines.
The circle shows the region
where you would expect to
find the pelvis and pelvic
spines; the arrows point to
a set of bones that are not
part of the pelvis.
Modeling the Regulatory Switches of the
Pitx1
Gene in Stickleback Fish
Natural Selection
Updated October 2019
www.BioInteractive.org
Page 8 of 11
PART 3: MODELING GENE SWITCHES IN STICKLEBACK FISH
1. Based on the information from the film, what is the difference in the DNA around the Pitx1 gene region
between marine and freshwater stickleback fish?
The
Pitx1
DNA of marine stickleback fish contains a pelvic switch, while the
Pitx1
DNA of freshwater
stickleback fish does not.
2. Make four models of the Pitx1 gene region. Two models will represent the Pitx1 gene region of the marine
stickleback. Two models will represent the Pitx1 gene region of the
freshwater stickleback.
Students should have two sets of pipe cleaners.
•
The marine stickleback DNA should have the pelvic, jaw, and pituitary switch, as well as promoter and
Pitx1
coding region designated in the appropriate colors.
•
The freshwater stickleback DNA should have all the components listed above but designate an absent or
mutated pelvic switch. All of the following representations are acceptable.
(1) No pelvic switch at all (no green area).
(2) A smaller pelvic switch (green area will be smaller than 2.5 cm) to represent a deletion.
(3) Pelvic switch is present (2.5–5 cm green area) but indicates that it is inactive, possibly with a large X
over the switch.
The exact sizes of and distance between the regions are unimportant.
Pitx1 coding region
Pelvic
switch
Pituitary
switch
Jaw switch
Marine Stickleback
Freshwater
Stickleback
(acceptable
representations)
(1)
(2)
(3)
Promoter
For Questions 3–9, the student models should contain the following:
• The Pitx1 coding region and promoter are represented.
• The regulatory switches are included and placed correctly.
• Freshwater stickleback fish have freshwater Pitx1 DNA containing no pelvic switch, and marine stickleback
fish have marine Pitx1 DNA with all three switches.
• DNA and proteins are clearly and accurately labeled.
• The appropriate activator proteins are in the appropriate tissue. (For example, there should be no jaw-
specific activators in the pelvic region.)
• The activators are associated with the appropriate switches.
• RNA polymerase is placed correctly on the DNA.
• Students correctly indicate whether Pitx1 is transcribed.
Optional:
• General transcription factors are labeled and clearly represented.
• Mediators are labeled and clearly represented.
Modeling the Regulatory Switches of the
Pitx1
Gene in Stickleback Fish
Natural Selection
Updated October 2019
www.BioInteractive.org
Page 9 of 11
Sample models are shown below.
Marine Stickleback — Pelvis; Transcription is ON
Pelvic switch
activator
Pelvic switch
Jaw switch
Pituitary switch
Pitx1 coding region Promoter
RNA
Polymerase
Note: Students may include general transcription factors and mediators in their model. If so, models like the one
shown below are also acceptable
.
Pelvic switch
activator
Pelvic switch
Jaw switch
Pituitary switch
Pitx1 coding region Promoter
General
Transcription
Factors
RNA
Polymerase
Mediators
Modeling the Regulatory Switches of the
Pitx1
Gene in Stickleback Fish
Natural Selection
Updated October 2019
www.BioInteractive.org
Page 10 of 11
Marine Stickleback — Jaw; Transcription is ON
RNA Polymerase
Pelvic switch
Jaw switch
Pituitary switch
Pitx1 coding region
Jaw switch
activator
Promoter
Freshwater Stickleback — Pelvis; Transcription is OFF
RNA Polymerase
Pelvic switch
activator
Jaw switch
Pituitary switch
Pitx1 coding region Promoter
Note: Make sure students use the appropriate freshwater stickleback DNA.
Freshwater Stickleback — Jaw; Transcription is ON
RNA Polymerase
Jaw switch
Pituitary switch
Pitx1 coding region Promoter
Jaw switch
activator
Modeling the Regulatory Switches of the
Pitx1
Gene in Stickleback Fish
Natural Selection
Updated October 2019
www.BioInteractive.org
Page 11 of 11
PART 3 Analysis Questions
1. Explain the role that regulatory switches play in determining whether stickleback embryos will develop pelvic
spines.
A mutation in the pelvic switch shuts off the
Pitx1
gene in the pelvis. This prevents the development of pelvic
spines.
2. According to the film, what is the selective pressure that led to freshwater stickleback fish losing their pelvic
spines?
Freshwater stickleback fish without pelvic spines had a selective advantage over stickleback fish with pelvic
spines because dragonfly larvae would capture young stickleback fish by grabbing onto their protruding
spines.
3. You isolate the DNA from the heart of the freshwater stickleback that lack pelvic spines. In the space provided
below, draw what the Pitx1 gene region looks like in the heart tissue of that freshwater stickleback. Be sure to
include the appropriate switches and Pitx1 coding region and label your drawing.
Sample student drawing: Make sure that students show or designate an absence of the pelvic switch and that
there are no switches other than the jaw or pituitary switch. Some students may be tempted to draw a “heart
switch.” Take this opportunity to reiterate that the genetic code is the same in all tissues other than the
exceptions listed in the Key Concepts and Learning Objectives.
Pitx1 coding region
Pituitary
switch
Jaw switch
Promoter
4. Models serve many purposes. In this activity, you used a model to visualize a process that is too small to see.
Most models have some limitations and don’t include all the details of a complex process. List three
limitations that your models have in representing the molecular process of Pitx1 gene transcription.
Student answers will vary. Have students discuss in groups and make a list. Possible answers could include:
•
There are no histones represented in the model.
•
There are other proteins involved in transcription that are not shown in the model (including proteins
involved in moving or modifying histones and unwinding DNA).
•
The entire stickleback genome is not represented in the model.
•
In the cell, there are many different transcription factors interacting in different ways.
•
The general transcription factor is made up of a number of different proteins, not all of which are
represented in the model.
REFERENCES
Carroll, Sean B., Benjamin Prud’homme, and Nicolas Gompel. “Regulating Evolution.” Scientific American 298, 5
(2008): 60–67. https://doi.org/10.1038/scientificamerican0508-60
.
AUTHORS
Written by Eriko Clements, PhD, HHMI, and Ann Brokaw, Rocky River High School
Edited by Susan Dodge and Laura Bonetta, PhD, HHMI
Field Tested by Amy Peterson, Gananda High School; Cindy Gay, Steamboat Springs High School; Dave Kenyon, Paw Paw
High School; David Knuffke, Deer Park High School; David Prescott, St. John's Ravenscourt School; Jeannie Cuervo, Cleveland
High School; Jeremy Barlow, Uniondale High School; Jessica Ullrich, Brentwood High School; Robert Bolen, Eastport South
Manor Junior / Senior High School; Sarah Freilich, Kehillah Jewish High School; Valerie May, Woodstock Academy