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Overexpression of Drosophila
Nanog homolog creates giants
Sonata Kirk1*+ and Kin Ti Sandstone1*+
N
ANOG is thought to be a key factor
in maintaining pluripotency in
embryonic stem cells (ESCs). Its
concerted expression with other factors such
as POU5F1 and SOX2 promotes ESC
identity. These factors offer an important area
of study because of their ability to establish
and maintain pluripotency. ESCs have the
ability to produce virtually any cell type of
all three germ layers (endoderm, ectoderm,
mesoderm). It is for this reason that
understanding the mechanisms that maintain
a cell's pluripotency is critical for researchers
to understand how stem cells work; and may
lead to future advances in treating
degenerative disease. The lack of a Nanog
homolog in Drosophila and C. elegans was
long thought to be the result of evolutionary
changes in pluripotency maintenance
mechanisms and/or the divergence of the
factors involved. The fact that the
development of Drosophila and C. elegans
also does not involve the blastocyst stage, a
development stage found mainly in
mammals, corroborated this hypothesis. We
have identified the Drosophila homolog of
the mammalian nanog gene and
overexpressed it in a conditionally active
Notch background during the prepupal stage.
Adult flies have a significantly larger trunk
and somatic features never seen before.
These flies are amenable to husbandry and
can be kept in stock as usual. Thus, we
propose that this might be indirect evidence
for the existence of gigantic creatures as
described in Greek mythology.
Coordinated transcription factor networks
have emerged as the master regulatory
mechanisms of stem cell pluripotency and
differentiation. Many stem cell-specific
transcription factors, including the
pluripotency transcription factors, OCT4,
NANOG, and SOX2 function in
combinatorial complexes to regulate the
expression of loci, which are involved in
embryonic stem (ES) cell pluripotency and
cellular differentiation (1, 4). In mammals,
embryonic stem cells are able to differentiate
and give rise to derivatives of the three
germinal layers (ectoderm, endoderm, and
mesoderm) and functional gametes (3). This
property of cells is referred to as pluripotency.
The pluripotent status of preimplantation
embryo cells and embryonic stem cells is
maintained by a complicated system of
molecular signaling pathways and
transcription factors. The key regulators in
this system are the transcription factors OCT4
and NANOG (2,6).
We sought to determine what the absence of
Nanog in Drosophila meant. Therefore, we
looked thoroughly into the genome of the fly
trying to identify alternative genes that could
potentially play Nanog’s role. To our surprise,
we were able to match a previously
uncharacterized gene (CG10411) to Nanog
with an expected value lower than 10-5 (see
Supplementary Material). In situ
hybridizations and luciferase assays
confirmed that the aforementioned gene
corresponded to a Drosophila Nanog homolog
(data not shown). However, overexpression
and knock-down studies failed to reveal any
detectable phenotype.
Since Notch is well known for its leading
role in practically every biological process,
from lateral inhibition to maintenance of
pluripotency in specific cell types, we decided
to conditionally overexpress both of the “N”
genes at the prepupal stage, which is
associated with the peak of pluripotent cell
number in Drosophila. The result was beyond
any imagination (Figure 1).
Adult flies were ridiculously large. Their
trunk was approximately 5 times bigger than a
wild type Drosophila and their dorsal bristles
were significantly enlarged. The plumose
(feathery) arista, the bristling of the head and
thorax, as well as the wing venation were
identical to that of the wild types, but scaled
up in size. Furthermore, it came as a surprise
that we were actually capable of crossing the
mutant flies to wild type ones and getting
fertile and viable progeny. Our findings
clearly suggest a path for the single step
production of giants, forcibly questioning the
gradual evolution theory and evoking that
Greek mythology might be far away from
only myths.
References and Notes
1. S. Kirk, K.T. Sandstone. Nature 121, 647 (2009).
2. S. Kirk. Development 22, 185 (2011).
3. U.L. Salvo et al. PNAS 102, 7888 (2005)
4. F. Chivalries et al. Curr Biol 20, 757 (1993)
5. Materials and methods are available as
supporting material on Science Online.
6. K.T. Sandstone. PLoS One 634, 1888 (2010).
7. We thank Foam Chivalries for granting us lab
space for our experiments. We would also like to
thank members of the Chivalries and Las Kiddie
labs for useful discussions. We finally thank Ali
Task and Sou Bassi for suggestions and critical
reading of the manuscript. This research was
funded by a Father Bank grant to KTS and by a
NMAA scholarship to SK.
Supporting Online Material
Fig. 1. Double “N”
mutant exhibiting the
gigantic phenotype.
Such individuals can
reach a body
length of 15mm.
Black arrows depict
the Notch dependent
wing phenotype.
Despite their size,
weight and the Notch
phenotype, these
flies are outstanding
flyers.
Red arrow indicates
WT Drosophila.
Photos in scale.
104!
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VOL 333!
SCIENCE!
www.sciencemag.org/cgi/content/full/333/20/104/DC2
Materials and Methods
SOM text
Figs. S1 to S5
Tables S1 to S3
References
28 December 2010; accepted 14 March 2011
10.1127/science.1199683
1 KoKogene
Center, Institute of Amazing
Research (IAR), Down That Road,
Massachusetts 10411, United States of America.
* These authors contributed equally to this work.
+To whom correspondence should be addressed.
E - m a i l : s o n a t a . k i r k @ k k c . i a r. u s ( S K ) ;
[email protected] (KTS).
www.sciencemag.org
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