NIH Public Access

Author Manuscript
Chem Biol. Author manuscript; available in PMC 2011 June 25.
Published in final edited form as:
NIH-PA Author Manuscript

Chem Biol. 2010 June 25; 17(6): 590–606. doi:10.1016/j.chembiol.2010.04.013.

Synthetic strategies for studying embryonic development

Xiaohu Ouyang and James K. Chen*
Department of Chemical and Systems Biology, Stanford University School of Medicine 269
Campus Drive, CCSR 3155, Stanford, CA 94305, USA

Abstract
Developmental biology has evolved from a descriptive science to one based on genetic principles
and molecular mechanisms. While molecular biology and genetic technologies have been the
primary drivers of this transformation, synthetic strategies have been increasingly utilized to
interrogate the mechanisms of embryonic patterning with spatial and temporal precision. In this
review, we survey how chemical tools and engineered proteins have been used to perturb
developmental processes at the DNA, RNA, protein, and cellular levels. We discuss the design
principles, experimental capabilities, and limitations of each method, as well as future challenges
NIH-PA Author Manuscript

for the chemical and developmental biology communities.

INTRODUCTION
The transformation of a single, totipotent cell into a multicellular organism requires a
complex ensemble of molecular programs that are executed with spatiotemporal fidelity.
Understanding how these chemical mechanisms give rise to form and function is a major
objective of modern embryology, which has evolved from a descriptive science based on
tissue manipulations and morphological observations to one that integrates genomic
technologies and quantitative, molecular models. For example, vertebrate limb development
along the proximal-distal axis requires the “apical ectodermal ridge,” epithelial tissue
overlying the limb bud tip, and it is now known that fibroblast growth factors (FGFs)
expressed by these cells are key mediators of this growth (Fallon et al., 1994; Niswander et
al., 1993). Digit identity is similarly dictated by a posterior “zone of polarizing activity” that
is now associated with limb bud cells that secrete the morphogen Sonic Hedgehog (Shh)
(Niswander et al., 1994), and dorsal-ventral patterning of the limb is regulated by the dorsal
ectoderm, which produces another morphogen called Wnt7a (Parr and McMahon, 1995).
NIH-PA Author Manuscript

Genetic analyses and computational models have further established that these signaling
pathways do not act independently; rather, they interact through feedback mechanisms to
coordinate vertebrate limb patterning in space and time (Mackem and Lewandoski, 2009;
Zeller et al., 2009).

The integration of molecular principles into embryological theory can be primarily attributed
to advances in molecular biology and genomic technologies. Mutagenesis screens of model
organisms such as worms (Caenorhabditis elegans), fruit flies (Drosophila melanogaster),
zebrafish (Danio rerio), and mice (Mus musculus) have identified thousands of genetic loci
that are required for embryonic patterning, many of which have now been mapped by
positional cloning. Methods for targeted gene disruption in whole organisms, such as

Correspondence: James K. Chen, Telephone: (650) 725-3582, Fax: (650) 723-2253, [email protected].
Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our
customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of
the resulting proof before it is published in its final citable form. Please note that during the production process errors may be
discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
 Ouyang and Chen Page 2

knockouts generated by homologous recombination or non-homologous end-joining, have

allowed biologists to study embryonic patterning genes in a more deliberate manner, and
collectively these studies have revealed several evolutionarily conserved genes and their
NIH-PA Author Manuscript

downstream effectors—FGFs, Hedgehogs (Hhs), Wnts, bone morphogenetic proteins

(BMPs), and Notch receptors, to name a few—that are used iteratively and combinatorially
to control cell behavior and fate during embryogenesis. The completion of multiple genome-
sequencing efforts has further revolutionized the study of organismal development by
providing a comprehensive list of patterning molecules and their cis-regulatory elements,
and whole-genome approaches are now commonly applied in developmental biology
research. These latter technologies include the use of DNA microarrays or massively parallel
sequencing to identify genes that are expressed in specific embryonic contexts, as well as the
coupling of these methods with chromatin immunoprecipitation to uncover trans-regulatory
mechanisms. High-throughput strategies are also being used to conduct genome-wide
phenotypic screens in cell-based models and even whole organisms, typically through RNA
interference (RNAi) methods.

While genetic methodologies have dominated developmental biology for the past three
decades, it has become increasingly clear that further advances in the field will require fresh
ideas and new approaches. Like many biological disciplines, embryology is becoming a
quantitative science that strives to explain complex phenotypes in terms of molecular
components, thermodynamic equilibria, and kinetic rates (Lewis, 2008). In addition to the
NIH-PA Author Manuscript

vertebrate limb development studies described above, Drosophila body segmentation has
been predictively modeled according to the anterior-posterior distribution of specific
transcriptional activators and repressors and their cooperative affinities for binding sites
within segmentation gene enhancers (Segal et al., 2008). Dorsal-ventral patterning of the
frog embryo (Xenopus laevis and Xenopus tropicalis) has also been quantitatively described
as opposing concentration gradients of BMPs and their extracellular antagonists that are
scaled relative to embryo size through a molecular shuttling mechanism (Ben-Zvi et al.,
2008). Establishing, testing, and refining new models will require an ability to perturb
developmental signaling mechanisms with a spatial and temporal precision that is difficult to
achieve through genetic manipulations alone. Conventional knockout technologies have
limited temporal control, RNAi usually requires hours to sufficiently deplete targeted
transcripts, and post-translational processes are inherently difficult to target through genetic
methods. Approaches that utilize chemical concepts and synthetic elements, however, are
less constrained by Nature’s molecular architecture. As a result, the developmental
biologist’s toolbox now includes a growing number of chemistry-based technologies.

In this review, we provide an overview of how synthetic strategies have been used to study
embryonic development in a variety of model organisms. We categorize these technologies
NIH-PA Author Manuscript

according to their mechanisms of action, including targeted perturbations at the

transcriptional, RNA, protein, and cellular levels. The use of small-molecule modulators in
developmental biology has been reviewed elsewhere and will not be revisited here
(Firestone and Chen, 2010; Yeh and Crews, 2003). Rather, we focus on rationally designed
technologies, examine their underlying chemical and biological principles, and consider
their advantages and limitations. We also discuss emerging chemical approaches that have
not yet been applied in model organisms but could prove to be valuable tools for in vivo
studies. We hope that this review will not only provide its readers with a synopsis of this
nascent field but also inspire further exploration of embryonic development through the lens
of chemical biology.

Chem Biol. Author manuscript; available in PMC 2011 June 25.

 Ouyang and Chen Page 3

CHEMICAL CONTROL OF GENE TRANSCRIPTION

Dynamic, stereotypic gene expression is a primary driver of embryonic patterning, and
NIH-PA Author Manuscript

developmental biologists have therefore focused on technologies that enable in vivo

transcriptional control. Commonly used gene inactivation strategies include knockouts
through homologous recombination (Mansour et al., 1988), TILLING (Targeting Induced
Local Lesions in Genomes) (McCallum et al., 2000), and RNAi-dependent gene silencing
(Fire et al., 1998). The transcription of exogenous genes throughout the organism or in a
cell-specific manner can also be readily achieved through the transient introduction or
genomic integration of oligonucleotide constructs. While these approaches have yielded key
insights into the genetic programs that give rise to tissue form, they are typically constitutive
or dependent on endogenous trans-regulatory mechanisms. Genetic recombination
technologies such as the Cre/loxP and FLP/FRT systems can provide greater conditionality
in some cases (Dymecki, 1996; Gu et al., 1994), but even these methods are constrained by
the promoters used to activate Cre or FLP recombinase expression. Small molecules,
however, can alter embryonic gene transcription with precise spatial, temporal, and/or dose
control. In particular, ligand-inducible transcription factors and recombinases have been
used to achieve transcriptional regulation in vivo, combining the rapidity and tunability of
chemical modulation with the specificity of genetic targeting.

Small molecule-dependent transcription factors

NIH-PA Author Manuscript

Like several chemical approaches, the development of synthetic transcriptional activators

that are ligand-actuated has been largely inspired by Nature’s regulatory mechanisms.
Signaling proteins that mediate hormonal responses were first co-opted as tools for
transcriptional control, as it was found that the ligand-binding domains of the glucorticoid,
estrogen, and ecdysone receptors (GR, ER, and EcR) can be fused to heterologous proteins
to modulate their function (Figure 1a) (Christopherson et al., 1992; Eilers et al., 1989; Green
and Chambon, 1987). As with endogenous hormone receptors, these chimeric transactivators
are complexed with cytosolic proteins such as heat shock protein 90 (Hsp90), and ligand
binding induces a protein conformational change that disrupts this cytosolic complex and
allows the factor to translocate into the nucleus. Coupling these ligand-binding domains to
DNA-binding and transactivating polypeptides therefore enables the small molecule-
dependent transcription of genes driven by the corresponding response elements.

Since their introduction during the late 1980s, chimeric hormone receptors have been widely
used to regulate gene expression in cultured cells and embryos. For example, Kolm and Sive
fused the myogenic transcription factor MyoD to the ligand-binding domains of either GR or
ER, and frog embryos expressing these chimeric transactivators exhibited ectopic muscle
cells upon treatment with dexamethasone or estradiol, respectively (Kolm and Sive, 1995).
NIH-PA Author Manuscript

The Zivkovic laboratory generalized this approach by coupling the GR ligand-binding

domain to the DNA and activation domains of the yeast transcription factor Gal4, enabling
the dexamethasone-dependent expression of any UAS-driven transgene (de Graaf et al.,
1998). Using the Gal4-GR transactivator, they chemically induced the expression of Xwnt1
and XactivinbB in zebrafish embryos, resulting in morphological phenotypes associated with
the ectopic action of these morphogens. Due to potential crosstalk between these
transactivator systems and endogenous GR or ER signaling, our laboratory subsequently
developed a ligand-gated transcription factor containing the EcR ligand-binding domain,
exploiting the orthogonality of this insect-specific hormone receptor to vertebrate signaling
mechanisms (Esengil et al., 2007). By fusing this polypeptide to the Gal4 DNA-binding
region and a minimal activation domain from herpes simplex virus protein VP16 and
expressing this transactivator under the control of tissue-specific promoters, we were able to
achieve heart and skeletal muscle-specific green fluorescent protein (GFP) expression in
zebrafish embryos upon exposure to the synthetic EcR ligand tebufenozide.

Chem Biol. Author manuscript; available in PMC 2011 June 25.

 Ouyang and Chen Page 4

The other commonly used technology for chemically inducible gene expression takes
advantage of the tetracycline resistance mechanism in Escherichia coli (Thaker et al.).
Bacterial resistance to this antibiotic involves the expression of a transmembrane efflux
NIH-PA Author Manuscript

pump (TetA), which is regulated by a transcriptional repressor (TetR). In the absence of

tetracycline (or its synthetic derivative doxycycline), TetR binds to operator sequences
(tetO) in the tetA regulatory domain; the binding of tetracycline to TetR, however, promotes
its dissociation from tetO, enabling tetA transcription and antibiotic clearance from the
bacteria. Based on this system, Gossen and Bujard established two ligand-inducible gene
transcription strategies that can be applied in mammals (Figure 1b). In the “Tet-Off” system,
the DNA- and ligand-binding domains of TetR are fused with the VP16 activation domain to
form a constitutive transcriptional activator (tTA) that is inhibited by tetracycline (Gossen
and Bujard, 1992). The “Tet-On” system utilizes a “reverse” form of tTA (rtTA) that
actually requires tetracycline for tetO binding (Gossen et al., 1995). Using these
complementary methods, transgene expression can be inhibited or activated in a ligand-
dependent manner, and it has been adapted for use in fruitflies (Bieschke et al., 1998),
zebrafish (Huang et al., 2005), frogs (Ridgway et al., 2000), and mice (Furth et al., 1994;
Kistner et al., 1996) with varying degrees of success. In one particularly elegant application
of this technology, the Tilghman laboratory studied the role of endothelial receptor B
(EDNRB) in murine embryogenesis by integrating either tTA or rtTA into one Ednrb locus
and tetO responsive elements into the other allele (Shin et al., 1999). Administration of the
tetracycline derivative doxycycline to EdnrbtTA/EdnrbtetO or EdnrbrtTA/EdnrbtetO embryos
NIH-PA Author Manuscript

therefore suppressed or induced Ednrb transcription, respectively, while maintaining the

tissue specificity of the endogenous Endrb gene. By varying the timing of doxycycline
treatment, Tilghman and her co-workers were able to determine the developmental stages
during which EDNRB signaling is required for the differentiation of neural crest cells into
melanoblasts and enteric neurons.

Collectively, these examples demonstrate the temporal control of embryonic transcription

that can be achieved through small-molecule ligands and synthetic receptors. They also
illustrate the reliance of these systems on endogenous cis-regulatory elements for spatial
control. While recapitulating native gene expression patterns is adequate for certain
applications, promoter elements that will restrict transcription to targeted cell populations
are not always available, and in some cases it would be advantageous to target tissues
according to morphological cues alone. Synthetic chemistry has enabled developmental
biologists to circumvent these limitations. In particular, the conjugation of caging groups to
hormone or tetracycline receptor ligands provides an extra level of conditionality that is
orthogonal to the timing of ligand addition and the spatiotemporal dynamics of
transactivator expression. The precision of optical techniques makes photoactivatable
ligands especially versatile reagents, and a number of light-activatable hormone receptor
NIH-PA Author Manuscript

ligands have been reported (Link et al., 2004; Link et al., 2005). Although the control of in
vivo gene transcription with these synthetic compounds has not yet been described, caged
doxycycline has been successfully used to induce embryonic gene expression in a tissue-
specific manner.

As a proof of principle, Cambridge and co-workers generated transgenic mice that

ubiquitously express an rtTA-based transactivator and carry a tetO-dependent GFP reporter
(Cambridge et al., 2009). Embryos surgically removed from these mice and cultured with
the 1-(4,5-dimethoxy-2-nitrophenyl)ethyl ether of doxycycline (DMNPE-doxycycline)
exhibited light-dependent GFP expression that could be induced throughout the organism or
localized to a targeted area such as the dorsal root ganglia (Figure 1c). Transgenic Xenopus
tadpoles that expressed rtTA and carried tetO-GFP reporter constructs responded to
DMPNE-caged doxycycline in a qualitatively similar manner, suggesting the generality of
this approach. However, the lower levels of GFP fluorescence observed in irradiated,

Chem Biol. Author manuscript; available in PMC 2011 June 25.

 Ouyang and Chen Page 5

DMPNE-doxycycline-treated tadpoles in comparison to those treated with unmodified

doxycycline indicate that organismal penetrance of the caged ligand or its diffusion after
photoactivation may be limiting factors in some experimental contexts.
NIH-PA Author Manuscript

Small molecule-dependent recombinases

Although hormone receptor ligand-binding domains are used endogenously to gate
transcriptional activity, in principle they could be used to control the function of any protein
that acts within the nucleus. Realizing this potential, the Chambon laboratory fused the
bacteriophage P1 recombinase Cre to a mutated ER ligand-binding domain that responds to
the synthetic inducer 4-hydroxytamoxifen but not endogenous estrogens (Cre-ERT; G525R
human ER), thereby creating a small molecule-dependent genetic recombination system
(Feil et al., 1996). Cells expressing the Cre-ERT chimera will undergo 4-hydroxytamoxifen-
dependent excision or inversion of genomic DNA flanked by loxP sites, depending on
whether these 34-base pair regions have the same or opposite orientations, respectively
(Figure 1d). McMahon and co-workers provided the first demonstration that the Cre-ERT
system can be used to effect genomic recombination in mouse embryos in utero, generating
transgenic mice with Wnt1 enhancer-dependent Cre-ERT expression and a β-galactosidase
reporter whose transcription requires the excision of loxP site-flanked cassette (Danielian et
al., 1998). Injection of 4-hydroxytamoxifen into pregnant transgenic mice induced Cre-
mediated recombination and β-galactosidase expression in the embryonic central nervous
system, which coincides with the Wnt1 expression pattern.
NIH-PA Author Manuscript

The utility of this system for studying vertebrate development has been further established
by the Lamonerie laboratory (Fossat et al., 2006). Mirroring Tilghman’s tTA/rtTA strategy
for the Ednrb gene, they generated transgenic mice in which one allele of the Otx2 gene was
replaced with cDNA encoding Cre-ERT2, another 4-hydroxytamoxifen-specific inducible
recombinase (G400V/M543A/L544A mouse ER) (Feil et al., 1997). The other Otx2 allele
was modified to have unidirectional loxP sites flanking the second exon (“floxed” Otx2).
One major advantage of this “self-knockout” approach is that the Cre-ERT2 transgene will
disrupt the remaining Otx2 allele only in cells that would normally express this homeobox
transcription factor at the time of 4-hydroxytamoxifen exposure; cells that would express
Otx2 at later developmental stages are spared. Accordingly, Otx2flox/Cre-ERT2 embryos in
pregnant mice injected with tamoxifen prodrug 7.5 days post conception (E7.5) exhibited
head phenotypes consistent with a loss of Otx2 function (Figure 1e). By varying the timing
of tamoxifen treatment, Lamonerie and co-workers were able to determine that Otx2 is
required for craniofacial development before E10.5 and midbrain patterning between E10.5
and E16.5. The Cre-ERT2/loxP system has also been validated in zebrafish embryos, and
tamoxifen has been used to induce the excision and inversion of transgenes in this model
organism (Hans et al., 2009).
NIH-PA Author Manuscript

In analogy to DMNPE-caged doxycycline, photoactivatable versions of tamoxifen could

broaden the utility of Cre-ERT2 as a tool for embryological research. Toward this goal,
Jullien and co-workers have synthesized and evaluated a number of caged tamoxifen
derivatives (Sinha et al., 2010). Since ultraviolet light irradiation of 4-hydroxytamoxifen
promotes its photoisomerization and decomposition, they established that 4-
hydroxycyclofen, a photostable structural analog, can interact with the ERT2 ligand-binding
domain with comparable potency (Figure 1d). 4,5-dimethoxy-2-nitrobenzyl (DMNB)-caged
4-hydroxycyclofen can therefore convey light-dependent nuclear translocation of proteins
containing the ERT2 ligand-binding domain, as demonstrated in cultured cells and zebrafish
embryos expressing a GFP-ERT2 chimera containing a nuclear localization sequence.
However, whether DMNB-4-hydroxycyclofen will be an effective reagent for light-
controlled genomic recombination in vivo remains uncertain. The success of this approach
will depend on how the kinetics of 4-hydroxycyclofen efflux from irradiated cells compares

Chem Biol. Author manuscript; available in PMC 2011 June 25.

 Ouyang and Chen Page 6

to the rate of Cre-ERT2-mediated DNA recombination. Consistent with this potential

limitation, Koh and co-workers have observed that even caged 4-hydroxytamoxifen
derivatives that covalently modify the ER ligand-binding domain cannot effectively induce
NIH-PA Author Manuscript

Cre-ERT-mediated recombination in cultured cells after photoactivation (Link et al., 2005).

In principle, these approaches could also be applied to the FLP/FRT recombination system,
which has been widely used in fruit flies and to a more limited extent in frogs, zebrafish, and
mice. The versatility of Cre/loxP-based technologies has also been expanded with the
development of heterologous loxP sequences that efficiently recombine with themselves but
not the wildtype loxP site (Siegel et al., 2001). More sophisticated genomic modifications
can be achieved by combining these orthogonal recombination platforms, including serial
inversion and excision steps to switch from the expression of one gene to another (known as
the FLEx switch method) (Schnutgen et al., 2003). Nested, orthogonal recombination sites
can also be used to allow sequential inversion steps (Boniface et al., 2009), potentially
allowing genes to be switched on and off. Combining these strategies with ligand-regulated
recombinases will allow developmental biologists to manipulate gene function in vivo with
improved dexterity.

CHEMICAL CONTROL OF RNA FUNCTION

Although embryonic gene expression has been traditionally studied at the transcriptional
NIH-PA Author Manuscript

level, tissue patterning also involves the regulation of RNA splicing, translation, and
degradation. For example, splice variants of Delta-C, a Notch receptor ligand, have been
found to have distinct signaling capabilities during embryogenesis, differentially affecting
midline development and somitogenesis in zebrafish (Mara et al., 2008). Dozens of
microRNAs (miRNAs) are expressed in a stereotypic manner to modulate RNA function in
specific cell populations, and individual miRNAs have been implicated in the specification
of muscle, lymphocytes, neurons, germ cells, and other cell types (Shi and Jin, 2009).
Chemical technologies that enable the targeting of specific RNAs therefore constitute
another effective means for studying the molecular mechanisms of embryonic patterning.

One strategy has been to use ligand-inducible transcription to control the expression of
exogenous short hairpin RNAs (shRNAs), which are processed by endogenous miRNA
pathway machinery into short interfering RNAs (siRNAs) and consequently induce the
degradation of targeted transcripts (Paddison et al., 2002). The tamoxifen/Cre-ERT platform
has been used to selectively silence expression of the pluripotency factor Nanog in
primordial germ cells (Yamaguchi et al., 2009) and to knockdown FGF receptor 2 (Fgfr2)
expression in the developing limb (Coumoul et al., 2005). Reversible shRNA-mediated gene
silencing has been achieved in mice through the doxycycline/Tet-on system (Seibler et al.,
NIH-PA Author Manuscript

2007), and EcR-based transactivators have similarly employed in cultured cells (Gupta et al.,
2004; Rangasamy et al., 2008). Since these methods are straightforward applications of the
transcriptional technologies described above, we do not discuss them further here. Rather we
highlight in this section approaches that utilize synthetic chemistry to directly modulate
RNA function in embryos. We also discuss the application of ligand-gated riboswitches for
inducible gene expression in vivo.

Caged RNA molecules

The microinjection of exogenous mRNAs and short interfering RNAs (siRNAs) into
embryos is a common technique for studying gene function, especially at early
developmental stages. In particular, mRNAs are frequently injected into early-stage ascidian
(Ciona intestinalis), sea urchin (Lytechinus variegatus and other species), zebrafish, and
frog embryos to assess gain-of-function phenotypes, and siRNAs are typically introduced
into worms and fruitflies to induce loss-of-function phenotypes. Although these methods are

Chem Biol. Author manuscript; available in PMC 2011 June 25.

 Ouyang and Chen Page 7

simple and effective, their lack of conditionality limits their utility for studying genes that
have pleiotropic functions during embryogenesis.
NIH-PA Author Manuscript

Since RNAs are not membrane-permeable, introducing mRNAs and siRNAs into embryos at
later developmental time points presents a major technical hurdle. Developmental and
chemical biologists have therefore pursued the caging of these molecules with light-sensitive
groups, as in principle these latent RNAs could be microinjected at the one-cell stage and
subsequently photoactivated in targeted cell populations. Blocking RNA function through
chemical modifications, however, is associated with its own technical issues. For example,
exogenous mRNAs are typically generated by in vitro transcription, necessitating the
addition of caging groups to the full-length transcript rather in a site-specific manner.

The Okomoto group attempted to tackle this challenge by reacting in vitro transcribed
mRNAs with 6-bromo-4-diazomethyl-7-hydroxycoumarin (diazo-BHC), thereby modifying
the phosphodiester backbone with photocleavable groups (Figure 2a) (Ando et al., 2001). As
a proof of principle, mRNAs encoding GFP and β-galactosidase reporters were caged with
diazo-BHC and then injected into one-cell-stage zebrafish embryos. Subsequent exposure of
the embryos to 365-nm light induced reporter expression as expected, yet the resulting levels
of GFP fluorescence or β-galactosidase activity were approximately one-eighth that
achieved with the microinjection of equivalent doses of unmodified mRNAs. In addition, the
fold-change in reporter expression induced by photolysis was only four- to five-fold, a
NIH-PA Author Manuscript

dynamic range that will be insufficient for many experimental applications. This low
efficacy undoubtedly reflects the requirement of multiple caging groups to sufficiently
abrogate mRNA translation (approximately 30 modified phosphodiesters per kilobase of
RNA) and the low efficiency by which they can all be removed. Another limiting factor is
the chemical instability of the caged mRNAs, as each BHC phosphotriester is susceptible to
intramolecular attack by the ribose 2′-OH and subsequent hydrolysis. Similar shortcomings
were observed by the Friedman group when they sought to cage siRNAs with 1-(1-
diazoethyl)-4,5-dimethoxy-2-nitrobenzene (diazo-DMNB) (Shah et al., 2005), indicating
that these issues are inherent to the general approach.

The amenability of siRNAs to chemical synthesis does provide some possible ways forward.
For example, siRNA analogs composed of 2′-deoxy-2′-fluoro nucleic acids (siFNAs) still
promote the degradation of targeted transcripts, but their 2′-fluoro modification renders them
less susceptible to hydrolysis (Figure 2a). siFNAs caged with diazo-DMNB consequently
exhibit greater stability in vivo, and Monroe and co-workers have achieved light-inducible
silencing of GFP expression in zebrafish embryos using these reagents (Blidner et al., 2008).
The Friedman laboratory has recently reported an alternative approach, in which the
terminal phosphates of blunt-ended siRNA precursors were selectively modified by diazo-
NIH-PA Author Manuscript

DMNB to yield stable caged duplexes (Figure 2a) (Shah et al., 2009). Photoactivation of
these oligonucleotides in cultured cells led to a six-fold decrease in gene expression, a
dynamic range three times greater than that observed for siRNAs with caged phosphotriester
linkages. Since siRNA- and siFNA-dependent toxicity has reported in zebrafish embryos,
the promise of these new synthetic oligonucleotides as functional genomic tools may be best
realized in worms, fruit flies, and other organisms that are more tractable to RNAi.

Non-natural, RNA-targeting oligonucleotides

Although RNAi is an effective method for loss-of-function studies in worms and fruit flies,
it is less commonly used in other model organisms such as zebrafish and frogs. In addition
to their possible cytotoxic effects, double-stranded RNAs have limited perdurance in vivo,
effectively restricting their application to early developmental genes. Synthetic chemistry
has yielded non-natural oligonucleotides that could potentially overcome some of these
limitations, including 2′-OMe RNAs, phosphorothioate (PS) DNAs, methoxyethylamine

Chem Biol. Author manuscript; available in PMC 2011 June 25.

 Ouyang and Chen Page 8

(MEA) and N,N-diethylethylenediamine (DEED) phosphoramidates, locked nucleic acids

(LNAs), peptide nucleic acids (PNAs), morpholinos (MOs), and chimeric oligomers
composed of more than one nucleoside class (Figure 2a). Each oligonucleotide utilizes
NIH-PA Author Manuscript

standard DNA bases while substituting the ribose backbone and/or phosphodiester linkages
with other chemical substituents, resulting in enhanced in vivo stability. Depending on the
specific structural modifications, hybridization of the oligomers to their complementary
RNA targets either promotes RNase H-dependent transcript degradation or acts as a steric
block at the site of hybridization.

Although there have been a few reports of gene silencing in frog embryos using PS DNAs
(Woolf et al., 1990), MEA or DEED phosphoramidate/DNA hybrids (Dagle et al., 1990;
Dagle et al., 2000; Veenstra et al., 2000), and LNA/PS DNA chimeras (Lennox et al., 2006),
these RNase H-active antisense agents are not widely used by the developmental biology
community. Similarly, PS DNAs and 2′-OMe RNAs are not generally used to study
zebrafish embryogenesis, as they are both cytotoxic at doses sufficient to alter RNA function
(Tomasini et al., 2009). RNAse H-inactive antisense oligonucleotides such as MOs have
proven to be more effective reverse-genetic tools in vivo, and they have been used to
interrogate gene function in the embryos of ascidians (Satou et al., 2001), sea urchins
(Coffman et al., 2004), zebrafish (Nasevicius and Ekker, 2000), frogs (Heasman et al.,
2000), chickens (Gallus gallus) (Kos et al., 2001), and mice (Coonrod et al., 2001). These
oligomers are composed of a nuclease-resistant morpholine-phosphorodiamidate backbone,
NIH-PA Author Manuscript

and typically 25-base oligonucleotides are injected into embryos at the one-cell stage, after
which they can remain functional for days (Nasevicius and Ekker, 2000; Summerton and
Weller, 1997). MOs that hybridize to the start site or its flanking 5′ untranslated region
(UTR) can inhibit RNA translation, allowing them to block both maternal and zygotic RNA
function (Summerton, 1999); those that target intron-exon junctions can yield misspliced
zygotic transcripts that that include introns or skip exons, frequently introducing a reading-
frame shift and a premature stop codon (Draper et al., 2001). MOs have also been used to
target miRNAs or their precursors to investigate the functions of these non-coding RNAs
(Flynt et al., 2007; Kloosterman et al., 2007). In fact, thousands of MOs have been used to
study zebrafish development alone, targeting genes such as the morphogen wnt8a, the Hh
pathway component suppressor of fused (sufu), and the mesodermal transcription factor no
tail-a (ntla).

PNAs also inhibit RNA function through RNase H-independent steric blockade, but they
have been applied in vivo to a much lesser degree than MOs. This limited use is due in part
to the low water solubility of PNA oligomers, making it difficult to achieve the micromolar
concentrations required to block gene expression in organisms. Farber and co-workers
circumvented this technical hurdle by utilizing negatively charged PNAs (ncPNAs; also
NIH-PA Author Manuscript

known commercially as gripNAs™), which have a backbone composed of trans-4-hydroxy-

L-proline/phosphonate polyamide subunits (Urtishak et al., 2003). ncPNAs used for in vivo
studies are typically shorter in length than MOs (18 bases) since they hybridize to RNA with
stronger affinity. ncPNAs also exhibit greater sensitivity to base-pair mismatches than MOs.
In practice, both RNase H-inactive antisense oligonucleotides can be effective gene-
silencing tools in embryos, and in some cases one reagent is more efficacious than the other
even when targeting the same RNA sequence. For example, the Farber laboratory observed
that an ncPNA targeting the homeobox gene dharma (dha) start site was able to recapitulate
dha mutant phenotypes, whereas a MO against the same region was ineffective. Yet only a
handful of ncPNAs targeting embryonic transcripts have been reported in the scientific
literature, and all of these genes are expressed early in development. It is therefore possible
that the amide bond-containing ncPNAs are more labile in vivo than the
phosphorodiamidate-based MOs.

Chem Biol. Author manuscript; available in PMC 2011 June 25.

 Ouyang and Chen Page 9

MOs and ncPNAs do have certain limitations, however. Although synthetic oligonucleotides
have proven to be valuable tools for studying developmental biology, phenotypes induced
by these reagents must be interpreted with caution. Both reagents can have off-target effects
NIH-PA Author Manuscript

that lead to embryonic defects, including p53-dependent cell death. Appropriate control
experiments therefore must be conducted to confirm the specificity of these antisense
reagents in vivo, including recapitulation of the phenotype with multiple MOs or ncPNAs
against the same gene and the use of base pair-mismatch control oligonucleotides (Eisen and
Smith, 2008). The Farber and Ekker labs have also demonstrated that MO and ncPNA off-
target effects can be minimized by concurrently inhibiting p53 expression using a p53 MO,
thereby unmasking the specific function of the gene of interest (Robu et al., 2007).

In addition, the constitutive activities of conventional MOs and ncPNAs renders them less
effective for studying genes that are expressed in more than one tissue and/or at multiple
developmental stages. Caged antisense oligonucleotides could therefore be more versatile
probes of gene function, and those actuated by light would be particularly useful in optically
transparent embryos such as those produced by ascidians, sea urchins, and zebrafish.
Toward this goal, our laboratory caged MOs by using a DMNB-based photocleavable linker
to tether the 25-base gene-targeting oligonucleotide to a complementary inhibitor composed
of MO nucleosides (Figure 2b) (Shestopalov et al., 2007). Intramolecular Watson-Crick
base-pairing produces a MO hairpin that cannot effectively hybridize to its RNA target, and
linker photolysis with 365-nm light liberates the targeting MO to allow RNA function
NIH-PA Author Manuscript

blockade. Using this approach, we have conditionally regulated ntla expression in zebrafish
embryos with spatial and temporal precision, demonstrating that this T-box family
transcription factor is required for notochord cell fate commitment, migration, and
differentiation. We have also generated caged MOs against several other developmental
patterning genes, including floating head (flh), heart of glass (heg), and ets varient gene 2
(etv2) (Figure 2c) (Ouyang et al., 2009).

A similar ncPNA caging strategy was concurrently reported by Dmochowski and coworkers,
in this case using 18-base ncPNAs, complementary 2′-OMe RNA inhibitors, and a
nitrobenzyl-based linker (Tang et al., 2007). By microinjecting caged ncPNAs targeting dha
and the BMP antagonist chordin (chd) into zebrafish embryos and irradiating them globally
with ultraviolet light, they were able to induce mutant phenotypes. Interpreting these
developmental defects, however, is complicated by the release of 2′-OMe RNA upon linker
photolysis, as these synthetic oligonucleotides have been shown to be toxic to zebrafish
embryos (Tomasini et al., 2009).

More recently, the Mayer laboratory in collaboration with SuperNova Life Sciences
developed an alternative caging strategy that employs MO/caged RNA duplexes called
NIH-PA Author Manuscript

PhotoMorphs™ (Figure 2b) (Tomasini et al., 2009). Like the caged MOs and ncPNAs
described above, these reagents utilize a single light-sensitive group, in this case a
nitrobenzyl-based linker that joins two 12-base RNA oligonucleotides. The MO/caged RNA
duplex resists strand exchange with endogenous transcripts, likely reflecting the fact that
RNA-binding proteins and RNA tertiary structure restrict the accessibility of the latter.
Photolysis of the caged RNA shifts the binding energetics in favor of MO/mRNA
hybridization, and PhotoMorphs™ have been used to silence the expression of Ntla, E-
cadherin, and Rheb in zebrafish embryos. MO activity, however, is not fully restored upon
irradiation, as the caged RNA must be used in five- to ten-fold molar excess to compensate
for its gradual degradation by endogenous nucleases. Unfortunately, other synthetic
oligonucleotides tested by the Mayer laboratory were found to be toxic to zebrafish
embryos, preventing their use as caged inhibitors. The PhotoMorph™ system is therefore
conceptually similar to having multiple caging groups per oligonucleotide; more than one

Chem Biol. Author manuscript; available in PMC 2011 June 25.

 Ouyang and Chen Page 10

caged RNA must be photolyzed to activate each MO molecule, and the dynamic range of
inducible activity is therefore limited.
NIH-PA Author Manuscript

Small molecule-dependent riboswitches

While synthetic oligonucleotides and their caged derivatives have proven to be valuable
tools for the developmental biology community, their versatility is offset by their
irreversible activities. Conventional MO and ncPNAs block RNA function in a constitutive
manner, and caged versions reported to date cannot be turned off after they have been
photoactivated. Since embryogenesis utilizes a limited number of signaling molecules in a
dynamic, iterative manner, technologies that enable reversible RNA control would advance
our understanding of patterning mechanisms. As with the hormone receptor family of
transcriptional modulators, Nature has provided us with examples for how conditional RNA
regulation can be achieved.

For instance, certain bacteria, fungi, and plant transcripts contain cis-regulatory elements
that respond to endogenous metabolites, such as thiamine pyrophosphate, glycine, and
glucosamine-6-phosphate. Upon metabolite-binding, these RNA sequences undergo
conformational changes to adopt structures that either terminate transcription prematurely,
prevent ribosomal docking, or generate self-cleaving ribozymes. Such mechanisms enable
microbes and plants to modulate their expression of biosynthetic enzymes in response to
changes in metabolite concentrations. The Mulligan laboratory pioneered the use of
NIH-PA Author Manuscript

riboswitches to control mammalian gene expression by inserting a number of candidate

ribozyme sequences into various regions of a β-galactoside reporter gene (Yen et al., 2004).
Through this survey and subsequent optimization studies, they determined that a mutant
form of the self-cleaving Sm1 ribozyme from the trematode Shistosoma mansoni can
prevent reporter expression when inserted into the 5′ UTR (Figure 2d). By conducting a
chemical library screen, they also discovered that the nucleoside analog toyocamycin
inhibits Sm1 self-cleavage and rescues gene expression (Figure 2d) (Yen et al., 2006; Yen et
al., 2004). Toyocamycin-dependent RNA translation could be achieved in both cultured
cells and in mice infected with an adeno-associated virus encoding a ribozyme-firefly
luciferase transgene, and in principle this technology could be used to control embryonic
gene expression. Subsequent studies by the Mulligan group revealed that inhibition of RNA
self-cleavage by toyocamycin actually requires its covalent incorporation into the transcript
itself rather than simply RNA/toyocamycin binding (Yen et al., 2006). The kinetics by
which transgene expression is lost upon toyocamycin removal will consequently depend
upon the degradation rate of transcripts containing this nucleoside derivative. In principle,
greater temporal control could therefore be achieved with small molecules that recapitulate
the non-covalent modulation of riboswitch function by endogenous metabolites.
NIH-PA Author Manuscript

Deiters and co-workers have extended the capabilities of this approach by synthesizing a
nitrobenzyl dioxolane derivative of toyocamycin that liberates the nucleoside upon 365-nm
light exposure and intracellular hydrolysis (Figure 2d) (Young et al., 2009). Thus, light-
activated gene expression could be achieved by combining these methodologies. It is also
possible that orthogonal small molecule/riboswitch systems could be used in parallel to
independently control the expression of multiple genes in a developing organism.

CHEMICAL CONTROL OF PROTEIN FUNCTION

The majority of technologies described in the preceding sections combine the specificity of
genetically encoded components with the rapid kinetics of small-molecule binding or
photochemical transformations. In most cases, however, the temporal precision by which
these systems can perturb embryonic gene function is limited by endogenous processes. For
techniques that activate gene expression, possible rate-determining steps include gene

Chem Biol. Author manuscript; available in PMC 2011 June 25.

 Ouyang and Chen Page 11

transcription, RNA splicing or translation, or post-translational modifications required for

protein function. Methods for inhibiting oligonucleotide function are contingent on RNA
and/or protein degradation rates. Technologies that act directly on signaling proteins could
NIH-PA Author Manuscript

modulate patterning mechanisms with greater speed, in principle enabling the study of
developmental processes that occur on the minute timescale. Indeed, the widespread
application of small molecules that selectively target Hh, Wnt, BMP, or Notch pathway
components exemplifies the power of this approach. Since implementing small-molecule
modulators in a genome-wide manner has certain technical challenges—the serendipitous
nature of compound discovery and the difficulty of establishing target specificity—we focus
here on strategies for protein regulation that are potentially generalizable. Current
technologies can be grouped into two categories: (1) methods control protein splicing or
stability; and (2) those that are designed to regulate individual members of specific protein
families.

Small molecule-dependent protein splicing

Rather than regulating protein function at the translational level, an alternative strategy
would be to induce the assembly of active proteins from latent polypeptides. One
mechanism that Nature has evolved for post-translational protein engineering is the use of
intein domains that catalyze their self-excision from polypeptide precursors and the
simultaneous ligation of flanking sequences (exteins) (Paulus, 2000). Most endogenous
inteins mediate cis-splicing within single-chain polypeptides, but at least one intein
NIH-PA Author Manuscript

subfamily that can couple two protein domains in trans has been discovered (Wu et al.,
1998). In the latter reaction, the intein domain is split into two inactive fragments, each of
which is linked to a portion of another protein. Reconstitution of the intein domain leads to
its activation and trans-splicing of the tethered polypeptide chains.

These remarkable reactions have prompted chemical biologists to create synthetic congeners
that enable small-molecule control of protein maturation. For example, the Muir laboratory
developed an artificial split-intein system by separating the cis-splicing VMA intein of
Saccharomyces cerevisiae into two halves and coupling them to individual, complementary
fragments of a heterologous protein (Figure 3a) (Mootz and Muir, 2002). One split-intein
fusion protein was then tagged with FK506-binding protein 12 (FKBP12) and the other with
the FKBP12/rapamycin-binding domain of mammalian TOR (FRB). Co-expression of the
split-intein system in cells and treatment with rapamycin leads to formation of the FKBP12/
rapamycin/FRB complex, reconstitution of the VMA intein, and trans-splicing of the
heterologous polypeptides. Using this technology, Muir and co-workers have achieved
rapamycin-induced splicing of inactive firefly luciferase fragments in fruit flies, which can
be tuned by varying the rapamycin dose or co-administering the FKBP12 ligand ascomycin
to competitively inhibit FKBP12/rapamycin/FRB complex formation (Schwartz et al.,
NIH-PA Author Manuscript

2007). In principle, this approach could be broadly applied to study embryonic patterning
mechanisms through the use of rapamycin analog/FRB mutant pairs that are orthogonal to
endogenous signaling proteins (see below).

Liu and co-workers have reported a complementary method for conditional protein splicing
that involves insertion of the ER ligand-binding domain into the RecA intein from
Mycobacterium tuberculosis (Buskirk et al., 2004). By subjecting this synthetic construct to
several cycles of mutagenesis and selection, they identified chimeric inteins that can mediate
cis-splicing of flanking polypeptides in a 4-hydroxytamoxifen-dependent manner. The
technology was subsequently used to regulate the Gli family of Hh pathway transcription
factors in cultured cells (Yuen et al., 2006). Although the 4-hydroxytamoxifen-liganded
intein fusions could generate only about one-third of the Gli transcriptional response
observed with wildtype Gli1, this level of efficacy was sufficient to differentiate pluripotent

Chem Biol. Author manuscript; available in PMC 2011 June 25.

 Ouyang and Chen Page 12

mesenchymal cells into osteoblasts. Whether the intein-ER system in its current form will be
applicable in vivo awaits further study.
NIH-PA Author Manuscript

Small molecule-dependent protein degradation

Rapamycin-mediated hetero-dimerization of FKBP12- and FRB-tagged proteins has also
been exploited to attain conditional protein degradation. For instance, the Muir laboratory
has developed a methodology that utilizes split ubiquitin for the rescue of function (SURF),
in which the protein of interest is fused to a degron via the FRB domain and half of the
ubiquitin polypeptide (Pratt et al., 2007). The targeted protein is therefore constitutively
degraded by the proteosome, but when co-expressed in cells with another chimera composed
of FKBP12 and the other half of ubiquitin, it is stabilized in a rapamycin-dependent manner.
This is because the reconstituted ubiquitin that forms upon rapamycin binding is cleaved
from the protein of interest by dedicated proteases, taking the degron along with it. The
FKBP12/rapamycin/FRB system can be co-opted to achieve small molecule-inducible
protein degradation as well, as demonstrated by Church and coworkers (Janse et al., 2004).
In this case, the S. cerevisiae proteasome subunits Rpn10 or Pre10 were fused to the yeast
ortholog of FKBP12, and the protein of interest was tagged with the yeast equivalent of the
FRB domain. Rapamycin treatment of yeast expressing these chimeras then leads to the
recruitment of the targeted protein to the proteosome, which is sufficient to promote its
degradation. Finally, the Kanemaki laboratory has successfully transferred the auxin-based
degron system from plants into multiple animal cell lines, allowing proteins tagged with
NIH-PA Author Manuscript

auxin-induced degrons to be rapidly proteolyzed in a small molecule-dependent manner

(Nishimura et al., 2009).

Although these systems can regulate protein degradation in cultured cells, their efficacy in
vivo has not yet been established. To date, only two technologies for small molecule-
dependent protein stability have been validated in whole organisms, and both utilize similar
biochemical mechanisms. The first approach emerged serendipitously from collaborative
efforts by the Crabtree and Wandless laboratories to develop rapamycin analog/FRB mutant
pairs that would avoid crosstalk with endogenous TOR signaling (Stankunas et al., 2003).
During the course of these studies, they observed that proteins fused to one FRB mutant
containing three amino acid substitutions (FRB*; K2095P/T2098L/W2101F human TOR)
were rapidly degraded by the proteosome, presumably because these mutations destabilize
the FRB fold and consequently the entire chimeric protein. The addition of rapamycin or its
C20-methyallyl derivative (MaRap) stabilized these fusion proteins by recruiting
endogenous FKBP12 (Figure 3b).

The Crabtree and Longaker laboratories then generated mouse embryos homozygous for a
glycogen synthase kinase 3β (GSK3β)-FRB* transgene knocked into the endogenous
NIH-PA Author Manuscript

GSK3β locus (Liu et al., 2007). In the absence of rapamycin, these transgenic embryos
phenocopied conventional GSK3β knockouts, exhibiting cleft palates and skeletal defects.
Since rapamycin is teratogenic when administered to pregnant mice bearing wildtype
embryos prior to the E10.5 stage but does not cause overt developmental abnormalities at
later time points, they explored the phenotypic consequences of 48-hour drug treatments
starting at E13.5. Through this temporal analysis, it was determined that GSK3β is required
between E13.5 and E15 for palatogenesis and between E15.5 and E17 for skeletal
development (Figure 3c). Unfortunately effective doses of the non-teratogenic compound
MaRap could not be achieved in utero, so using this methodology to investigate protein
functions at earlier developmental stages will require new rapamycin analogs/FRB mutant
pairs.

The Wandless laboratory has further simplified this approach by creating a new small
molecule-sensitive destabilization domain that does not require formation of a ternary

Chem Biol. Author manuscript; available in PMC 2011 June 25.

 Ouyang and Chen Page 13

complex (Banaszynski et al., 2006). In this “single-ligand, single-domain” system, a mutant,

unstable form of human FKBP12 (F36V) is fused to proteins of interest, promoting their
degradation in a FKBP12 ligand-dependent manner (Figure 3d). The availability of non-
NIH-PA Author Manuscript

teratogenic FKBP12 ligands such as Shield-1 and the lack of endogenous binding partners
help ensure that this method can be applied to whole organisms with uniform efficacy
between cell types. Indeed, preliminary studies indicate that the Shield-1/FKBP12 mutant
technology can be used to conditionally and reversibly control protein levels in zebrafish
embryos without collateral developmental defects (T. J. Wandless, personal
communication).

Small molecule-based targeting of kinases

One drawback of chemically modulated protein homeostasis is that functional perturbation
of the targeted gene is not coincident with ligand binding; rather, synthesis or degradation of
the mature protein is the rate-determining step. Direct activation or inhibition of protein
function by small molecules can occur on faster timescales, but only a fraction of gene
products have known chemical regulators. Nevertheless, certain gene families can now be
systematically targeted by chemical inhibitors through structure-based protein engineering
and ligand design. This strategy is exemplified by the Shokat laboratory’s development of
kinase mutant-targeted inhibitors that achieve genome-wide specificity. By changing a large
conserved residue in the ATP binding site (typically isoleucine, threonine, or phenylalanine)
to either glycine or alanine, a “hole” can be introduced into the kinase without significantly
NIH-PA Author Manuscript

altering its catalytic activity. Shokat and co-workers then designed kinase inhibitors with a
complementary “bump” by adding naphthyl or naphthylmethyl groups to the Src family
kinase inhibitor PP1 (Bishop et al., 1998). Kinase mutants with these “holes” can be
inhibited by these “bump”-containing PP1 analogs while wildtype kinase activities are left
intact (Figure 3e). Using this approach, individual tyrosine or serine/threonine kinases can
be specifically and reversibly inhibited in whole organisms, even those that have closely
related homologs. For example, Ginty and co-workers studied individual members of the
Trk receptor kinase family, which respond to neurotrophins to regulate nervous system
development (Chen et al., 2005). They knocked in naphthylmethyl-PP1-sensitive forms of
each Trk receptor (TrkAF592A, TrkBF616A, and TrkCF617A) into the corresponding wildtype
allele, and treated homozygous mutant mouse embryos in utero with the PP1 derivative.
Drug treatment of the TrkAF592A, TrkBF616A, and TrkCF617A embryos led to loss of superior
cervical ganglia, nodose ganglia, and parvalbumin-positive dorsal root ganglia neurons,
respectively. The homozygous mutants developed normally, and wildtype embryos were
insensitive to naphthylmethyl-PP1 treatment.

Optogenetic control of ion channels

NIH-PA Author Manuscript

In most cases, small molecules can be used to modulate gene function in physiologically
relevant timescales. However, certain biological processes such as neuronal depolarization
occur within milliseconds, and faster-acting technologies are necessary to study these
systems. Modulating ion channel function during embryogenesis is an area of particular
interest, as it is now widely appreciated that electrical activity influences multiple stages of
neuronal development (Spitzer, 2006). Several synthetic strategies for controlling neuronal
action potentials have been developed and applied in whole organisms, permitting the
activation of specific neurons with millisecond precision. All of these methods involve light-
gated channels that contain either synthetic or endogenous photoisomerizable cofactors.
Although the application of these tools to investigate embryonic patterning has not yet been
reported, the examples below illustrate their basic design principles and their utility for in
vivo studies.

Chem Biol. Author manuscript; available in PMC 2011 June 25.

 Ouyang and Chen Page 14

The use of synthetic photoswitchable compounds to modulate ion channel activity state
dates back nearly four decades, when Erlanger and co-workers found that electrophilic,
azobenzene-containing quaternary amines were able to alkylate the nicotinic acetylcholine
NIH-PA Author Manuscript

receptor (nAChR) and render it light-sensitive (Bartels et al., 1971). The trans isomer of the
azobenzene-containing agonist locked the channel in an open state, and formation of the cis
isomer upon exposure to 330-nm light caused channel inactivation (Figure 3f). Since this
seminal work, several laboratories have applied this general strategy to other ion channels,
including ligand-gated, voltage-gated, and mechanosensitive proteins. For example, the
Trauner and Isacoff laboratories engineered an ionotropic glutamate receptor (iGluR6) to
incorporate a modifiable cysteine proximal to the glutamate binding site (Volgraf et al.,
2006). They then covalently attached a tether agonist composed of maleimide, azobenzene,
and glutamate groups (MAG-1) to the iGluR6 mutant. The resulting synthetic receptor
rendered cells permeable to ion currents in a light-dependent manner; irradiation with 380-
nm light promoted cis isomerization of MAG-1 and current influx, and exposure to 500-nm
light favored the trans MAG-1 isomer and ion channel closure. Photoswitching of the light-
gated iGluR6 receptor (LiGluR6) occurred on the millisecond timescale, and the degree of
channel activation could be modulated by changing the wavelength of light.

Even though the MAG-1/LiGluR6 technology requires channel adduct formation with an
electrophilic reagent, Trauner and Isacoff have successfully used this approach to explore
neuronal function in whole organisms (Wyart et al., 2009). They created a zebrafish line
NIH-PA Author Manuscript

containing a UAS-dependent LiGluR6 transgene and then crossed it with several transgenic
lines expressing Gal4 in different subsets of spinal neurons. The progeny were then treated
with MAG-1 and stimulated with pulses of 390-nm light to depolarize the targeted cells,
resulting in neuron-specific behaviors. For example, light activation of caudal Kolmer-
Agduhr neurons induced tail oscillations that mimicked forward swimming, while bilaterial
illumination of caudal Rohon-Beard neurons induced tail bends characteristic of the touch-
escape response.

Light-gated channels found in Nature have been more widely applied to interrogate neuronal
function, owing to their reliance on endogenous retinal as a cofactor rather than reactive
synthetic ligands (Figure 3f) (Zhang et al., 2007a). Although several photo-responsive
channels have been identified, channelrhodopsin-2 (ChR2) from the green algae
Chlamydomonas reinhardtii and halorhodopsin from the halobacterium Natronomonas
pharaonis (NpHR) are the two most commonly used proteins (Lanyi et al., 1990; Nagel et
al., 2003). ChR2 is cation channel that allows Na+ ions to enter the cell in response to 470-
nm light, and NpHR is a chloride pump that is activated by 580-nm light. Neuronal firing
can therefore be induced and suppressed by ChR2 and NpHR, respectively, permitting the
generation of specific action potential profiles with millisecond resolution. These light-gated
NIH-PA Author Manuscript

channels have been used to modulate neuron function in worms (Nagel et al., 2005; Zhang et
al., 2007b), fruit flies (Schroll et al., 2006), zebrafish (Baier and Scott, 2009; Douglass et al.,
2008), mice (Bi et al., 2006; Zhang et al., 2007b), and even primates (Han et al., 2009),
demonstrating their efficacy across neuronal subtypes and animal species. The Deisseroth
laboratory’s development of photoactivatable rhodopsin-GPCR chimeras that actuate
intracellular signaling pathways promises to extend this approach to other developmental
processes (Airan et al., 2009).

Optogenetic control of small GTPases

Nature has created other photo-responsive systems that could be re-engineered into light-
actuated probes of patterning mechanisms. For example, plants control phototropism and
stomatal opening through light-sensing kinases called phototropins that contain tandem
light-oxygen-voltage (LOV) domains (Christie, 2007). When exposed to blue light, the LOV
domain reacts with its flavin mononucleotide (FMN) cofactor, generating a cysteinyl

Chem Biol. Author manuscript; available in PMC 2011 June 25.

 Ouyang and Chen Page 15

covalent adduct that is stable for seconds and then reverts to the non-covalent haloprotein
(Figure 3f). In the Avena sative Phototropin1 protein, formation of this covalent intermediate
is associated with a conformational change that disrupts interactions between one of its LOV
NIH-PA Author Manuscript

domains and a C-terminal helical extension called Jα, causing the latter to unfold (Harper et
al., 2003). By conjugating the LOV-Jα polypeptide to a constitutively active mutant of Rac1,
a GTPase that regulates actin cytoskeletal dynamics, Hahn and coworkers were able to
produce a photoactivatable Rac1 chimera (PA-Rac1) (Wu et al., 2009). In the absence of
light, the LOV-Jα polypeptide prevented the binding of Rac1 to its downstream effector
PAK, whereas exposure to 458-nm light induced unwinding of the Jα helix, restored Rac1/
PAK interactions, and promoted lamellipodial protrusions and membrane ruffling. PA-Rac1
photoactivation is reversible, repeatable, and can be conducted in whole organisms, as
demonstrated by the Huttenlocher laboratory (Yoo et al., 2010). In collaboration with the
Hahn group, they created transgenic zebrafish that specifically expressed the PA-Rac1
fusion protein in neutrophils, and by activating PA-Rac1 with a spatiotemporally controlled
beam of 458-nm light, they were able to direct neutrophil migration in the transgenic
embryos.

Another LOV domain-containing polypeptide, the FKF1 protein that controls flowering in
Arabidopsis thaliana, has been re-engineered to achieve light-activated protein-protein
interactions in live cells. In this case, FKF1 associates with another flowering regulator
called GIGANTEA (GI) in a light-dependent manner (Sawa et al., 2007), and the Dolmetsch
NIH-PA Author Manuscript

laboratory has demonstrated that fusion proteins containing these polypeptides can photo-
dimerize in a similar manner (Yazawa et al., 2009). For instance, cells co-expressing FKF1-
Rac1 and farnesylated GI exhibit plasma membrane translocation of Rac1 activity upon
exposure to 450-nm light, and focal illumination can induce the formation of lamellipodia.
Dolmetsch and coworkers also achieved light-activated gene transcription by fusing FKF1 to
the VP16 activation domain and GI to the Gal4 DNA binding domain. Unlike the
Phototropin1-derived LOV-Jα system, the light-induced cysteinyl-flavin adduct in FKF1
persists for hours rather than seconds. Short illumination times can therefore induce long-
lasting protein-protein interactions, making the FKF1/GI-based approach preferable for
certain research applications.

CHEMICAL CONTROL OF CELL FUNCTION

As detailed in the sections above, our efforts to understand embryonic patterning at the
molecular level is now matched with synthetic tools that enable perturbations of DNA,
RNA, or protein function with spatiotemporal precision. It should be noted, however, that
the surgical manipulations and ablations that characterized embryology prior to its molecular
revolution are still valuable research tools. Genetic technologies that enable conditional and
NIH-PA Author Manuscript

targeted cell ablation have also been developed, in particular methods that involve the
tissue-specific expression of a diphtheria toxin-derived polypeptide (Breitman et al., 1987;
Palmiter et al., 1987). Although this approach has provided important insights into metazoan
development and physiology, its reliance on genomic regulatory elements limits its
generality. The precise timing and efficacy of cell ablation are subject to the activities of
these promoters, and due to the high toxicity of this diphtheria toxin fragment, even low
levels of promoter activity can lead to unintended cell death and embryonic mispatterning.
These problems can be mitigated to some extent by delivering full-length diphtheria toxin
into transgenic organisms expressing the receptor for this polypeptide in targeted tissues
(Saito et al., 2001); however, the proteolytic susceptibility of diphtheria toxin and its short
biological half-life are other potential drawbacks.

Cell ablation techniques that rely upon exogenous small molecules could provide greater
experimental flexibility, particularly with respect to the timing, tunability, and sustainability

Chem Biol. Author manuscript; available in PMC 2011 June 25.

 Ouyang and Chen Page 16

of these perturbations. One such method takes advantage of nitro group-containing prodrugs,
which are converted into potent cytotoxic agents by their nitroreductase-expressing bacteria
targets (Roldan et al., 2008). The first demonstration of this approach was conducted by
NIH-PA Author Manuscript

Clark and his co-workers, who generated transgenic mice that express E. coli nitroreductase
in luminal cells of the mammary gland (Clark et al., 1997). Treatment of these mice with the
prodrug CB1954 resulted in the rapid apoptosis of these cells, while neighboring
myoepithelial cells were not affected. More recently, nitroreductase-mediated cell ablation
has been executed in zebrafish by the Parsons and Stainier laboratories (Curado et al., 2007;
Pisharath et al., 2007). In these studies, zebrafish lines that selectively express the bacterial
enzyme in pancreatic β-cells, cardiomyocytes, or hepatocytes were generated, and the
transgenic embryos were cultured in medium containing the prodrug metronidazole at
various time points. Spatially and temporally controlled cell ablation was achieved in each
case, and the targeted tissues were able to recover upon washout of the nitroreductase
substrate (Figure 4). While tissue-dependent drug penetrance and ablation rates have been
observed (Curado et al., 2007), the nitroreductase/metronidazole technique appears to be a
generalizable strategy for studying tissue patterning and regeneration.

CONCLUDING REMARKS
The examples highlighted in this review illustrate how synthetic systems created by
chemistry and protein engineering have changed the way we study embryogenesis and other
NIH-PA Author Manuscript

organismal processes. As we try to understand increasingly complex systems, new

approaches will be needed to interrogate patterning mechanisms with greater experimental
finesse and spatiotemporal precision. For the immediate future, the developmental biology
community would benefit from the extension of existing technologies. Just as the “bump-
and-hole” approach has enabled the inhibition of individual kinases with genome-wide
specificity, this method could be applied to other gene families, particularly those that share
a common cofactor and/or structural motif. For instance, allele-specific inhibitors of
methyltransferases or acetyltransferases could provide critical insights into the epigenetic
mechanisms that regulate cell fate. Orthogonal riboswitches could enable simultaneous
control of multiple transcripts, and parallel “single-ligand, single-domain” degradation
systems could achieve the same versatility at the protein level. Both approaches would allow
biologists to determine how developmental signaling molecules work in concert to effect
specific morphological changes.

There are also a number of technical challenges facing the chemical and developmental
biology communities as we seek a more molecular, quantitative understanding of
embryogenesis. The increasing demand for conditional reagents requires that new caging
technologies be developed. Chromophores that are more sensitive to ultraviolet light or
NIH-PA Author Manuscript

compatible with two-photon irradiation would minimize cell damage and facilitate the
targeting of deeper tissues. Caging strategies for small molecules or oligomers that do not
involve light-sensitive groups could also be valuable advances since not all tissues are
readily targeted with focal or patterned light sources. In these cases, enzymatically triggered
reagents could be used in conjunction with transgenic animals, in analogy to the
nitroreductase-based cell ablation technology.

In addition, new methods for delivering and maintaining exogenous reagents in whole
organisms are needed. In the case of caged small molecules, their efficacy in vivo hinges
upon their rates of diffusion and the rates of the biological processes they actuate. Strategies
for limiting the former after uncaging would therefore broaden the functionality of these
compounds. The membrane impermeability of synthetic oligonucleotides poses a different
challenge, as their delivery into non-injectable embryos or at later developmental stages is a
major technical hurdle. Technologies that enable the uniform delivery of these compounds

Chem Biol. Author manuscript; available in PMC 2011 June 25.

 Ouyang and Chen Page 17

into embryos, ideally by simply adding them to the culture medium, would be a significant
breakthrough.
NIH-PA Author Manuscript

Given the evolutionary diversity of metazoans, it is unlikely that a single synthetic strategy
will be the “silver bullet” for deciphering the dynamic mechanisms that control
embryogenesis. For optically transparent embryos that develop ex utero, technologies
involving genetically encoded photoswitchable proteins hold particular promise. The direct
optical control of protein function is unsurpassed for its immediate impact on developmental
signaling, as well as its spatiotemporal precision. The reversibility of optogenetic systems is
also unparalleled by methods that target DNA or RNA function, as the basal state can be
restored seconds after the light source is removed. One can even envision a suite of
engineered, light-activatable domains that survey a broad range of deactivation rates,
allowing one to select protein activity lifetimes that match specific applications.
Photoactivatable domains with different spectral properties could also be engineered to
enable orthogonal control of two or more proteins, akin to the ChR2 and NpHR systems
described above. Yet how far these approaches can be extended to other model organisms
remains to be seen. Recent advances in fiber optic systems have enabled optogenetic
manipulations in live mice (Barretto et al., 2009; Deisseroth et al., 2006), but applying these
technologies in the womb will be experimentally challenging. In these cases, new non-
optical, small molecule-actuated tools will likely have a broader impact on developmental
biology research.
NIH-PA Author Manuscript

Just as molecular biology complemented the dissecting needle in the 1980s, small molecule-
and synthetic protein-based technologies are now emerging as important embryological
tools. Chemical biologists therefore have an opportunity to play leading roles in the post-
genomic era of developmental biology. Trained in chemical principles and empowered by
chemical synthesis, we are well positioned to investigate the thermodynamic and kinetic
processes that underlie embryogenesis and to create new research modalities unfettered by
Nature’s bonds. Through this unique perspective, chemists can help find new answers to the
age-old question of how function begets form.

Acknowledgments
We gratefully acknowledge financial support from the NIH Director’s Pioneer Award (DP1 OD003792), the NIH/
NIGMS (R01 GM072600), the March of Dimes Foundation (1-FY-08-433), and a Bio-X Stanford Interdisciplinary
Graduate Fellowship in Human Health.

References
Airan RD, Thompson KR, Fenno LE, Bernstein H, Deisseroth K. Temporally precise in vivo control of
NIH-PA Author Manuscript

intracellular signalling. Nature. 2009; 458:1025–1029. [PubMed: 19295515]

Ando H, Furuta T, Tsien RY, Okamoto H. Photo-mediated gene activation using caged RNA/DNA in
zebrafish embryos. Nat Genet. 2001; 28:317–325. [PubMed: 11479592]
Baier H, Scott EK. Genetic and optical targeting of neural circuits and behavior--zebrafish in the
spotlight. Curr Opin Neurobiol. 2009; 19:553–560. [PubMed: 19781935]
Banaszynski LA, Chen LC, Maynard-Smith LA, Ooi AG, Wandless TJ. A rapid, reversible, and
tunable method to regulate protein function in living cells using synthetic small molecules. Cell.
2006; 126:995–1004. [PubMed: 16959577]
Barretto RP, Messerschmidt B, Schnitzer MJ. In vivo fluorescence imaging with high-resolution
microlenses. Nat Methods. 2009; 6:511–512. [PubMed: 19525959]
Bartels E, Wassermann NH, Erlanger BF. Photochromic activators of the acetylcholine receptor. Proc
Natl Acad Sci U S A. 1971; 68:1820–1823. [PubMed: 5288770]
Ben-Zvi D, Shilo BZ, Fainsod A, Barkai N. Scaling of the BMP activation gradient in Xenopus
embryos. Nature. 2008; 453:1205–1211. [PubMed: 18580943]

Chem Biol. Author manuscript; available in PMC 2011 June 25.

 Ouyang and Chen Page 18

Bi A, Cui J, Ma YP, Olshevskaya E, Pu M, Dizhoor AM, Pan ZH. Ectopic expression of a microbial-
type rhodopsin restores visual responses in mice with photoreceptor degeneration. Neuron. 2006;
50:23–33. [PubMed: 16600853]
NIH-PA Author Manuscript

Bieschke ET, Wheeler JC, Tower J. Doxycycline-induced transgene expression during Drosophila
development and aging. Mol Gen Genet. 1998; 258:571–579. [PubMed: 9671025]
Bishop AC, Shah K, Liu Y, Witucki L, Kung C, Shokat KM. Design of allele-specific inhibitors to
probe protein kinase signaling. Curr Biol. 1998; 8:257–266. [PubMed: 9501066]
Blidner RA, Svoboda KR, Hammer RP, Monroe WT. Photoinduced RNA interference using DMNPE-
caged 2′-deoxy-2′-fluoro substituted nucleic acids in vitro and in vivo. Mol Biosyst. 2008; 4:431–
440. [PubMed: 18414741]
Boniface EJ, Lu J, Victoroff T, Zhu M, Chen W. FlEx-based transgenic reporter lines for visualization
of Cre and Flp activity in live zebrafish. Genesis. 2009; 47:484–491. [PubMed: 19415631]
Breitman ML, Clapoff S, Rossant J, Tsui LC, Glode LM, Maxwell IH, Bernstein A. Genetic ablation:
targeted expression of a toxin gene causes microphthalmia in transgenic mice. Science. 1987;
238:1563–1565. [PubMed: 3685993]
Buskirk AR, Ong YC, Gartner ZJ, Liu DR. Directed evolution of ligand dependence: small-molecule-
activated protein splicing. Proc Natl Acad Sci U S A. 2004; 101:10505–10510. [PubMed:
15247421]
Cambridge SB, Geissler D, Calegari F, Anastassiadis K, Hasan MT, Stewart AF, Huttner WB, Hagen
V, Bonhoeffer T. Doxycycline-dependent photoactivated gene expression in eukaryotic systems.
Nat Methods. 2009; 6:527–531. [PubMed: 19503080]
NIH-PA Author Manuscript

Chen X, Ye H, Kuruvilla R, Ramanan N, Scangos KW, Zhang C, Johnson NM, England PM, Shokat
KM, Ginty DD. A chemical-genetic approach to studying neurotrophin signaling. Neuron. 2005;
46:13–21. [PubMed: 15820690]
Christie JM. Phototropin blue-light receptors. Annu Rev Plant Biol. 2007; 58:21–45. [PubMed:
17067285]
Christopherson KS, Mark MR, Bajaj V, Godowski PJ. Ecdysteroid-dependent regulation of genes in
mammalian cells by a Drosophila ecdysone receptor and chimeric transactivators. Proc Natl Acad
Sci U S A. 1992; 89:6314–6318. [PubMed: 1631124]
Clark AJ, Iwobi M, Cui W, Crompton M, Harold G, Hobbs S, Kamalati T, Knox R, Neil C, Yull F, et
al. Selective cell ablation in transgenic mice expression E. coli nitroreductase. Gene Ther. 1997;
4:101–110. [PubMed: 9081700]
Coffman JA, Dickey-Sims C, Haug JS, McCarthy JJ, Robertson AJ. Evaluation of developmental
phenotypes produced by morpholino antisense targeting of a sea urchin Runx gene. BMC Biol.
2004; 2:6. [PubMed: 15132741]
Coonrod SA, Bolling LC, Wright PW, Visconti PE, Herr JC. A morpholino phenocopy of the mouse
mos mutation. Genesis. 2001; 30:198–200. [PubMed: 11477708]
Coumoul X, Shukla V, Li C, Wang RH, Deng CX. Conditional knockdown of Fgfr2 in mice using
Cre-LoxP induced RNA interference. Nucleic Acids Res. 2005; 33:e102. [PubMed: 15987787]
NIH-PA Author Manuscript

Curado S, Anderson RM, Jungblut B, Mumm J, Schroeter E, Stainier DY. Conditional targeted cell
ablation in zebrafish: a new tool for regeneration studies. Dev Dyn. 2007; 236:1025–1035.
[PubMed: 17326133]
Dagle JM, Littig JL, Sutherland LB, Weeks DL. Targeted elimination of zygotic messages in Xenopus
laevis embryos by modified oligonucleotides possessing terminal cationic linkages. Nucleic Acids
Res. 2000; 28:2153–2157. [PubMed: 10773085]
Dagle JM, Walder JA, Weeks DL. Targeted degradation of mRNA in Xenopus oocytes and embryos
directed by modified oligonucleotides: studies of An2 and cyclin in embryogenesis. Nucleic Acids
Res. 1990; 18:4751–4757. [PubMed: 1697675]
Danielian PS, Muccino D, Rowitch DH, Michael SK, McMahon AP. Modification of gene activity in
mouse embryos in utero by a tamoxifen-inducible form of Cre recombinase. Curr Biol. 1998;
8:1323–1326. [PubMed: 9843687]
de Graaf M, Zivkovic D, Joore J. Hormone-inducible expression of secreted factors in zebrafish
embryos. Dev Growth Differ. 1998; 40:577–582. [PubMed: 9865967]

Chem Biol. Author manuscript; available in PMC 2011 June 25.

 Ouyang and Chen Page 19

Deisseroth K, Feng G, Majewska AK, Miesenbock G, Ting A, Schnitzer MJ. Next-generation optical
technologies for illuminating genetically targeted brain circuits. J Neurosci. 2006; 26:10380–
10386. [PubMed: 17035522]
NIH-PA Author Manuscript

Douglass AD, Kraves S, Deisseroth K, Schier AF, Engert F. Escape behavior elicited by single,
channelrhodopsin-2-evoked spikes in zebrafish somatosensory neurons. Curr Biol. 2008; 18:1133–
1137. [PubMed: 18682213]
Draper BW, Morcos PA, Kimmel CB. Inhibition of zebrafish fgf8 pre-mRNA splicing with
morpholino oligos: a quantifiable method for gene knockdown. Genesis. 2001; 30:154–156.
[PubMed: 11477696]
Dymecki SM. Flp recombinase promotes site-specific DNA recombination in embryonic stem cells
and transgenic mice. Proc Natl Acad Sci U S A. 1996; 93:6191–6196. [PubMed: 8650242]
Eilers M, Picard D, Yamamoto KR, Bishop JM. Chimaeras of myc oncoprotein and steroid receptors
cause hormone-dependent transformation of cells. Nature. 1989; 340:66–68. [PubMed: 2662015]
Eisen JS, Smith JC. Controlling morpholino experiments: don’t stop making antisense. Development.
2008; 135:1735–1743. [PubMed: 18403413]
Esengil H, Chang V, Mich JK, Chen JK. Small-molecule regulation of zebrafish gene expression. Nat
Chem Biol. 2007; 3:154–155. [PubMed: 17237798]
Fallon JF, Lopez A, Ros MA, Savage MP, Olwin BB, Simandl BK. FGF-2: apical ectodermal ridge
growth signal for chick limb development. Science. 1994; 264:104–107. [PubMed: 7908145]
Feil R, Brocard J, Mascrez B, LeMeur M, Metzger D, Chambon P. Ligand-activated site-specific
recombination in mice. Proc Natl Acad Sci U S A. 1996; 93:10887–10890. [PubMed: 8855277]
NIH-PA Author Manuscript

Feil R, Wagner J, Metzger D, Chambon P. Regulation of Cre recombinase activity by mutated

estrogen receptor ligand-binding domains. Biochem Biophys Res Commun. 1997; 237:752–757.
[PubMed: 9299439]
Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE, Mello CC. Potent and specific genetic
interference by double-stranded RNA in Caenorhabditis elegans. Nature. 1998; 391:806–811.
[PubMed: 9486653]
Firestone AJ, Chen JK. Controlling destiny through chemistry: small-molecule regulators of cell fate.
ACS Chem Biol. 2010; 5:15–34. [PubMed: 20000447]
Flynt AS, Li N, Thatcher EJ, Solnica-Krezel L, Patton JG. Zebrafish miR-214 modulates Hedgehog
signaling to specify muscle cell fate. Nat Genet. 2007; 39:259–263. [PubMed: 17220889]
Fossat N, Chatelain G, Brun G, Lamonerie T. Temporal and spatial delineation of mouse Otx2
functions by conditional self-knockout. EMBO Rep. 2006; 7:824–830. [PubMed: 16845372]
Furth PA, St Onge L, Boger H, Gruss P, Gossen M, Kistner A, Bujard H, Hennighausen L. Temporal
control of gene expression in transgenic mice by a tetracycline-responsive promoter. Proc Natl
Acad Sci U S A. 1994; 91:9302–9306. [PubMed: 7937760]
Gossen M, Bujard H. Tight control of gene expression in mammalian cells by tetracycline-responsive
promoters. Proc Natl Acad Sci U S A. 1992; 89:5547–5551. [PubMed: 1319065]
Gossen M, Freundlieb S, Bender G, Muller G, Hillen W, Bujard H. Transcriptional activation by
NIH-PA Author Manuscript

tetracyclines in mammalian cells. Science. 1995; 268:1766–1769. [PubMed: 7792603]

Green S, Chambon P. Oestradiol induction of a glucocorticoid-responsive gene by a chimaeric
receptor. Nature. 1987; 325:75–78. [PubMed: 3025750]
Gu H, Marth JD, Orban PC, Mossmann H, Rajewsky K. Deletion of a DNA polymerase beta gene
segment in T cells using cell type-specific gene targeting. Science. 1994; 265:103–106. [PubMed:
8016642]
Gupta S, Schoer RA, Egan JE, Hannon GJ, Mittal V. Inducible, reversible, and stable RNA
interference in mammalian cells. Proc Natl Acad Sci U S A. 2004; 101:1927–1932. [PubMed:
14762164]
Han X, Qian X, Bernstein JG, Zhou HH, Franzesi GT, Stern P, Bronson RT, Graybiel AM, Desimone
R, Boyden ES. Millisecond-timescale optical control of neural dynamics in the nonhuman primate
brain. Neuron. 2009; 62:191–198. [PubMed: 19409264]
Hans S, Kaslin J, Freudenreich D, Brand M. Temporally-controlled site-specific recombination in
zebrafish. PLoS One. 2009; 4:e4640. [PubMed: 19247481]

Chem Biol. Author manuscript; available in PMC 2011 June 25.

 Ouyang and Chen Page 20

Harper SM, Neil LC, Gardner KH. Structural basis of a phototropin light switch. Science. 2003;
301:1541–1544. [PubMed: 12970567]
Heasman J, Kofron M, Wylie C. Beta-catenin signaling activity dissected in the early Xenopus
NIH-PA Author Manuscript

embryo: a novel antisense approach. Dev Biol. 2000; 222:124–134. [PubMed: 10885751]
Huang CJ, Jou TS, Ho YL, Lee WH, Jeng YT, Hsieh FJ, Tsai HJ. Conditional expression of a
myocardium-specific transgene in zebrafish transgenic lines. Dev Dyn. 2005; 233:1294–1303.
[PubMed: 15977161]
Janse DM, Crosas B, Finley D, Church GM. Localization to the proteasome is sufficient for
degradation. J Biol Chem. 2004; 279:21415–21420. [PubMed: 15039430]
Kistner A, Gossen M, Zimmermann F, Jerecic J, Ullmer C, Lubbert H, Bujard H. Doxycycline-
mediated quantitative and tissue-specific control of gene expression in transgenic mice. Proc Natl
Acad Sci U S A. 1996; 93:10933–10938. [PubMed: 8855286]
Kloosterman WP, Lagendijk AK, Ketting RF, Moulton JD, Plasterk RH. Targeted inhibition of
miRNA maturation with morpholinos reveals a role for miR-375 in pancreatic islet development.
PLoS Biol. 2007; 5:e203. [PubMed: 17676975]
Kolm PJ, Sive HL. Efficient hormone-inducible protein function in Xenopus laevis. Dev Biol. 1995;
171:267–272. [PubMed: 7556904]
Kos R, Reedy MV, Johnson RL, Erickson CA. The winged-helix transcription factor FoxD3 is
important for establishing the neural crest lineage and repressing melanogenesis in avian embryos.
Development. 2001; 128:1467–1479. [PubMed: 11262245]
Lanyi JK, Duschl A, Hatfield GW, May K, Oesterhelt D. The primary structure of a halorhodopsin
NIH-PA Author Manuscript

from Natronobacterium pharaonis. Structural, functional and evolutionary implications for

bacterial rhodopsins and halorhodopsins. J Biol Chem. 1990; 265:1253–1260. [PubMed: 2104837]
Lennox KA, Sabel JL, Johnson MJ, Moreira BG, Fletcher CA, Rose SD, Behlke MA, Laikhter AL,
Walder JA, Dagle JM. Characterization of modified antisense oligonucleotides in Xenopus laevis
embryos. Oligonucleotides. 2006; 16:26–42. [PubMed: 16584293]
Lewis J. From signals to patterns: space, time, and mathematics in developmental biology. Science.
2008; 322:399–403. [PubMed: 18927385]
Link KH, Cruz FG, Ye HF, O’Reilly K E, Dowdell S, Koh JT. Photo-caged agonists of the nuclear
receptors RARgamma and TRbeta provide unique time-dependent gene expression profiles for
light-activated gene patterning. Bioorg Med Chem. 2004; 12:5949–5959. [PubMed: 15498671]
Link KH, Shi Y, Koh JT. Light activated recombination. J Am Chem Soc. 2005; 127:13088–13089.
[PubMed: 16173704]
Liu KJ, Arron JR, Stankunas K, Crabtree GR, Longaker MT. Chemical rescue of cleft palate and
midline defects in conditional GSK-3beta mice. Nature. 2007; 446:79–82. [PubMed: 17293880]
Mackem S, Lewandoski M. Limb development takes a measured step toward systems analysis. Sci
Signal. 2009; 2:pe33. [PubMed: 19454648]
Mansour SL, Thomas KR, Capecchi MR. Disruption of the proto-oncogene int-2 in mouse embryo-
derived stem cells: a general strategy for targeting mutations to non-selectable genes. Nature.
NIH-PA Author Manuscript

1988; 336:348–352. [PubMed: 3194019]

Mara A, Schroeder J, Holley SA. Two deltaC splice-variants have distinct signaling abilities during
somitogenesis and midline patterning. Dev Biol. 2008; 318:126–132. [PubMed: 18430417]
McCallum CM, Comai L, Greene EA, Henikoff S. Targeted screening for induced mutations. Nat
Biotechnol. 2000; 18:455–457. [PubMed: 10748531]
Mootz HD, Muir TW. Protein splicing triggered by a small molecule. J Am Chem Soc. 2002;
124:9044–9045. [PubMed: 12148996]
Nagel G, Brauner M, Liewald JF, Adeishvili N, Bamberg E, Gottschalk A. Light activation of
channelrhodopsin-2 in excitable cells of Caenorhabditis elegans triggers rapid behavioral
responses. Curr Biol. 2005; 15:2279–2284. [PubMed: 16360690]
Nagel G, Szellas T, Huhn W, Kateriya S, Adeishvili N, Berthold P, Ollig D, Hegemann P, Bamberg E.
Channelrhodopsin-2, a directly light-gated cation-selective membrane channel. Proc Natl Acad Sci
U S A. 2003; 100:13940–13945. [PubMed: 14615590]
Nasevicius A, Ekker SC. Effective targeted gene ‘knockdown’ in zebrafish. Nat Genet. 2000; 26:216–
220. [PubMed: 11017081]

Chem Biol. Author manuscript; available in PMC 2011 June 25.

 Ouyang and Chen Page 21

Nishimura K, Fukagawa T, Takisawa H, Kakimoto T, Kanemaki M. An auxin-based degron system

for the rapid depletion of proteins in nonplant cells. Nat Methods. 2009; 6:917–922. [PubMed:
19915560]
NIH-PA Author Manuscript

Niswander L, Jeffrey S, Martin GR, Tickle C. A positive feedback loop coordinates growth and
patterning in the vertebrate limb. Nature. 1994; 371:609–612. [PubMed: 7935794]
Niswander L, Tickle C, Vogel A, Booth I, Martin GR. FGF-4 replaces the apical ectodermal ridge and
directs outgrowth and patterning of the limb. Cell. 1993; 75:579–587. [PubMed: 8221896]
Ouyang X, Shestopalov IA, Sinha S, Zheng G, Pitt CL, Li WH, Olson AJ, Chen JK. Versatile
synthesis and rational design of caged morpholinos. J Am Chem Soc. 2009; 131:13255–13269.
[PubMed: 19708646]
Paddison PJ, Caudy AA, Bernstein E, Hannon GJ, Conklin DS. Short hairpin RNAs (shRNAs) induce
sequence-specific silencing in mammalian cells. Genes Dev. 2002; 16:948–958. [PubMed:
11959843]
Palmiter RD, Behringer RR, Quaife CJ, Maxwell F, Maxwell IH, Brinster RL. Cell lineage ablation in
transgenic mice by cell-specific expression of a toxin gene. Cell. 1987; 50:435–443. [PubMed:
3649277]
Parr BA, McMahon AP. Dorsalizing signal Wnt-7a required for normal polarity of D-V and A-P axes
of mouse limb. Nature. 1995; 374:350–353. [PubMed: 7885472]
Paulus H. Protein splicing and related forms of protein autoprocessing. Annu Rev Biochem. 2000;
69:447–496. [PubMed: 10966466]
Pisharath H, Rhee JM, Swanson MA, Leach SD, Parsons MJ. Targeted ablation of beta cells in the
NIH-PA Author Manuscript

embryonic zebrafish pancreas using E. coli nitroreductase. Mech Dev. 2007; 124:218–229.
[PubMed: 17223324]
Pratt MR, Schwartz EC, Muir TW. Small-molecule-mediated rescue of protein function by an
inducible proteolytic shunt. Proc Natl Acad Sci U S A. 2007; 104:11209–11214. [PubMed:
17563385]
Rangasamy D, Tremethick DJ, Greaves IK. Gene knockdown by ecdysone-based inducible RNAi in
stable mammalian cell lines. Nat Protoc. 2008; 3:79–88. [PubMed: 18193024]
Ridgway P, Quivy JP, Almouzni G. Tetracycline-regulated gene expression switch in Xenopus laevis.
Exp Cell Res. 2000; 256:392–399. [PubMed: 10772812]
Robu ME, Larson JD, Nasevicius A, Beiraghi S, Brenner C, Farber SA, Ekker SC. p53 activation by
knockdown technologies. PLoS Genet. 2007; 3:e78. [PubMed: 17530925]
Roldan MD, Perez-Reinado E, Castillo F, Moreno-Vivian C. Reduction of polynitroaromatic
compounds: the bacterial nitroreductases. FEMS Microbiol Rev. 2008; 32:474–500. [PubMed:
18355273]
Saito M, Iwawaki T, Taya C, Yonekawa H, Noda M, Inui Y, Mekada E, Kimata Y, Tsuru A, Kohno K.
Diphtheria toxin receptor-mediated conditional and targeted cell ablation in transgenic mice. Nat
Biotechnol. 2001; 19:746–750. [PubMed: 11479567]
Satou Y, Imai KS, Satoh N. Action of morpholinos in Ciona embryos. Genesis. 2001; 30:103–106.
NIH-PA Author Manuscript

[PubMed: 11477683]
Sawa M, Nusinow DA, Kay SA, Imaizumi T. FKF1 and GIGANTEA complex formation is required
for day-length measurement in Arabidopsis. Science. 2007; 318:261–265. [PubMed: 17872410]
Schnutgen F, Doerflinger N, Calleja C, Wendling O, Chambon P, Ghyselinck NB. A directional
strategy for monitoring Cre-mediated recombination at the cellular level in the mouse. Nat
Biotechnol. 2003; 21:562–565. [PubMed: 12665802]
Schroll C, Riemensperger T, Bucher D, Ehmer J, Voller T, Erbguth K, Gerber B, Hendel T, Nagel G,
Buchner E, et al. Light-induced activation of distinct modulatory neurons triggers appetitive or
aversive learning in Drosophila larvae. Curr Biol. 2006; 16:1741–1747. [PubMed: 16950113]
Schwartz EC, Saez L, Young MW, Muir TW. Post-translational enzyme activation in an animal via
optimized conditional protein splicing. Nat Chem Biol. 2007; 3:50–54. [PubMed: 17128262]
Segal E, Raveh-Sadka T, Schroeder M, Unnerstall U, Gaul U. Predicting expression patterns from
regulatory sequence in Drosophila segmentation. Nature. 2008; 451:535–540. [PubMed:
18172436]

Chem Biol. Author manuscript; available in PMC 2011 June 25.

 Ouyang and Chen Page 22

Seibler J, Kleinridders A, Kuter-Luks B, Niehaves S, Bruning JC, Schwenk F. Reversible gene

knockdown in mice using a tight, inducible shRNA expression system. Nucleic Acids Res. 2007;
35:e54. [PubMed: 17376804]
NIH-PA Author Manuscript

Shah S, Jain PK, Kala A, Karunakaran D, Friedman SH. Light-activated RNA interference using
double-stranded siRNA precursors modified using a remarkable regiospecificity of diazo-based
photolabile groups. Nucleic Acids Res. 2009; 37:4508–4517. [PubMed: 19477960]
Shah S, Rangarajan S, Friedman SH. Light-activated RNA interference. Angew Chem Int Ed Engl.
2005; 44:1328–1332. [PubMed: 15643658]
Shestopalov IA, Sinha S, Chen JK. Light-controlled gene silencing in zebrafish embryos. Nat Chem
Biol. 2007; 3:650–651. [PubMed: 17717538]
Shi Y, Jin Y. MicroRNA in cell differentiation and development. Sci China C Life Sci. 2009; 52:205–
211. [PubMed: 19294345]
Shin MK, Levorse JM, Ingram RS, Tilghman SM. The temporal requirement for endothelin receptor-B
signalling during neural crest development. Nature. 1999; 402:496–501. [PubMed: 10591209]
Siegel RW, Jain R, Bradbury A. Using an in vivo phagemid system to identify non-compatible loxP
sequences. FEBS Lett. 2001; 505:467–473. [PubMed: 11576551]
Sinha DK, Neveu P, Gagey N, Aujard I, Benbrahim-Bouzidi C, Le Saux T, Rampon C, Gauron C,
Goetz B, Dubruille S, et al. Photocontrol of Protein Activity in Cultured Cells and Zebrafish with
One- and Two-Photon Illumination. Chembiochem. 2010; 11:653–663. [PubMed: 20187057]
Spitzer NC. Electrical activity in early neuronal development. Nature. 2006; 444:707–712. [PubMed:
17151658]
NIH-PA Author Manuscript

Stankunas K, Bayle JH, Gestwicki JE, Lin YM, Wandless TJ, Crabtree GR. Conditional protein alleles
using knockin mice and a chemical inducer of dimerization. Mol Cell. 2003; 12:1615–1624.
[PubMed: 14690613]
Summerton J. Morpholino antisense oligomers: the case for an RNase H-independent structural type.
Biochim Biophys Acta. 1999; 1489:141–158. [PubMed: 10807004]
Summerton J, Weller D. Morpholino antisense oligomers: design, preparation, and properties.
Antisense Nucleic Acid Drug Dev. 1997; 7:187–195. [PubMed: 9212909]
Tang X, Maegawa S, Weinberg ES, Dmochowski IJ. Regulating gene expression in zebrafish embryos
using light-activated, negatively charged peptide nucleic acids. J Am Chem Soc. 2007;
129:11000–11001. [PubMed: 17711280]
Thaker M, Spanogiannopoulos P, Wright GD. The tetracycline resistome. Cell Mol Life Sci. 2010;
67:419–431. [PubMed: 19862477]
Tomasini AJ, Schuler AD, Zebala JA, Mayer AN. PhotoMorphs: a novel light-activated reagent for
controlling gene expression in zebrafish. Genesis. 2009; 47:736–743. [PubMed: 19644983]
Urtishak KA, Choob M, Tian X, Sternheim N, Talbot WS, Wickstrom E, Farber SA. Targeted gene
knockdown in zebrafish using negatively charged peptide nucleic acid mimics. Dev Dyn. 2003;
228:405–413. [PubMed: 14579379]
Veenstra GJ, Weeks DL, Wolffe AP. Distinct roles for TBP and TBP-like factor in early embryonic
NIH-PA Author Manuscript

gene transcription in Xenopus. Science. 2000; 290:2312–2315. [PubMed: 11125147]

Volgraf M, Gorostiza P, Numano R, Kramer RH, Isacoff EY, Trauner D. Allosteric control of an
ionotropic glutamate receptor with an optical switch. Nat Chem Biol. 2006; 2:47–52. [PubMed:
16408092]
Woolf TM, Jennings CG, Rebagliati M, Melton DA. The stability, toxicity and effectiveness of
unmodified and phosphorothioate antisense oligodeoxynucleotides in Xenopus oocytes and
embryos. Nucleic Acids Res. 1990; 18:1763–1769. [PubMed: 1692405]
Wu H, Hu Z, Liu XQ. Protein trans-splicing by a split intein encoded in a split DnaE gene of
Synechocystis sp. PCC6803. Proc Natl Acad Sci U S A. 1998; 95:9226–9231. [PubMed:
9689062]
Wu YI, Frey D, Lungu OI, Jaehrig A, Schlichting I, Kuhlman B, Hahn KM. A genetically encoded
photoactivatable Rac controls the motility of living cells. Nature. 2009; 461:104–108. [PubMed:
19693014]

Chem Biol. Author manuscript; available in PMC 2011 June 25.

 Ouyang and Chen Page 23

Wyart C, Del Bene F, Warp E, Scott EK, Trauner D, Baier H, Isacoff EY. Optogenetic dissection of a
behavioural module in the vertebrate spinal cord. Nature. 2009; 461:407–410. [PubMed:
19759620]
NIH-PA Author Manuscript

Yamaguchi S, Kurimoto K, Yabuta Y, Sasaki H, Nakatsuji N, Saitou M, Tada T. Conditional

knockdown of Nanog induces apoptotic cell death in mouse migrating primordial germ cells.
Development. 2009; 136:4011–4020. [PubMed: 19906868]
Yazawa M, Sadaghiani AM, Hsueh B, Dolmetsch RE. Induction of protein-protein interactions in live
cells using light. Nat Biotechnol. 2009; 27:941–945. [PubMed: 19801976]
Yeh JR, Crews CM. Chemical genetics: adding to the developmental biology toolbox. Dev Cell. 2003;
5:11–19. [PubMed: 12852848]
Yen L, Magnier M, Weissleder R, Stockwell BR, Mulligan RC. Identification of inhibitors of
ribozyme self-cleavage in mammalian cells via high-throughput screening of chemical libraries.
RNA. 2006; 12:797–806. [PubMed: 16556935]
Yen L, Svendsen J, Lee JS, Gray JT, Magnier M, Baba T, D’Amato RJ, Mulligan RC. Exogenous
control of mammalian gene expression through modulation of RNA self-cleavage. Nature. 2004;
431:471–476. [PubMed: 15386015]
Yoo SK, Deng Q, Cavnar PJ, Wu YI, Hahn KM, Huttenlocher A. Differential Regulation of Protrusion
and Polarity by PI(3)K during Neutrophil Motility in Live Zebrafish. Dev Cell. 2010; 18:226–
236. [PubMed: 20159593]
Young DD, Garner RA, Yoder JA, Deiters A. Light-activation of gene function in mammalian cells
via ribozymes. Chem Commun (Camb). 2009:568–570. [PubMed: 19283293]
NIH-PA Author Manuscript

Yuen CM, Rodda SJ, Vokes SA, McMahon AP, Liu DR. Control of transcription factor activity and
osteoblast differentiation in mammalian cells using an evolved small-molecule-dependent intein.
J Am Chem Soc. 2006; 128:8939–8946. [PubMed: 16819890]
Zeller R, Lopez-Rios J, Zuniga A. Vertebrate limb bud development: moving towards integrative
analysis of organogenesis. Nat Rev Genet. 2009; 10:845–858. [PubMed: 19920852]
Zhang F, Aravanis AM, Adamantidis A, de Lecea L, Deisseroth K. Circuit-breakers: optical
technologies for probing neural signals and systems. Nat Rev Neurosci. 2007a; 8:577–581.
[PubMed: 17643087]
Zhang F, Wang LP, Brauner M, Liewald JF, Kay K, Watzke N, Wood PG, Bamberg E, Nagel G,
Gottschalk A, et al. Multimodal fast optical interrogation of neural circuitry. Nature. 2007b;
446:633–639. [PubMed: 17410168]
NIH-PA Author Manuscript

Chem Biol. Author manuscript; available in PMC 2011 June 25.

 Ouyang and Chen Page 24
NIH-PA Author Manuscript
NIH-PA Author Manuscript

Figure 1. Synthetic control of gene transcription

(a) Schematic representation of hormone receptor-based transactivator systems. Chemical
structures for representative agonists are shown. TF, transcription factor; HR LBD, hormone
receptor ligand-binding domain; HRE, hormone response element. (b) Schematic
representation of tetracycline repressor-based transcription systems, including the “Tet-Off”
and “Tet-On” configurations. Chemical structures of tetracycline and its synthetic
derivatives are shown. Tet-R, Tet repressor; rTet-R, reverse Tet transactivator; AD,
activation domain; TetO, Tet repressor operon. Although hormone receptor-and tetracycline
repressor-based transactivators can each form protein dimers, monomeric forms of these
NIH-PA Author Manuscript

proteins are depicted for schematic simplicity. (c) Control of GFP expression in mice
embryos using the “Tet-On” system and caged doxycycline. Both global and localized
(dashed box and inset) irradiation are shown. Adapted with permission (Cambridge et al.,
2009; Copyright 2009, Nature America, Inc). (d) Schematic representation of tamoxifen-
induced, Cre-dependent recombination, including excision and inversion reactions. The
recombinase homodimer is depicted as a monomer for schematic simplicity, and chemical
structures of tamoxifen and its derivatives are shown. ER LBD, estrogen receptor ligand-
binding domain. (e) Tamoxifen-induced “self knockout” of the Otx2 gene in mouse embryos
using the Cre-ERT2 system. Abnormal craniofacial and brain development is observed only
in Otx2flox/Cre-ERT2 embryos treated with the ER agonist. Adapted with permission (Fossat et
al., 2006; Copyright 2006, European Molecular Biology Organization).

Chem Biol. Author manuscript; available in PMC 2011 June 25.

 Ouyang and Chen Page 25
NIH-PA Author Manuscript

Figure 2. Synthetic control of RNA function

(a) Chemical structures for natural oligonucleotides, their modified analogs, and non-natural
oligonucleotides. Caging groups are shown in red. (b) Schematic representation of the caged
MO hairpin (left) and PhotoMorphs™ technologies (right). (c) Light-controlled silencing of
flh and heg in zebrafish embryos using caged MO hairpins. Adapted with permission
(Ouyang et al., 2009; Copyright 2009, American Chemical Society). (d) Schematic
representation of a small molecule-dependent riboswitch (orange) that regulates transcript
stability. Chemical structures of toyocamycin and its caged derivative are shown.
NIH-PA Author Manuscript
NIH-PA Author Manuscript

Chem Biol. Author manuscript; available in PMC 2011 June 25.

 Ouyang and Chen Page 26
NIH-PA Author Manuscript

Figure 3. Synthetic control of protein function

(a) Schematic representation of rapamycin-induced polypeptide splicing using fusion
proteins composed of FRB, FKBP, and split inteins. (b) Schematic representation of ligand-
and FKBP-dependent stabilization of FRB*-containing fusion proteins and the chemical
structure of the FRB*-specific rapamycin analog C20-MaRap. POI, protein of interest. (c)
Rapamycin-dependent rescue of GSK3β activity in homozygous GSK3β-FRB* knock-in
mouse embryos. Rapamycin has no effect on wildtype embryos during the two-day drug
regimen, but the majority of GSK3β-FRB* mice are fully rescued from cleft palate defects
(white arrowhead) upon rapamycin treatment. Partial rescues were also observed (data not
shown). Adapted with permission (Liu et al., 2007; Copyright 2007, Nature Publishing
Group). (d) Schematic representation of ligand-dependent stabilization of FKBP mutant-
containing fusion proteins and the chemical structure of Shield-1. (e) Schematic
representation of the “bump-and-hole” strategy for achieving targeted protein inhibition with
NIH-PA Author Manuscript

genome-wide specificity. Chemical structures of promiscuous (PP1) and mutant kinase-

specific (naphthylmethyl-PP1) inhibitors are shown. (f) Schematic representation of light-
regulated protein conformation and function using synthetic and endogenous cofactors.
Azobenzene-, retinal-, and flavin mononucleotide-based approaches are shown, with the
azobenzene-linked small-molecule modulator depicted as a solid black circle.
NIH-PA Author Manuscript

Chem Biol. Author manuscript; available in PMC 2011 June 25.

 Ouyang and Chen Page 27
NIH-PA Author Manuscript
NIH-PA Author Manuscript

Figure 4. Chemically induced cell ablation

(a) Schematic representation of metronidazole-dependent apoptosis of nitroreductase-
expressing cells (red) without collateral damage to adjacent cells (green). (b) Metronidazole-
induced ablation of pancreatic β cells (red) in transgenic zebrafish larvae with insulin
promoter-driven nitroreductase-mCherry expression (ins:NTR-mCherry). Exocrine pancreas
cells (green) were not affected, and no drug-dependent cell ablation was observed in
ins:mCherry larvae. Adapted with permission (Pisharath et al., 2007; Copyright 2006,
Elsevier Ireland Ltd.).
NIH-PA Author Manuscript

Chem Biol. Author manuscript; available in PMC 2011 June 25.