REFLECTIONS This paper is available online at www.jbc.

org
THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 284, NO. 20, pp. 13277–13283, May 15, 2009
Printed in the U.S.A.

Differential Gene Expression in

Vertebrate Embryos
Published, JBC Papers in Press, January 21, 2009, DOI 10.1074/jbc.X800018200
Igor B. Dawid
From the Laboratory of Molecular Genetics, Eunice Kennedy Shriver NICHD, National Institutes of
Health, Bethesda, Maryland 20892

T
he invitation to contribute to the Reflections series in the Journal of Biological Chem-
istry is an honor that I greatly appreciate. It is humbling to find oneself in such distin-
guished company and a challenge to the literary limitations reflected all too obviously
in the scientific writing I usually engage in. What I would like to do is to describe my

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own professional path in the context of the evolution of the field. In this I would like to focus on the
story of how embryology, after joining forces with biochemistry and molecular biology, became to
be usually called developmental biology and how the focus of my work and that of much of the field
revolved around differential gene expression in different cell types, tissues, and developmental
stages. As always in biology, it is a tale of new questions and concepts and of advancing technology
that allowed old questions to be answered and new ones to be posed in a rational and approachable
manner.

Becoming a Developmental Biologist: The Carnegie Years

I studied chemistry in Vienna, proceeding to a thesis in biochemistry that led to a Ph.D. in 1960.
The way in which I reached the Department of Embryology of the Carnegie Institution after a
postdoctoral stay at the Massachusetts Institute of Technology was both fortuitous and fortunate,
as I have related elsewhere (1). The Carnegie Department in Baltimore, then headed by Jim Ebert,
was a great place to be for a biochemist who wanted to enter developmental biology and establish
his research in this field. Many factors made this an outstanding environment, first and foremost
the wonderful colleagues at the Department, especially Don Brown, who had arrived a few years
earlier and provided me with much guidance and stimulation. The problem that fascinated us was
how does a fertilized egg develop into a complex organism with its varied cell types and complex
shape? Clearly, genes had something to do with it, but how did they act to generate developmental
variety? One point of interest was if and to what extent the genome itself might change during
differentiation. Although the constancy of the genome through development was generally
accepted at the time, exceptions did come up. My earliest work in the field dealt with “extra” DNA
that was suspected to occur in the egg cytoplasm of our favorite model system, the frog Xenopus
laevis. I could show that it is mitochondrial DNA, resolving the riddle of egg DNA and contribut-
ing to the work in several laboratories that at the time established the existence of DNA in this
organelle (2, 3). A few years later, Don and I (4) and Joe Gall (5) independently showed that genes
encoding ribosomal RNA were amplified in oocytes, presumably to be able to support the prodi-
gious rate of rRNA synthesis in these cells. This was a developmental result, showing that, under
some circumstances, gene amplification did occur to support developmental needs. Although
amplification was later demonstrated for certain protein-coding genes in Drosophila oocytes as
well (6), gene amplification is not a widespread mechanism for differential gene expression in
development.
The generalization that different cells and tissues carry a constant genome but a variable pop-
ulation of proteins implies differential gene expression. Looking directly at individual protein-

MAY 15, 2009 • VOLUME 284 • NUMBER 20 JOURNAL OF BIOLOGICAL CHEMISTRY 13277
REFLECTIONS: Differential Gene Expression
coding genes, notably in animal cells, seemed impossible
during the first decade of my time at the Carnegie Institu-
tion. Still, progress was made in identifying and character-
izing individual abundant mRNAs from animal sources by
translating globin mRNA in cell-free extracts or by injec-
tion into frog oocytes (7, 8) or by taking advantage of the
beginnings of sequencing technology in the case of silk
protein mRNA (9). The grand breakthrough came, of
course, with cloning technology. This is not the place to
attempt a history of this development but to relate how it
affected me. I recall that I first learned about the successful
cloning of vertebrate DNA in bacteria because the Xeno-
pus ribosomal DNA used in this experiment had be
donated by Don Brown to the group at Stanford who car-
ried out this work (10). In the context of subsequent work
on differential gene expression, the next major advance
was the introduction of cDNA cloning (11). This tech-
nique made it possible to isolate individual mRNAs but
also to generate a library representing the entire mRNA
population in a cell or tissue, in other words, to look at the
expressed sequences in the genome in a global way. Clon-
ing and sequencing (12, 13) changed biology. My work, as
that of so many others, was changed and enhanced by
these developments in the most profound way. These
techniques allowed me to transfer my interest from mito-
chondrial and ribosomal genes to protein-coding genes.
Moving to the National Institutes of Health:
Beginning to Work with cDNA Libraries
By that time, in the late 1970s, I moved from the Carne-
gie Department of Embryology in Baltimore to the
National Institutes of Health (NIH). Thanks to the efforts
of Bob Goldberger, I joined the Laboratory of Biochemis-
try at the National Cancer Institute and a few years later
switched to the National Institute of Child Health and
Human Development (NICHD), which has remained my
home ever since. The greatly appreciated support from the
Institute has made it possible for me and my colleagues to
pursue our interests for all this time. Right around the time
of our move to NIH, we started to study differential gene
expression in development in a general way by applying
cDNA cloning technology to look at changing mRNA
populations in the frog embryo. As the methods for pre-
paring cDNA libraries improved, the idea to interrogate
various libraries with probes derived from RNA popula-
tions of different developmental stages, multiple tissues, or
cells exposed to various conditions was bound to arise. It
took a few years to turn this idea into praxis. Although
cDNA cloning was announced in 1976 (11), comparisons
of RNA populations continued to be made by using
hybridization kinetics throughout much of the decade.
13278 JOURNAL OF BIOLOGICAL CHEMISTRY
Starting in 1979 and 1980, several publications appeared
that made use of cDNA libraries to investigate develop-
mental or physiological changes in RNA populations. A
variety of biological systems were subjected to this
approach: developmental changes in Dictyostelium (14,
15) and Aspergillus (16), effects of starvation in yeast (17),
and changes induced by auxin in plants (18) and in embry-
onic development in sea urchins (19) and in Xenopus (20).
Our work with cDNA cloning began with studies on
vitellogenin mRNA in Xenopus by Walter Wahli (21) and,
directly relevant to the theme of this article, with the work
initiated by Mark Dworkin after he joined the lab as a
postdoctoral fellow in 1978 (20). Later, Mark continued
his research at Columbia University and in Vienna, Aus-
tria, but died of cancer at a tragically young age. At the
time Mark came to the lab, it was known that eggs and
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embryos of frogs and other animals contained a large pop-
ulation of mRNAs whose general properties had been
measured by renaturation kinetics, as summarized ele-
gantly in Eric Davidson’s book Gene Activity in Early
Development (22). These studies revealed the total com-
plexity of the embryonic mRNA population and the over-
all distribution and developmental changes of classes of
RNAs of certain ranges of abundance; yet for all the power
of these global analyses, it seemed likely that they masked
variations in the behavior of individual RNAs that could be
revealed only by looking at them one at a time. To do this,
we generated cDNA libraries from different embryonic
stages and then randomly selected about 800 clones for
further study. These clones were grown and transferred to
filters (23) to obtain what we would now call in hindsight a
macroarray. To probe these arrays, we prepared RNA
from five embryonic stages and two tissues, in some cases
using different cell fractions for RNA isolation, and syn-
thesized radioactively labeled cDNA from each RNA sam-
ple. Hybridization of the arrays with these probes allowed
us to compare the RNA populations in the different
embryonic stages in a semiquantitative way. Given the
technical limitations of these procedures, only moderately
to highly abundant RNAs/cDNAs yielded a detectable sig-
nal, and as a result, only ⬃30% of the cDNA clones could
be visualized with any of the probes that we used. Never-
theless, the experiment led to some interesting conclu-
sions, as illustrated in three panels taken from the original
publication (Fig. 1). First, it was clear that many of the
clones that could be visualized were present at all stages
tested, presumably representing widely expressed or so-
called housekeeping genes. Second, some clones were dif-
ferentially expressed; although this was expected, it dem-
onstrated the feasibility of identifying many differentially
VOLUME 284 • NUMBER 20 • MAY 15, 2009

FIGURE 1. Differential gene expression in eggs and stage 10 and 41
embryos of X. laevis. The figure has been rearranged from Ref. 20.
Clones from two embryonic cDNA libraries were transferred to nitrocel-
lulose membranes. The numbers at the top and bottom refer to columns
of clones that are not precisely aligned on the different filters; lines indi-
cate the corresponding positions on the three filters. The three replicate
membranes containing transferred colonies were hybridized with
labeled cDNA probes generated from RNA of the stages (St) indicated.
Similarities and differences in the expression patterns at different stages
can be seen. For further explanations, see text.
expressed genes in this way. Third, although no precise
measurements could be made at this point, it was clear
that many different developmental patterns were present
in the RNA population, confirming the expectation that
classification of mRNA in low, middle, and high abun-
dance classes was useful but a simplification of complex
patterns.
Subtractive Cloning: An Effective Approach to
Studying Differential Gene Expression
The approach of hybridizing cDNA libraries with
labeled cDNA preparations was capable of revealing dif-
ferences between RNA populations and could lead to the
isolation of differentially expressed genes, but a major
limitation was the fact that only moderately to highly
abundant RNAs could be visualized in this method. Thus,
the patterns observed were dominated by abundant
RNAs, often by widely expressed species corresponding to
housekeeping genes that are represented in multiple cop-
ies in standard cDNA libraries. A major advance that
removed this limitation came through a combination of
cDNA cloning with methods based on the kinetics of
hybridization of nucleic acids in solution that had been
worked out mostly by Roy Britten, Eric Davidson, and
their colleagues (22, 24, 25). When complementary
nucleic acid strands are annealed, the rate of duplex for-
mation is proportional to the concentration of the two
MAY 15, 2009 • VOLUME 284 • NUMBER 20
REFLECTIONS: Differential Gene Expression
strands for each particular sequence. Solution hybridiza-
tion in combination with cDNA synthesis and cloning is
the basis of subtractive cloning for the isolation of differ-
entially expressed genes. The method was developed inde-
pendently in our lab by Tom Sargent, who continues to be
my colleague as a Senior Investigator at NICHD, and by
Mark Davis, at that time also at NIH, and colleagues (26 –
28). In our case, it involved the synthesis of cDNA from
mRNA isolated from gastrula stage Xenopus embryos, fol-
lowed by hybridization of the cDNA in solution with an
excess of RNA from eggs. cDNA that failed to hybridize,
i.e. that is not or is very rarely represented in egg RNA, was
isolated, rendered double-stranded, and cloned in a plas-
mid vector. The great advantage of this approach com-
pared with direct interrogation of conventional cDNA
libraries is that copies of genes expressed in both stages are
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eliminated or greatly reduced, more effectively the more
highly expressed they are. This is important as abundant
RNAs dominate standard libraries, confounding efforts to
identify low abundance differentially expressed genes.
Thus, the subtractive method not only gives direct access
to differentially expressed genes but at the same time
expands the range of genes that can be effectively screened
to the class of low abundance transcripts.
By now, subtractive cloning appears to be an obvious
and simple method, but it presented substantial technical
challenges at the time. The successful resolution of these
challenges gives an illustration of two major uses for this
technique. One aim is to clone a particular gene known to
be differentially expressed; this was the goal that Mark
Davis and colleagues achieved in isolating the T cell recep-
tor (27, 28). In Tom Sargent’s studies in our lab (26, 29), the
aim was to achieve a broad comparison of gene expression
in different stages of frog embryogenesis, trying to char-
acterize patterns of gene activity and to identify genes
that have a role in development. Our initial studies
showed that frog gastrulas contain many gene transcripts
not represented in the maternal RNA pool (Fig. 2); we
named them DG genes, for differentially expressed in gas-
trula. Although some of the gastrula RNAs continue to be
expressed in later development, others persist for a short
only, suggesting that they have a time-limited role in the
embryo. Among the RNAs that attracted our attention in
the initial work, some were studied extensively in subse-
quent years. Of these, DG42 seemed of developmental
interest because of its regionally and temporally specific
expression (30), but its identity remained unclear for a
long time. Eventually, through the efforts of others labo-
ratories, it proved to represent the gene encoding verte-
brate hyaluronan synthase, an important gene that had
JOURNAL OF BIOLOGICAL CHEMISTRY 13279
REFLECTIONS: Differential Gene Expression
FIGURE 2. Characterization of the DG library. Clones from the sub-
tracted cDNA library containing DG genes of X. laevis were spotted onto
nitrocellulose membranes and hybridized with labeled cDNA probe
from egg and gastrula. Greatly enriched representation of genes
expressed in the gastrula is seen. The figure has been rearranged from
Ref. 26. For further explanations, see text.
long resisted cloning efforts (31–33). Our own work was
focused for several years on a group of genes with differ-
ential expression patterns that proved to encode cytokera-
tins. Some cytokeratins in this group are specifically
expressed during embryonic and tadpole development in
the frog, and they were useful as epidermal markers, which
we explored in work involving Milan Jamrich, Jeff Win-
kles, and others (34, 35). A particularly interesting appli-
cation of the DG library was its use in isolating Mix.1, the
first gene known to be induced by a signaling factor in the
embryo (36). In this experiment, the subtractive approach
was used twice. Explants from Xenopus embryos were
treated with activin, a factor that is able to induce
endoderm and mesoderm in the naïve explant, and the
genes induced by activin were enriched by subtraction of
the cDNA generated from induced explants with ovarian
RNA. The enriched cDNA was used to probe the DG
library, allowing the isolation of several activin-induced
genes, among which the homeobox gene Mix.1 was the
primary focus of attention. A similar approach for the
identification of inducible genes has generated useful
results in subsequent years.
The subtractive cloning method has been applied
widely in the intervening time, and some modifications
13280 JOURNAL OF BIOLOGICAL CHEMISTRY
have been introduced, taking advantage of PCR and other
technical developments. An important adaptation and
modification of the subtractive method is “normalization,”
a procedure in which cDNA is “subtracted” with the RNA
population from which it originates. Ultimately, of course,
that would remove all cDNAs from the sample, but if car-
ried out under controlled conditions, it is possible to
reduce most abundant components to the level of the
average rare component, producing a cDNA library that
contains similar numbers of copies of each RNA (37, 38).
Such libraries are very useful in random sequencing
projects (expressed sequence tag (EST) projects) as the
level of re-sequencing of abundant mRNAs is greatly
reduced. The availability of large collections of ESTs for
many organisms has been an indispensable complement
to genome sequencing and a critical ingredient in any
work on the molecular basis of development. Additional
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methods for comparative expression analysis have been
introduced and found wide use, including differential dis-
play (39) and serial analysis of gene expression (40). Serial
analysis of gene expression offers the opportunity for
quantitative assessment of gene expression levels, an
important feature in many applications.
In Situ Hybridization as a Means to Find
Differentially Expressed Genes
Many ideas and many techniques have contributed to
the rise of biology over the past half-century. One of the
most widely useful techniques is in situ hybridization, first
applied to localize specific DNA sequences by Gall and
Pardue (41, 42) and subsequently used to localize individ-
ual RNAs in cytological sections and in three-dimensional
objects such as embryos by whole mount techniques (43–
45). Fine cytological detail is achievable in this technique,
making it possible to study gene expression in an embryo
at a cellular or even subcellular level. The approach usually
taken is to isolate a gene of interest in one of a number of
ways and then study its expression pattern by the in situ
hybridization technique. Using in situ hybridization as a
method to identify genes of developmental interest and to
survey the broad range of expression patterns that occur
was first done, to my knowledge, by Niehrs and colleagues
using Xenopus embryos (46). Our laboratory, in particular
Tetsu Kudoh, Michael Tsang, Neil Hukriede, and others
(47), and the laboratory of Thisse and Thisse (ZFIN
Zebrafish Model Organism Database ID ZDB-PUB-
010810-1) applied this approach to the zebrafish embryo.
Given the considerable spatial resolution that can be
obtained (for illustration, see Fig. 3), the method reveals a
wealth of patterning data that are useful at different levels
of analysis. First, a catalog of expression patterns is
VOLUME 284 • NUMBER 20 • MAY 15, 2009

FIGURE 3. Montage of expression patterns as visualized by in situ
hybridization for a selection of cDNA clones from the zebrafish.
Each image represents an embryo hybridized with a different cDNA
clone. Different stages from the gastrula to 3-day-old larvae are repre-
sented in this montage, which was generated to illustrate the wide vari-
ety of patterns that are obtained. For example, genes expressed in the
head or tail, the somites, or the nervous system and in multiple distinct
patterns within the nervous system have been visualized. The figure
was assembled by Michael Tsang based on data obtained in our screen
(the entire data set is available at the ZFIN Zebrafish Model Organism
Database) (47).
obtained that accelerates research: when you start work-
ing on a new gene, its expression pattern may already be
available. Second, the approach generates new markers for
different cell types and tissues. The wealth of molecular
markers now available, be they in situ probes or antibod-
ies, has ushered in a higher level of anatomical definition
than had been possible previously. Third, this is an
approach to functional genomics, i.e. to infer functions of
novel genes. Although pattern alone cannot provide reli-
able functional information, it can provide guidance to
further experiments. Fourth, access to functional informa-
tion is facilitated in the particular situation in which a
novel gene is recognized as a member of a synexpression
group (46). This term refers to a group of genes that have
similar complex expression patterns in development, and
in many instances, members of a synexpression group
prove to be components of a common functional pathway.
This allows identification of novel components in various
pathways. For example, a feedback inhibitor of Fgf signal-
ing named Sef was discovered in this way (48, 49).
The results of in situ hybridization screens can be help-
ful in drawing attention to previously unexplored relation-
ships, even when the genes under study have been de-
scribed in earlier work. Such an example is provided by
Tetsu Kudoh’s work in our laboratory that took its cue
from observing the expression of the cyp26 gene in the
anterior neural ectoderm of the zebrafish embryo (50).
This gene encodes all-trans-retinoic acid 4-hydroxylase,
an enzyme that degrades retinoic acid. As retinoic acid is
MAY 15, 2009 • VOLUME 284 • NUMBER 20
REFLECTIONS: Differential Gene Expression
known to affect brain development in guiding differentia-
tion along the anterior-posterior axis, the expression pat-
tern of cyp26 suggested to us that this gene had a role in
defining the retinoic acid gradient along this axis. This idea
led to a study in which we explored the role of retinoic
acid, Wnt, and Fgf in the organization of the early nervous
system in the zebrafish, with a particular focus on the inte-
gration of these different signaling pathways in the process
(50).
Application of DNA Microarray Technology to the
Study of Embryogenesis
Hybridization of a nucleic acid probe to nucleic acid
fixed on a support has a long history, from the early efforts
of Bolton and McCarthy (51) to the “paradigm changing”
introduction of nitrocellulose as support by Nygaard and
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Hall (52) and to the introduction of gel-to-membrane
transfer by Southern (53). The idea of applying target
DNAs in the form of dots to a solid support likewise was
introduced some time ago (54, 55), but the approach
changed dramatically when, based on the exploitation of
genome and EST sequencing efforts, the modern microar-
ray procedure was introduced (56, 57). Given the high
density of probes on the slide or chip, it becomes possible
to compare gene expression patterns in different cells for
many (ultimately all) of an organism’s genes at the same
time. Multiple wide reaching applications have been
developed for this technology.
Our use of the microarray technology got started in ear-
nest when Affymetrix威 generated chips for X. laevis and
the zebrafish. Although the genome sequences of the
zebrafish and Xenopus tropicalis, a species closely related
to X. laevis, were at the time incomplete (and continue to
be so) and the arrays therefore represent only about half of
the genes in the two species, a great deal of information
can be generated in such experiments (58 – 61). We car-
ried out several types of analyses on embryonic material
from frogs and fish. In particular, experiments that com-
pared different regions of Xenopus embryos proved useful.
Hui Zhao and Kosuke Tanegashima separated gastrula
and early neurula embryos into several parts by microdis-
section, compared the different RNA populations by
microarray analysis, and subjected individual genes with
differential expression to further analysis. This approach
yielded information on a guanine nucleotide exchange fac-
tor with a role in the Wnt-planar cell signaling pathway
that controls cell movements in gastrulation (62) and
revealed the role of the transmembrane protein Lrig3 in
modulating signaling pathways to control neural crest for-
mation in the Xenopus embryo (63).
JOURNAL OF BIOLOGICAL CHEMISTRY 13281
REFLECTIONS: Differential Gene Expression
Using the zebrafish, we have carried out several
microarray experiments, including an as yet unpublished
study describing the transcriptome of the pineal gland in a
global way (R. Toyama, X. Chen, N. Jhawar, J. A. Epstein,
Y. Gothilf, D. Klein, and I. B. Dawid, unpublished data). In
another project, we focused on targets of the Fgf signaling
pathway in early development. Sung-Kook Hong injected
embryos with mRNA encoding Fgf or a dominant-nega-
tive form of the Fgf receptor, and the RNA populations
isolated from both classes of embryos were compared with
controls by microarray analysis. As a result of these exper-
iments, we identified two factors, Ier2 and Fibp1, whose
possible functions in embryogenesis had not been charac-
terized. Ier2 and Fibp1 proved to be involved downstream
of Fgf signaling in the establishment of left/right asymme-
try in the zebrafish (S.-K. Hong and I. B. Dawid, unpub-
lished data). This example again illustrates how differen-
tial expression analysis can assist in functional genomics
to help identify novel factors and associate novel functions
with previously described proteins.
The Future of Differential Expression Analysis
The pace of progress in biology over the past half-cen-
tury has been so rapid and the advances so profound that
predictions are bound to be wrong, especially coming
form someone whose general inclination to caution has
invariably produced overly conservative estimates of
future developments. Still, there is little doubt that differ-
ential expression analysis, increasing in range, resolution,
and precision, will continue to be an important approach
in many fields of biological research, notably in develop-
mental biology but also in other applications such as diag-
nosis of diseases and searches for targets for pharmacolog-
ical intervention. Among currently used techniques,
subtractive cloning remains a useful tool 25 years after its
initiation. DNA microarray methods have generated a
broad range of applications that likely will grow further for
the foreseeable future, especially as uses in diagnosis, hap-
lotype mapping, and other areas expand. When applied to
cDNA copies of RNA populations, new ultrahigh through-
put sequencing methods have the potential to supply the
most complete information about the genes that are
expressed in the material of interest (64). Effective com-
parisons can then be carried out between different cells
and tissues or embryonic stages and between different
metabolic or differentiation states of cells and embryos. It
seems that with this methodology we are poised to achieve
the ultimate aim of being able to describe the differential
expression of all genes in a developing organism. The con-
tinuous refinement of the description of differential gene
expression will enrich efforts to elucidate the transcrip-
13282 JOURNAL OF BIOLOGICAL CHEMISTRY
tional control mechanisms and intercellular signaling
pathways that combine to promote the differentiation
and patterning of the embryo.
A Personal Conclusion
On rereading what I have written, it appears less clearly
a memoir than some of the articles in the Reflections
series, having more the character of a thematic piece, yet I
am trying to focus on the work we have done in the area of
differential gene expression while placing our work into a
broader context. To achieve some degree of thematic
unity, I mentioned briefly or not at all other areas that
occupied my interest over the years. This also accounts for
the fact that I did not mention so far several colleagues
who featured in the development of my laboratory’s
research, such as Peter Wellauer, whose elegant electron
microscopy explored ribosomal RNA and DNA structure
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(65, 66); Susan Haynes, Brian Mozer, and Alex Mazo, who
first isolated the Drosophila trx and fsh genes (67– 69);
Paolo Di Nocera and Brian Kay, who studied repetitive
DNA elements (70, 71); Masanori Taira, who developed
our work on Lim homeodomain transcription factors
(72, 73); and Jean-Pierre Saint-Jeannet and more
recently Raymond Habas, who studied Wnt signaling
(74 –76). These and many more former and present col-
leagues have greatly enriched my life in science and
beyond.
I feel that I have been most fortunate in having the
opportunity to engage in a career of research in biology.
Looking at it from the current vantage point of single-digit
pay lines, one might think that scientists of my generation
had an easy time of it. In some ways that may well be true,
even though at the time the field seemed highly competi-
tive, and the mountain to climb toward an independent
research career appeared steep. It certainly has been a pro-
pitious time to work in biology, developmental biology in
particular; the field was in a sustained growth phase, with
exciting advances following each other in rapid succes-
sion, and broad support was available for the work in this
country and elsewhere. Now, biological research has more
the appearance of a “mature” enterprise, with obvious lim-
itations on growth; yet the excitement has not left the field,
progress is as rapid as ever, and many opportunities for
fundamental insights and for practical applications arise
continuously. It remains, I believe, a great field for a young
scientist to pursue a career as a principle investigator,
albeit not for every person who enters the field; a series of
talents is needed to succeed in this age, some of them
different from those demanded of us. The basic require-
ments for a research career, an interest in science, an
understanding of what the important questions are, and
VOLUME 284 • NUMBER 20 • MAY 15, 2009
 REFLECTIONS: Differential Gene Expression

an ability to do the “right” experiment, have not changed. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 9228 –9232
39. Liang, P., and Pardee, A. B. (1992) Science 257, 967–971
For those who have these talents, a career in science 40. Velculescu, V. E., Zhang, L., Vogelstein, B., and Kinzler, K. W. (1995) Science
remains a great choice. It certainly has been for me. 270, 484 – 487
41. Gall, J. G., and Pardue, M. L. (1969) Proc. Natl. Acad. Sci. U. S. A. 63, 378 –383
42. Pardue, M. L., and Gall, J. G. (1970) Science 168, 1356 –1358
Acknowledgments—This work was supported by the National Institutes 43. Hafen, E., Levine, M., Garber, R. L., and Gehring, W. J. (1983) EMBO J. 2,
of Health Intramural Research Program of the NICHD. I thank Michael 617– 623
Tsang for the assembly of Fig. 3. 44. Jowett, T., and Lettice, L. (1994) Trends Genet. 10, 73–74
45. Hauptmann, G., and Gerster, T. (1994) Trends Genet. 10, 266
Address correspondence to: idawid@nih.gov. 46. Gawantka, V., Pollet, N., Delius, H., Vingron, M., Pfister, R., Nitsch, R., Blu-
menstock, C., and Niehrs, C. (1998) Mech. Dev. 77, 95–141
47. Kudoh, T., Tsang, M., Hukriede, N. A., Chen, X., Dedekian, M., Clarke, C. J.,
REFERENCES Kiang, A., Schultz, S., Epstein, J. A., Toyama, R., and Dawid, I. B. (2001) Genome
1. Dawid, I. B. (2006) Curr. Biol. 16, R391–R392 Res. 11, 1979 –1987
2. Dawid, I. B. (1965) J. Mol. Biol. 12, 581–599 48. Tsang, M., Friesel, R., Kudoh, T., and Dawid, I. B. (2002) Nat. Cell Biol. 4,
3. Dawid, I. B. (1966) Proc. Natl. Acad. Sci. U. S. A. 56, 269 –276 165–169
4. Brown, D. D., and Dawid, I. B. (1968) Science 160, 272–280 49. Furthauer, M., Lin, W., Ang, S. L., Thisse, B., and Thisse, C. (2002) Nat. Cell
5. Gall, J. G. (1968) Proc. Natl. Acad. Sci. U. S. A. 60, 553–560 Biol. 4, 170 –174
6. Spradling, A. C., and Mahowald, A. P. (1980) Proc. Natl. Acad. Sci. U. S. A. 77, 50. Kudoh, T., Wilson, S. W., and Dawid, I. B. (2002) Development (Camb.) 129,
1096 –1100 4335– 4346
7. Lockard, R. E., and Lingrel, J. B. (1969) Biochem. Biophys. Res. Commun. 37, 51. Bolton, E. T., and McCarthy, B. J. (1962) Proc. Natl. Acad. Sci. U. S. A. 48,
204 –212 1390 –1397

Downloaded from www.jbc.org by guest, on October 16, 2010

8. Lane, C. D., Marbaix, G., and Gurdon, J. B. (1971) J. Mol. Biol. 61, 73–91 52. Nygaard, A. P., and Hall, B. D. (1963) Biochem. Biophys. Res. Commun. 12,
9. Suzuki, Y., and Brown, D. D. (1972) J. Mol. Biol. 63, 409 – 429 98 –104
10. Morrow, J. F., Cohen, S. N., Chang, A. C., Boyer, H. W., Goodman, H. M., and 53. Southern, E. M. (1975) J. Mol. Biol. 98, 503–517
Helling, R. B. (1974) Proc. Natl. Acad. Sci. U. S. A. 71, 1743–1747 54. Wahli, W., Dawid, I. B., Wyler, T., Jaggi, R. B., Weber, R., and Ryffel, G. U.
11. Maniatis, T., Kee, S. G., Efstratiadis, A., and Kafatos, F. C. (1976) Cell 8, (1979) Cell 16, 535–549
163–182 55. Kafatos, F. C., Jones, C. W., and Efstratiadis, A. (1979) Nucleic Acids Res. 7,
12. Sanger, F., Nicklen, S., and Coulson, A. R. (1977) Proc. Natl. Acad. Sci. U. S. A. 1541–1552
74, 5463–5467 56. Schena, M., Shalon, D., Davis, R. W., and Brown, P. O. (1995) Science 270,
13. Maxam, A. M., and Gilbert, W. (1977) Proc. Natl. Acad. Sci. U. S. A. 74, 467– 470
560 –564 57. Lipshutz, R. J., Morris, D., Chee, M., Hubbell, E., Kozal, M. J., Shah, N., Shen, N.,
14. Williams, J. G., and Lloyd, M. M. (1979) J. Mol. Biol. 129, 19 –35 Yang, R., and Fodor, S. P. (1995) BioTechniques 19, 442– 447
15. Rowekamp, W., and Firtel, R. A. (1980) Dev. Biol. 79, 409 – 418 58. Baldessari, D., Shin, Y., Krebs, O., Konig, R., Koide, T., Vinayagam, A., Fenger,
16. Zimmermann, C. R., Orr, W. C., Leclerc, R. F., Barnard, E. C., and Timberlake, U., Mochii, M., Terasaka, C., Kitayama, A., Peiffer, D., Ueno, N., Eils, R., Cho,
W. E. (1980) Cell 21, 709 –715
K. W., and Niehrs, C. (2005) Mech. Dev. 122, 441– 475
17. Kramer, R. A., and Andersen, N. (1980) Proc. Natl. Acad. Sci. U. S. A. 77,
59. Sinner, D., Kirilenko, P., Rankin, S., Wei, E., Howard, L., Kofron, M., Heasman,
6541– 6545
J., Woodland, H. R., and Zorn, A. M. (2006) Development (Camb.) 133,
18. Baulcombe, D. C., and Key, J. L. (1980) J. Biol. Chem. 255, 88907– 88913
1955–1966
19. Lasky, L. A., Lev, Z., Xin, J. H., Britten, R. J., and Davidson, E. H. (1980) Proc.
60. Das, B., Cai, L., Carter, M. G., Piao, Y. L., Sharov, A. A., Ko, M. S., and Brown,
Natl. Acad. Sci. U. S. A. 77, 5317–5321
D. D. (2006) Dev. Biol. 291, 342–355
20. Dworkin, M. B., and Dawid, I. B. (1980) Dev. Biol. 76, 449 – 464
61. Mathavan, S., Lee, S. G., Mak, A., Miller, L. D., Murthy, K. R., Govindarajan,
21. Wahli, W., Ryffel, G. U., Wyler, T., Jaggi, F. B., Weber, R., and Dawid, I. B. (1978)
K. R., Tong, Y., Wu, Y. L., Lam, S. H., Yang, H., Ruan, Y., Korzh, V., Gong, Z.,
Dev. Biol. 67, 371–383
Liu, E. T., and Lufkin, T. (2005) PLoS Genet. 1, 260 –276
22. Davidson, E. H. (1986) Gene Activity in Early Development, Academic Press,
62. Tanegashima, K., Zhao, H., and Dawid, I. B. (2008) EMBO J. 27, 606 – 617
New York
63. Zhao, H., Tanegashima, K., Ro, H., and Dawid, I. B. (2008) Development
23. Grunstein, M., and Hogness, D. S. (1975) Proc. Natl. Acad. Sci. U. S. A. 72,
(Camb.) 135, 1283–1293
3961–3965
24. Britten, R. J., and Davidson, E. H. (1976) Proc. Natl. Acad. Sci. U. S. A. 73, 64. Mardis, E. R. (2008) Annu. Rev. Genomics Hum. Genet. 9, 387– 402
415– 419 65. Wellauer, P. K., and Dawid, I. B. (1978) J. Mol. Biol. 126, 769 –782
25. Britten, R. J., and Kohne, D. E. (1968) Science 161, 529 –540 66. Wellauer, P. K., and Dawid, I. B. (1973) Proc. Natl. Acad. Sci. U. S. A. 70,
26. Sargent, T. D., and Dawid, I. B. (1983) Science 222, 135–139 2827–2831
27. Hedrick, S. M., Cohen, D. I., Nielsen, E. A., and Davis, M. M. (1984) Nature 308, 67. Haynes, S. R., Dollard, C., Winston, F., Beck, S., Trowsdale, J., and Dawid, I. B.
149 –153 (1992) Nucleic Acids Res. 20, 2603
28. Hedrick, S. M., Nielsen, E. A., Kavaler, J., Cohen, D. I., and Davis, M. M. (1984) 68. Haynes, S. R., Mozer, B. A., Bhatia-Dey, N., and Dawid, I. B. (1989) Dev. Biol.
Nature 308, 153–158 134, 246 –257
29. Sargent, T. D. (1987) Methods Enzymol. 152, 423– 432 69. Mazo, A. M., Huang, D. H., Mozer, B. A., and Dawid, I. B. (1990) Proc. Natl.
30. Rosa, F., Sargent, T. D., Rebbert, M. L., Michaels, G. S., Jamrich, M., Grunz, H., Acad. Sci. U. S. A. 87, 2112–2116
Jonas, E., Winkles, J. A., and Dawid, I. B. (1988) Dev. Biol. 129, 114 –123 70. Kay, B. K., and Dawid, I. B. (1983) J. Mol. Biol. 170, 583–596
31. Meyer, M. F., and Kreil, G. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 4543– 4547 71. Di Nocera, P. P., Digan, M. E., and Dawid, I. B. (1983) J. Mol. Biol. 168, 715–727
32. Varki, A. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 4523– 4525 72. Taira, M., Jamrich, M., Good, P. J., and Dawid, I. B. (1992) Genes Dev. 6,
33. Pummill, P. E., Achyuthan, A. M., and DeAngelis, P. L. (1998) J. Biol. Chem. 356 –366
273, 4976 – 4981 73. Taira, M., Otani, H., Saint-Jeannet, J.-P., and Dawid, I. B. (1994) Nature 372,
34. Jamrich, M., Sargent, T. D., and Dawid, I. B. (1987) Genes Dev. 1, 124 –132 677– 679
35. Winkles, J. A., Sargent, T. D., Parry, D. A., Jonas, E., and Dawid, I. B. (1985) Mol. 74. He, X., Saint-Jeannet, J.-P., Woodgett, J. R., Varmus, H. E., and Dawid, I. B.
Cell. Biol. 5, 2575–2581 (1995) Nature 374, 617– 622
36. Rosa, F. M. (1989) Cell 57, 965–974 75. Saint-Jeannet, J.-P., He, X., Varmus, H. E., and Dawid, I. B. (1997) Proc. Natl.
37. Ko, M. S. (1990) Nucleic Acids Res. 18, 5705–5711 Acad. Sci. U. S. A. 94, 13713–13718
38. Soares, M. B., Bonaldo, M. F., Jelene, P., Su, L., Lawton, L., and Efstratiadis, A. 76. Habas, R., Dawid, I. B., and He, X. (2003) Genes Dev. 17, 295–309

MAY 15, 2009 • VOLUME 284 • NUMBER 20 JOURNAL OF BIOLOGICAL CHEMISTRY 13283