SPOTLIGHT 4501
Development 139, 4501-4503 (2012) doi:10.1242/dev.085183
© 2012. Published by The Company of Biologists Ltd
The effort to make mosaic analysis a household tool
Tian Xu1,* and Gerald M. Rubin2,*
The analysis of genetic mosaics, in which an animal carries
populations of cells with differing genotypes, is a powerful tool
for understanding developmental and cell biology. In 1990, we
set out to improve the methods used to make genetic mosaics in
Drosophila by taking advantage of recently developed
approaches for genome engineering. These efforts led to the
work described in our 1993 Development paper.
Making mosaics: the early years
Mosaicism occurs naturally in many species. For example, Morgan
reported rare cases of gynandromorphy in Drosophila nearly 100
years ago (Morgan, 1914) and the random inactivation of one of the
X chromosomes in mammals was demonstrated 50 years ago
(Lyon, 1961). Mosaic animals can be generated by non-genetic
means, by transplantation of cells or tissues between animals of
differing genotypes. For example, Twitty and Schwind used
transplantation between salamanders of different sizes to show that
organ size is an intrinsic property (Twitty and Schwind, 1931).
However, such techniques are laborious and limited in the range of
biological questions that can be addressed.
The first intentional generation of genetic mosaics to study
development is attributed to Sturtevant (Sturtevant, 1929), who
used an unstable X chromosome in Drosophila to generate
individuals comprising X/O and X/X cells. Although Sturtevant
wrote that analysis of his data could “give considerable
information as to the cell lineage of Drosophila” (Sturtevant,
1929), the data were not fully analyzed until he provided them
several decades later to Garcia-Bellido and Merriam (GarciaBellido and Merriam, 1969), who then generated a developmental
fate map of the embryo. In the following 80 years, mosaic
analysis, which provides a way to mark a cell early in development
and then trace the fate of that cell and its progeny, has been utilized
to address questions ranging from developmental biology to
behavior, particularly in Drosophila. Landmark studies carried out
by Garcia-Bellido led to the idea of cellular compartments in
Drosophila imaginal discs (reviewed by Garcia-Bellido et al.,
1979). Hotta and Benzer utilized genetic mosaics to determine the
tissue focus of particular behaviors in the fly (Hotta and Benzer,
1972). Perhaps the most frequent application of mosaic analysis
has been in determining the cell autonomy of gene action. Morgan
and Bridges (Morgan and Bridges, 1919), using gynandromorphs,
showed that sex-linked genes are usually ‘autonomous’; that is,
each body part develops according to its genetic composition.
Sturtevant, through his studies of the vermillion gene, was the first
to use genetic mosaics to demonstrate the ‘non-autonomy’ of gene
function (Sturtevant, 1920). Mosaic analysis has been particularly
important as a method to predict the direction of signal
1
Department of Genetics, Howard Hughes Medical Institute, Yale University School
of Medicine, Boyer Center for Molecular Medicine, 295 Congress Avenue, New
Haven, CT 06510, USA. 2Janelia Farm Research Campus, Howard Hughes Medical
Institute, 19700 Helix Drive, Ashburn, VA 20147, USA.
*Authors for correspondence ([email protected]; [email protected])
transduction between cells during development (reviewed by
Rubin, 1989; Heitzler and Simpson, 1991).
The discovery by Curt Stern (Stern, 1936) of somatic crossingover between homologous chromosomes provided a reliable
method for generating mosaic tissues in Drosophila. A cell
heterozygous for a mutation (+/–) normally produces two identical
heterozygous daughter cells after mitosis. However, if a crossover
occurs between the two homologous chromosomes during mitosis,
a heterozygous somatic cell could produce a homozygous mutant
cell (–/–) and a twin homozygous wild-type cell (+/+), resulting in
a mosaic animal carrying cells with three distinct genotypes (+/–,
–/– and +/+). Such mitotic recombination occurs much less
frequently than meiotic recombination, but can be induced to a rate
of ~1% with ionizing radiation. Although this technique was
successfully used in many important studies, several technical
difficulties limited its application. First, the site of the mitotic
recombination event is not controlled. Second, the frequency of
mitotic recombination induced by radiation is low. Third, the high
radiation dose used to induce recombination causes extensive cell
and tissue damage. Lastly, to identify the clones of mutant cells,
the mutation of interest had to be closely linked with a marker gene
that produced a cell-autonomous, easily scored phenotype in the
cells of interest. Suitable marker genes, which needed to be located
near the mutation of interest on the same chromosome arm, were
often limiting.
The FLP/FRT system
Two key advances allowed us to overcome the limitations of the
ionizing radiation protocol and produce the highly efficient system
for mosaic analysis reported in our 1993 paper (Xu and Rubin,
1993): (1) the ability to engineer stable insertions of DNA
constructs in the fly genome using P-element transposon (Rubin
and Spradling, 1982) and (2) the demonstration that a site-specific
recombination system from yeast, comprising FLP recombinase
(FLPase) and its targets (FLP recombination targets, or FRTs), can
function in the Drosophila genome (Golic and Lindquist, 1989) and
catalyze mitotic recombination between FRTs located on
homologous chromosomes (Golic, 1991).
A Development classic
The year 2012 marks 25 years since the journal Development was
relaunched from its predecessor, the Journal of Embryology and
Experimental Morphology (JEEM). In 2008, we fully digitised our
Development and JEEM archives, and made them freely available
online. At the same time, we took the opportunity to revisit some
of the classic papers published in JEEM, in a series of commentaries
(see Alfred and Smith, 2008). Now, to mark a quarter century of
Development, we have been looking through our archives at some
of the most influential papers published in Development’s pages. In
this series of Spotlight articles, we have asked the authors of those
articles to tell us the back-story behind their work and how the
paper has influenced the development of their field. Look out for
more of these Spotlight papers in the next few issues.
DEVELOPMENT
Summary
In 1990 (when Tian Xu joined Gerry Rubin’s laboratory in
Berkeley as a postdoctoral fellow) we embarked on a project with
the aim of developing a widely applicable methodology that would
allow facile mosaic analysis for every gene in the Drosophila
genome (Fig. 1).
This set of lines enabled the generation of
mosaics for more than 95% of the genes in
the Drosophila genome
Since only the portion of the chromosome arm distal to the
mitotic recombination site becomes homozygous, it was important
to have an FRT site located close to the centromere on each of the
major chromosome arms. To achieve this, we first established a
large collection of strains each containing a different randomly
inserted FRT-containing P-element. We used a construct carrying
multiple tandem copies of the original long FRT sequence as we
(correctly) anticipated that this would increase the frequency of
FLPase-mediated mitotic recombination. With the help of Todd
Fig. 1. Genetic crosses used to produce clones of labeled cells
that are homozygous for a previously identified mutation.
Reproduction of figure 3 from the original paper (Xu and Rubin, 1993).
Chromosomes are illustrated with continuous or dashed lines and
centromeres are shown as circles. This scheme generates clones
homozygous for the l(3) mutation, marked by the absence of the w+
allele and hence an absence of pigmentation in the eye; the wild-type
twin-spot bears two copies of the w+ transgene, as revealed by darker
pigmentation relative to the surrounding heterozygous tissue. TM, third
multiply inverted balancer chromosome; w, white; y, yellow.
Development 139 (24)
Laverty and Wan Yu, the sites of insertion were mapped by in situ
hybridization to polytene chromosomes to identify those FRTcontaining P-elements inserted near centromeres. Inserted elements
that caused lethality or other phenotypes were rejected. Finally,
proximally located insertions on each chromosome arm were tested
for their ability to support mitotic recombination at high frequency.
In the end, we were able to identify a suitable FRT line for each of
the major chromosome arms. Together, this set of lines enabled the
generation of mosaics for more than 95% of the genes in the
Drosophila genome.
The design of the system provided many technical advantages
over radiation-induced mitotic recombination. First, mitotic
recombination only occurs at the FRT site, thus excluding the
possibility of segregation of the mutation and the marker used to
identify the cell clone (even when the two were not closely linked).
Second, the markers used to identify the cell clones could be
introduced as transgenic constructs. By placing the yellow+ and
white+ transgenes onto each of the FRT chromosome arms, mosaic
clones of any mutation in the genome could be marked with the
visible yellow or white marker. A mini-white+ transgene was also
placed onto these arms so that the mutant (–/–) and wild-type (+/+)
twin-spot clones could each be identified in the heterozygous
background (+/–); a clone of mutant cells in the eye would appear
unpigmented, whereas the wild-type twin-spot clone would be a
darker shade of red than the surrounding heterozygous tissue. The
ability to identify wild-type clones provides an internal control for
studying mutations that either result in growth advantage or cause
cell death. Most traditional cell markers could, however, only be
scored in terminally differentiated cells. Introducing epitope-tagged
markers in transgenic constructs allowed non-terminally
differentiated cells to be identified in mosaic clones, a capability
crucial to the study of genes involved in developmental decisions.
In addition, the drug-resistant gene neor was engineered into the Pelement constructs to genetically label the FRT sites and hence
facilitate strain construction. Third, the expression of FLPase has
little or no damaging effects on cells and tissues carrying these FRT
chromosomes. Moreover, FLPase expression can be controlled so
that clones of mutant cells can be generated at specific
developmental stages (via heat shock) or in a given tissue or cell
type. Finally, the frequency of mitotic recombination of these FRT
chromosomes is so high that almost every animal contains mosaic
clones. This not only facilitates mosaic analysis of external tissues,
but also made mosaic analysis of internal tissues or developing
tissues possible, as identification of animals containing internal
mutant clones cannot be accomplished by simple visual inspection
without dissection. More importantly, the exceptionally high
frequency of generating mosaic animals led to new genetic
applications, in particular allowing genetic screens to be conducted
in mosaic animals in which essential genes can be identified that
are required for the development and functions of adult tissues. The
desired phenotypes in this type of mosaic screen can be ascertained
in the F1 individuals, which is much more efficient than traditional
genetic screens involving three generations of breeding.
Subsequent technical developments
There have been continuous modifications and improvements of the
system for mosaic analysis since our 1993 publication that provide
additional sophistication in the generation and analysis of mutant
clones. One limitation with our original strains was that markers for
internal tissues could only be visualized after antibody staining. The
introduction of GFP-based markers in transgenic constructs allowed
live cells to be identified in mosaic clones of developing tissue (e.g.
DEVELOPMENT
4502 SPOTLIGHT
Martin et al., 2003), a crucial capability in the study of genes
involved in developmental decisions. Combination of the FLP/FRT
technology with the UAS/GAL4 system allows for the control and
visualization of gene expression in mosaic clones or for killing off
wild-type cells such that only mutant cells contribute to the final
organ or tissue of interest (Brand and Perrimon, 1993; Chou and
Perrimon, 1996; Stowers and Schwarz, 1999; Pagliarini and Xu,
2003; Yu et al., 2009). Methods for positively marking clones allow
detailed analysis of single-cell clones, greatly facilitating mosaic
studies in complex neural tissues (Lee and Luo, 1999).
Similar strategies for inducible mitotic
recombination in mice have been
developed and successfully utilized
Applications of the technique
The application of the FRT/FLP system in genome-wide mosaic
analyses and screens has had a significant impact in a variety of
fields in Drosophila research resulting in milestone discoveries. Such
studies have helped elucidate many signaling pathways and their
functions; for example, the regulation of growth and tissue size by
the Hippo/Lats and PTEN/TSC signaling pathways (Xu et al., 1995;
Huang et al., 1999; Potter et al., 2001; Potter et al., 2002; Harvey et
al., 2003; Huang et al., 2005). The system has also been instrumental
in the study of cancer biology in flies, and other biological processes
such as cell competition, including interactions between tissues of
differing genotypes (Pagliarini and Xu, 2003; Cova et al., 2004;
Moreno and Basler, 2004; Li and Baker, 2007; Wu et al., 2010). It
has led to important discoveries in defining stem cell niches (e.g. Xie
and Spradling, 1998; Xie and Spradling, 2000). The pervasiveness
of these Drosophila mosaic studies has also influenced the use of
genetic mosaics in other model organisms. Indeed, similar strategies
for inducible mitotic recombination in mice have been developed and
successfully utilized (Zong et al., 2005; Muzumdar et al., 2007;
Wang et al., 2007; Sun et al., 2008). We are pleased that the mosaic
analysis system that we developed two decades ago and its evolving
descendants continue to enable researchers to address many new
questions regarding the genetic regulation of cellular behavior and
function.
Acknowledgements
We thank Ulrike Heberlein, Tzumin Lee, Kevin Moses and Duc Nguyen for
comments on the manuscript.
Funding
T.X. and G.M.R. are supported by the Howard Hughes Medical Institute. This
work was also supported by a grant from the National Institutes of
Health/National Cancer Institute to T.X. Deposited in PMC for release after 12
months.
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DEVELOPMENT
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