1. Introduction: The Threespine Stickleback

In the previous tutorial, we learned about how a variety of factors have to come together for a transcription initiation complex to form, making the transcription of a eukaryotic gene possible. To review, complete the quiz below.

[qwiz qrecord_id=”sciencemusicvideosMeister1961-Review: Transcription in Eukaryotes”]

[h] Review: Transcription in eukaryotes


Enhancers of genes:

How transcription is controlled

In eukaryotes

[q] In the diagram below, RNA polymerase is shown at

[textentry single_char=”true”]x

[c]ID Y=[Qq]




[q] In the diagram below, newly transcribed RNA is shown at

[textentry single_char=”true”]x

[c]ID g=[Qq]




[q hotspot_user_interaction=”info_only” show_hotspots=”hover_show” hotspot_labels_stick=”hover_show keep”] Click around the diagram below. As you click around, you should be able to find six key features (which will show as labels). After you’ve found all six, study the diagram, then click “continue” to move on to the next question.

Enhancers/regulatory switch

 You found the enhancer

 HINT. The enhancer is…


protein-coding region


Transcription initiation complex (with RNA polymerase)


Transcription factors (g) and mediator proteins (h)


DNA bending protein



[q] In the diagram below, what number indicates coupled transcription-translation?

[textentry single_char=”true”]

[c]ID E=[Qq]









[q]In the diagram below, “b” is pointing to a segment of RNA that isn’t in the mRNA (at “a”). Therefore, “b” must be an [hangman].




To see the developmental and evolutionary consequences of transcriptional control of eukaryotic gene expression, we’re going to look at a small fish: the threespine stickleback (Gasterosteus aculeatus).

Threespine sticklebacks are found all around the Northern Hemisphere. On average, they’re between 3 and 4 centimeters long.  Their body form also varies. While what’s below is a bit of a simplification, you can think of the general pattern as follows.

  • In marine and deepwater lakes, you find Form A (shown above). Bony plates extend along the length of the body, and there’s a prominent pelvic spine on the fish’s underside. This spine can be locked in place, and that makes spiny sticklebacks difficult for a larger predatory fish to swallow.
  • Dragonfly larvae prey on freshwater sticklebacks

    Form B is found in shallow, freshwater lakes. This form lacks the bony plates and the pelvic spine. Why? In shallow freshwater environments, larger predatory fish are often lacking. Consequently, the pelvic spine, rather than being protective, is a wasted bit of tissue with no benefit. In addition, freshwater lakes have dragonfly larvae that prey on small fish. The attack comes from below, and the spines provide an easy target for the dragonfly larvae to grab onto. As a result, through natural selection, freshwater forms have lost their pelvic spines.

Evidence for parallel evolution. Note that the three inland freshwater populations (A, B, and C) are isolated in different lakes, yet each lacks the pelvic spine found in population D. Population D (unlike A, B, and C) migrates from the ocean upstream to lay its eggs. Click to enlarge.

If you consider the evolutionary history of sticklebacks without pelvic spines that live in lakes, the story becomes even more interesting. Like salmon, marine sticklebacks live at sea but migrate upstream to freshwater lakes to breed and lay eggs. All of the populations of freshwater threespine sticklebacks are the descendants of marine populations that became stranded in lakes after the last ice age. That means that (with a few possible exceptions) in every lake with pelvic-spineless sticklebacks, the adaptation evolved independently. In other words, this is a case of parallel evolution: similar environmental pressures causing different populations to independently evolve a similar evolutionary solution.

In the image above and to the right, fish from three unconnected Alaskan lakes have had their skeletons stained with a special dye. You can see that these fish all lack pelvic spines.

Changes in body form come about through changes in the genes that code for this form. As you’ll see in the video below, the genes involved in the loss of pelvic spines are not protein-coding genes, but enhancers: genes that regulate the transcription of protein-coding genes. In the video, enhancers are referred to as regulatory switches.

For an overview of this intersection of gene regulation and evolution, watch the video below.

2. Threespine stickleback evolution: checking understanding

The quiz below focuses on some of the key points from the video.

[qwiz qrecord_id=”sciencemusicvideosMeister1961-Stickleback Evolution: Quiz 1″]

[h]Stickleback evolution


[q] In shallow freshwater environments, sticklebacks have evolved which of the two forms below?

[textentry single_char=”true”]

[c]IE I=[Qq]






[q multiple_choice=”true”] Which of the following is the reason why populations of stickleback fish that live in lakes lost their pelvic spines?





[c]IFBlbHZpYyBzcGluZXMgYXJlIGVhc3kgZm9yIGluc2VjdCBwcm VkYXRvcnMgb2Ygc3RpY2tsZWJhY2tzIHRvIGdyYWIgb250by4=[Qq]




[q multiple_choice=”true”] The gene controlling the development of the pelvic spine is the Pitx1 gene, which codes for the Pitx1 protein. When the genes of fish with and without pelvic spines were compared, it was found that

[c]IFRoZSBzZXF1 ZW5jZSBvZiA=UGl0eDE=IHdhcyB0aGUgc2FtZSBpbiBmaXNoIHdpdGggcGVsdmljIHNwaW5lcyBhbmQgZmlzaCB3aXRob3V0IHBlbHZpYyBzcGluZXM=[Qq]






[q multiple_choice=”true”] Homologous structures are those that are inherited from a common ancestor. For example, a human being’s arm is homologous to a dog’s forelimb, a bird’s wing, and a whale’s flipper.

One reason why understanding the evolution of pelvic spine loss in sticklebacks is so important is that the pelvic spines are homologous to



[c]IHRoZSBoaW5kIGxpbWJzIG9mIGZvdX ItbGltYmVkIHZlcnRlYnJhdGVzLg==[Qq]




[q]Muscle cells transcribe large amounts of actin and myosin, two proteins involved in muscle contraction. The cells making up the lens of the eye transcribe little (if any) myosin or actin. Rather, eye-lens cells transcribe large amounts of a protein called crystallin. The difference between muscle cells and eye-lens cells is based on which of the following?



[c]RGlmZmVyZW50IGdlbmVzIGFyZSBhY3RpdmF0ZW QgaW4gZGlmZmVyZW50IHRpc3N1ZSB0eXBlcy4=[Qq]




[q]Another term for “enhancer” is regulatory [hangman].



3. Gene regulation at the transcriptional level and the threespine stickleback

From both the video and the text above, it should be clear that the Pitx1 gene is expressed in many — but not all — tissues throughout the body. Let’s look at a model of Pitx1 gene expression, and see how the body controls which tissues express the gene, and which one’s don’t.

Complete the interactive reading below.

[qwiz qrecord_id=”sciencemusicvideosMeister1961-Stickleback Gene Regulation: Interactive Reading”]

[h]Pitx1 regulation


[q labels=”top”]Start by labeling the diagram below.



[fx] No. Please try again.

[f*] Excellent!


[fx] No. Please try again.

[f*] Correct!

[l]protein-coding region

[fx] No. Please try again.

[f*] Correct!


[fx] No. Please try again.

[f*] Excellent!

[l]RNA polymerase

[fx] No, that’s not correct. Please try again.

[f*] Excellent!

[q]In the course of development, Pitx1 will be turned on in some tissues, but not others. How is this regulated? In each tissue where Pitx1 is expressed, there’s a corresponding enhancer or regulatory switch. At certain moments during development, regulatory proteins called activators will bind with these enhancers, allowing a transcription initiation complex to form.

In the diagram below, enhancers are shown, but not the activators.

[q] In embryonic tissue that will become the jaw, activators will bind with which enhancer/regulatory switch?

[textentry single_char=”true”]

[c]IE I=[Qq]





[q] In embryonic tissue that will become the pelvis, activators will bind with which enhancer/regulatory switch?

[textentry single_char=”true”]

[c]IE M=[Qq]





[q multiple_choice=”true”]Pitx1 is not expressed in the eye. The most likely reason for this is that

[c]VGhlcmUmIzgyMTc7cyBubyAmIzgyMjA7ZXll LWVuaGFuY2VyJiM4MjIxOyBmb3IgdGhlIA==UGl0eDE=IGdlbmUu[Qq]





4. How the spineless form of the stickleback evolved

Parallel evolution of sticklebacks without pelvic spines. Click to enlarge.

As described above, in populations of sticklebacks that were stranded in freshwater lakes, there were two forms of selective pressure against the pelvic spine.

  • Any fish with a pelvic spine would be at a disadvantage. That disadvantage comes from predation by dragonfly larvae, which grab onto the pelvic spines as a way of catching their stickleback prey.
  • Any fish without pelvic spines would have an advantage. In smaller, freshwater lakes, there were no large fish predators. Why? Because small lakes support smaller food webs, and there isn’t an adequate food supply to support large predators. So with no need to protect against being swallowed by larger fish, building a pelvic spine was a waste of energy. That meant that any stickleback that didn’t build a pelvic spine could use those resources to find food, find mates, etc.

The raw material for natural selection is mutation. We’ll discuss mutations in depth in a future module, but for now, just think of mutation as a change in DNA. Among the populations of stranded sticklebacks, any fish that had a heritable mutation that caused it to not build a pelvic spine would have an advantage that it could pass on to its offspring. That mutation couldn’t occur in the Pitx1 gene itself. That’s because Pitx is expressed in various regions of the body, and a mutation that stopped all expression of Pitx1 would be lethal. But a mutation in the pelvic switch would knock out expression only in the pelvis while allowing the gene to be expressed in other regions of the body.

As you observed in the video, scientists used a variety of techniques to find this mutation. These techniques included

  • experimental crosses between fish with pelvic spines and those without, and
  • Molecular genetic analysis of stickleback populations without pelvic spines.

As expected, the mutation found in different populations wasn’t exactly the same, but similar. In every case,  the mutation involved a loss of function in the same gene: the Pitx1 pelvic enhancer. A loss of function mutation is one that (not surprisingly) causes a gene to lose its function.

This can be seen in the image below, from Science Magazine (1/15/2010, volume 327). The horizontal bars, numbered 1 – 9, represent DNA sequences from individuals in nine populations of sticklebacks lacking pelvic spines. As you can see in the map (B), these populations range from Great Britain to Alaska (8 is Iceland). The red bars labeled Pel represents Pitx1 pelvic enhancers, which are located upstream from the Pitx1 gene on the same chromosome. Areas of the bar that are colored gray represent deletions. Note that in every population, the Pitx1 pelvic enhancer is completely or partially deleted. That means that in pelvic tissue, the Pitx1 gene is not switched on. As a result, no pelvic spine is built.

Complete the interactive reading below.

[qwiz style=”width: 628px !important;” qrecord_id=”sciencemusicvideosMeister1961-PitX 1 Expression in Sticklebacks”]

[h]Pitx1 expression in threespine sticklebacks: Final Quiz


[q] In the image below, a deletion in which DNA region would lead to fish without pelvic spines (but which were otherwise healthy and viable fish)?

[textentry single_char=”true”]

[c]IE E=[Qq]






[q] In the image below, a deletion in which DNA region would lead to fish that were not viable (couldn’t survive)?

[textentry single_char=”true”]

[c]IE Q=[Qq]





[q multiple_choice=”true”] If the image above this quiz included a representation of the pelvic enhancer region in a population of ocean-living sticklebacks, it would best be represented as

[c]IFNvbGlkIGJsdWUgZmxhbm tpbmcgc29saWQgcmVkOg==






[q labels=”top]Among the populations of stranded sticklebacks, any fish that had a heritable _____________ that caused it to not build a pelvic spine would have an _____________ that it could pass on to its offspring. That mutation couldn’t occur in the  __________-coding region of the  Pitx1 gene itself. That’s because Pitx is expressed in various regions of the body, and a mutation that stopped all expression of Pitx1 would be ____________. But a mutation in the pelvic ____________ would knock out expression only in the _____________, while allowing the gene to be _____________ in other regions of the body.


[fx] No. Please try again.

[f*] Good!


[fx] No. Please try again.

[f*] Great!


[fx] No, that’s not correct. Please try again.

[f*] Correct!


[fx] No. Please try again.

[f*] Excellent!


[fx] No. Please try again.

[f*] Correct!


[fx] No, that’s not correct. Please try again.

[f*] Good!


[fx] No. Please try again.

[f*] Excellent!


[q]Because the mutation in the Pitx1 enhancer arose independently in various populations, it can be thought of as an example of [hangman] evolution.


[q]The mutation that leads sticklebacks to not develop pelvic spine can be described as a [hangman] of function mutation.


[q multiple_choice=”true”]Based on the diagram below, which population lacks pelvic spines?



[c]Qg ==[Qq]


[q labels=”top”]Scientists designed an experiment to prove the claim that a loss of function mutation in the pelvic enhancer was the cause of the loss of pelvic spines in freshwater stickleback populations. To prove the claim, they inserted a fragment of DNA consisting of the Pitx1 gene plus the pelvic enhancer: . Which of the fish below is the genetically modified fish? Which is the unmodified fish?


[l]unmodified fish

[fx] No. Please try again.

[f*] Great!

[l]genetically modified fish

[fx] No. Please try again.

[f*] Excellent!


What’s next?

Now that you understand control of gene expression through transcription, you can move on to the last tutorial in this module: post-transcriptional eukaryotic gene regulation. 

Other options: