2012 SURF Application
The Role of Proto-Oncogenes in the Evolution of Animal Color Patterns, Using Drosophila guttifera as a Model
By: Zachary Johnson, Mentor: Dr. Thomas Werner
Background: Embryonic development of body plans in all animals is regulated by several families of genes,
collectively known as the toolkit, which have been conserved among highly diverse species through hundreds of
millions of years of evolution [1, 2]. The role of these genes does not end with embryonic development, but
continues later in life. When these important developmental genes malfunction, there are serious consequences;
toolkit genes are implicated as proto-oncogenes in multiple human cancers [3].
As was recently published in Nature, toolkit genes can be switched on again later during development to
decorate insect wings with complex color patterns [4]. Thus, toolkit genes appear not only to shape organisms, but
“paint” them as well. Werner et al. 2010 provided, for the first time, direct genetic evidence that the toolkit gene
wingless induces the expression of the yellow gene, which produces black melanin spots in the wings of Drosophila
guttifera (Figure 1a-b) [4].
Until recently, it was not known that different animals share most of their genes. However, genomic
sequencing of the first few model organisms allowed us to see that the genetic makeup of all animals is indeed very
similar; especially conserved are the toolkit genes. So conserved are these genes that it has been possible to
substitute orthologous genes in vivo between species separated by over one billion years of evolution [1]. For
example, the mouse Pax-6 gene, ultimately responsible for the development of eyes in mice, is sufficient to induce
the growth of ectopic eyes in Drosophila, showing the extreme level of conservation of toolkit genes over vast
ranges of evolutionary time [5].
D. guttifera is a species with beautiful pigmentation patterns on both wing and body (Figure 1a). The
wing pattern consists of black spots dispersed along the veins with grey shading in between. Four black stripes
adorn the thorax, oriented symmetrically along the midline atop a backdrop of cinnamon brown. Black spots are
Figure 1-yellow expression precedes expression of black melanin throughout D. guttifera. a, pigmentation pattern in
adult D. guttifera. b-d, In-situ hybridizations performed with a yellow probe displaying areas of yellow expression
(highlighted by black arrows) in the pupal wing (b), thorax (c), and abdomen (d) (Werner lab, unpublished).
found on the posterior edge of each abdominal segment on a similar cinnamon background, which darkens
posteriorly.
Because wingless was shown to induce black spots in D. guttifera wings, it is possible that this gene is also
responsible for other pigmentation patterns in the organism, such as the black spots of the abdomen. However,
other strong candidate genes exist as well, such as bric-à-brac 1 [6], bric-à-brac 2 [6], and Abdominal B [7]. All three
of these genes have been shown to induce abdominal pigmentation in Drosophila melanogaster, a species that
shares a common ancestor with D. guttifera that lived approximately 60 million years ago [8]. The toolkit genes
distal-less [9], engrailed [10], and spalt [10], implicated in the development of wing color patterns in butterflies, will
also serve as candidates in this study. Additional candidates include the toolkit genes: decapentaplegic [11] and
hedgehog [12], active in formation and pigmentation of the abdomen; and pannier, which has been shown to
function in thorax patterning [13].
Significance: The genetic interactions between toolkit genes and their target genes within developmental
pathways are poorly understood and more examples of their function are needed. Researching them further in a
model organism will illuminate genetic, developmental, and evolutionary mechanisms underlying novel traits in all
animals. The investigation of pigmentation patterns, traits critical to processes such as mimicry, camouflage, and
mate recognition, provides an attractive opportunity to advance our understanding of these mechanisms.
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2012 SURF Application
Question: Werner et al. 2010 showed toolkit-induced pigmentation patterning in the wings of D. guttifera. Most
conveniently, the same species also has complex pigmentation patterns on its abdomen and thorax, which await
investigation. This grants the opportunity to answer this proposal’s essential question:
Did pigmentation patterns throughout the body of D. guttifera evolve through the same genetic mechanism, or did
the complex pigmentation patterns on different body parts evolve independently by employing different toolkit
genes and developmental pathways? The tools needed to answer this question are at hand. Working transgenic
techniques have been developed for D. guttifera [4] and the necessary genomic sequences are available
(unpublished).
Hypothesis: wingless or any other of the proposed candidate genes will show an expression pattern in the pupal
epidermis that correlates with the stripe and spot patterns of the thorax and abdomen, respectively. I base this
assertion on previous findings showing that the expression of these genes correlates with adult pigment patterns
on wings and bodies of fruit flies [12, 14] and butterflies [10].
Specific Aims: My project will focus on pigmentation patterns in D. guttifera. The role of the genetic toolkit in this
organism’s wing pigmentation has been investigated previously [4]. It is my goal to expand this investigation into
other body parts of the fly. I wish to determine the genes ultimately responsible for inducing color patterns in the
abdomen and thorax of D. guttifera. More specifically, it is my goal to determine the correlation between toolkit
gene expression patterns in several stages of pupal development and the pigmentation patterns of the adult fly.
Methods: In order to determine which toolkit genes are at work in the production of color patterns in the thorax
and abdomen of D. guttifera, I will perform in situ hybridizations with DIG-labeled RNA probes in adult epithelial
tissues during pupal development. Probes for the toolkit genes listed in the background have already been made
and are available for my use in the lab of Dr. Thomas Werner. I will use these probes to perform hybridizations in
pupae at several stages of development.
It is already known that the yellow gene is expressed at the mid-pupal stage (Werner lab, unpublished).
Therefore, expression or co-expression of the toolkit genes in question should occur slightly earlier and is expected
to foreshadow the expression pattern of yellow and the adult melanin patterns (Figure 1). I will perform my
investigation in pupae from the time the adult epidermis forms through the mid-pupal stage.
Prior to starting the in situ hybridizations, D. guttifera pupae will be harvested from existing stocks,
dissected, and rinsed thoroughly to remove internal organs. Hybridizations will be performed according to
protocols in place in the Werner lab. A short outline of the hybridization protocol for thoraxes and abdomens can
be seen in Figure 2.
Day 1
 Prepare tissues via washes with
ethanol, methanol, and PBT.
 Permeabilize with Proteinase K.
 Pre-hybridize.
 Incubate with probe to allow for
binding with desired mRNA
transcripts.
Day 2
 Wash away unbound probe via 3
successive incubations in
hybridization solution and PBT.
 Incubate with anti-DIG alkaline
phosphatase-labeled antibody.
Day 3
 Prepare tissues for staining via
washes with PBT and Staining
Buffer.
 Stain tissues with Staining
Solution, checking regularly for
pattern development.
Figure 2- Outline of the in situ hybridization protocol
If the expression of a candidate gene in the pupal epidermis is found to correlate with the adult color
patterns, future research will aim to provide definitive genetic proof that the implicated gene is responsible for
inducing the pigmentation pattern with which it correlates. This can be accomplished via misexpression of the gene
in another area of the same body part in an attempt to induce ectopic pigmentation. It should be noted, however,
that providing this proof would require the use of advanced transgenic techniques, which are beyond the scope and
timeline of this proposal.
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2012 SURF Application
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