Cell, Vol.

100, 27–40, January 7, 2000, Copyright 2000 by Cell Press

Development: The Natural History of Genes Review

Matthew P. Scott astonishing natural biological order, creating optimism

Departments of Developmental Biology and Genetics about discovering new kinds of medicine. Natural bio-
Howard Hughes Medical Institute logical order is familiar from the near universality of
Stanford University School of Medicine metabolic pathways and the genetic code. Develop-
Stanford, California 94305-5329 mental mechanisms underlying the morphology of di-
verse creatures are also remarkably universal—more
than anyone had dreamed. There had been concern
Introduction that molecules and regulatory mechanisms would vary
Developmental biology is a powerful way to learn how tremendously through the animal kingdom, and that
genes and proteins operate in their natural habitats. In hundreds of separate signaling systems would be oper-
a growing animal, genes come to life. Just as animal ative, but neither idea appears to be true. Darwin quoted
structure becomes more understandable when ecology, von Baer: “The feet of lizards and mammals, the wings
physiology, and behavior are taken into account, genes and feet of birds, no less than the hands and feet of
and proteins are best understood in the context of devel- man, all arise from the same fundamental form” (Darwin,
opmental biology—a natural history of genes. 1871). In one sense von Baer’s “form” is the shape of
Developmental biology will help to exploit the current proteins such as FGF and Hox and Hedgehog. The de-
flood of descriptive knowledge from gene hunters. The velopmental mechanisms of each experimental organ-
gene flood is reminiscent of the first time plants and ism used to seem impressively distinct; recently the
animals were extensively described and classified. Car- emphasis has been on unifying themes and common
olus Linnaeus (1707–1778), famed as “God’s registrar,” genes. The field of development and evolution is recon-
was a botanist and professor of medicine at the Univer- ciling these two conflicting view by finding out how a
sity of Uppsala (Goerke, 1973). Linnaeus confronted a modest number of protein types can build a plethora of
chaotic natural world and organized it, inventing the animal types.
system of binomial nomenclature (Linnaeus, 1735). Not Linnaeus worked at a time when it was important (and
all the relationships were clear. Observing organisms in still news) to affirm that every animal comes from an egg.
terms of their ecology and physiology was helpful in We have come a long way. We consider here current
sorting them out. Similar organizing power comes from knowledge of developmental biology, focusing on mole-
learning how proteins work together in pathways and cules that have maintained their connection to a process
complexes, connecting proteins to particular tissues or or pathway for half a billion years or so. Main find-
changes in cell properties. Newfound genes can often ings include the following: (1) Different cell types tran-
be connected to known processes or pathways because scribe different genes. Cytologically indistinguishable
mutations in the new genes give rise to familiar pheno- cells can have elaborate spatial and temporal gene ex-
types. pression patterns. (2) Although DNA rearrangements
We have much to learn about the origins of families and deletions are used in specific instances for differen-
of proteins and how they became dedicated to their tial gene function, transcription is a more common type
present day tasks. Linnaeus, also facing daunting com- of gene regulation. (3) Transient bursts of transcription
plexity, began his classification with elegant simplicity: factor function, or of signals, lead to lasting changes
“Minerals grow, plants grow and live, animals grow, live, in cell phenotype, often affecting generations of cells.
and have feeling.” The first edition of Systema Naturae Changes in chromatin structure, often reversible, per-
in 1735 listed 549 species; by the eleventh edition, 5897 petuate earlier regulatory decisions. (4) Localized RNA
species were represented. Not all of them were in con- molecules create differences in egg cytoplasm and
tigs. Linnaeus’ students and colleagues formed a world- within cells for asymmetric cell divisions. Asymmetric
wide web ([Link]), supplying him with a constant flow cell division is crucial for both stem cells and differentia-
of seeds, plants, eggs, animal skins, skeletons, descrip- tion. (5) Transcription factors regulate batteries of genes.
tions, and lore. Animals were divided into six categories: Combinations of regulators govern most differentiation
quadrupeds, birds, amphibia (including reptiles), fishes, events, so the same regulator can be used repeatedly.
insects, and worms (including most invertebrates). An- The outcome depends on what other regulators are act-
other category, the “Paradoxa” contained animals of ing in concert or opposition. (6) Embryos are remarkably
uncertain type, including satyrs, phoenixes, and peli- flexible in adapting to different cell numbers, environ-
cans. Linnaeus promoted acceptance of his system by mental conditions, and altered gene function. Cells are
the clever political tactic of naming plants after eminent sensitive to the character of their neighbors and tend to
biologists. (Perhaps instead of naming categories of die or change if the neighboring cells are inappropriate.
proteins cadherins or cyclins, we could have varmins They are also sensitive to the number of neighbors,
and bishins, nussvolins and wieschins, and lewins.) the “community effect.” (7) Many signals act over short
Linnaeus was first to emphasize the relationship be- distances, perhaps reflecting their origins in organisms
tween humans and apes, causing an outcry. Much more with few cells. (8) Systems of checks and balances keep
startling relationships between humans and animals be- powerful signals in check. Intracellular and extracellular
came evident upon investigating the mechanisms that antagonists that limit the range or potency of the signals
build them. During the past 20 years developmental biol- affect most or all signals. Some antagonists are induc-
ogy has been enriched by a profound recognition of ible by their signals, thus providing feedback responses
Cell
28

in proportion to the initial intensity of the signal. Secreted cells, some nerve cells, and others remain reproductive
antagonists could have evolved as a form of chemical cells?” (Sander, 1985). To these issues we must add
warfare between organisms. (9) Cells are responsive to the problem of pattern formation: how can a right hand
signals or to transcription factors at some times but not reliably be made to mirror the left?
others, a property known as competence. Also, cells That transcription is an important level of control was
respond to a particular signal differently, depending on clear decades ago from studies of bacteria. The earliest
their history. History affects signal effectiveness, making views of differential transcription in an eukaryote came
certain genes responsive to signals by localizing mole- from watching polytene chromosomes from fly salivary
cules within cells or by polarizing, shaping, or moving glands change their puffing patterns with developmental
the cell. (10) Spatial and temporal regulation of the cell time (Beermann, 1952). The puff patterns, showing visi-
cycle is crucial to forming organized tissues, allowing ble changes at 194 loci, are indicators of differential
restorative events, and maintaining stem cells. (11) Cell activation of genes in time, in part under the control of
death and aging are genetically governed processes, steroid hormones (Ashburner, 1990). This was the first
precisely scripted and regulated during normal develop- direct demonstration of differential gene activity in re-
ment and alterable by disease or injury. (12) Most of the sponse to a steroid.
proteins that regulate development have been con- More insights into multicellular development came
served through evolution. Often related genes have simi- when steady-state populations of RNA molecules were
lar functions in vastly different animals. measured in different cell types using solution hybrid-
I have organized this review using the control of yeast ization. In “R0T” curve experiments, hybridization rates
mating–type to frame the discoveries of the past few were dependent on initial concentration (R0) and time
decades. The ramifications of the MAT locus encom- (T). Elegant kinetic analyses were used to measure the
pass phenomena common to many developing organ- complexity of the RNA and compare sequences from a
isms: cell differentiation, differential gene transcription, variety of cells (Galau et al., 1974; Hough et al., 1975).
DNA rearrangement, localized RNA molecules, coordi- This allowed the recognition of classes of RNAs that
nate regulation of gene batteries, inheritance of deter- varied tremendously in their abundance, the detection
mined cell states, gene silencing, signals and sex, cell of differences in RNA populations between cells of dif-
division, and cell asymmetry. Modern developmental ferent types, and detection of the transcription of repeti-
biology has its roots in pioneer subjects of molecular tive versus single-copy DNA. The favored human cell
biology. B. Polisky has proposed that the phage ␭ ge- mix, the brain, took honors for the most complex RNA
nome would be the most entertaining 50 kb of DNA to population.
have along if one was to be marooned on a desert island In the yeast Saccharomyces, transcription patterns
with nothing to do but experiments. The ␭ lifecycle (Hen- distinguish three cell types. The two haploid mating-
drix et al., 1983) is extraordinarily fascinating for a devel- types are named a and ␣; the third type of cell is a
opmental biologist, with beautifully balanced regulation diploid heterozygote, a/␣. The DNA sequence of the two
of operons, DNA-binding proteins, competing actions haploid genomes is nearly the same. Certain genes are
of different regulators, and the ordered assembly of a uniquely active in one or two of the three cell types,
complex infectious particle. Similarly, yeast biology is a constituting one basis for cell differentiation. RNA tran-
useful bridge to the processes of multicellular collabora- scription is one measure of the differences. Of 6164
tion. Much developmental biology is included in accom- genes analyzed, 32 genes were found to be differentially
panying reviews, including bacterial development, stem expressed according to mating-type (Galitski et al.,
1999). Seventeen genes changed with ploidy, regardless
cells, neurogenesis, axon pathfinding, plant biology, and
of mating-type. Haploid ␣ cells have 32 genes tran-
the cell cycle. A major review of induction, gradients,
scribed at more than 2-fold higher levels than in haploid
and organizers, among other topics, is provided in Fra-
a cells. Haploid a cells have 50 genes transcribed at
ser and Harland (2000 [this issue of Cell]). I will not
more than 2-fold higher levels than in haploid ␣ cells. The
emphasize those topics nor the revolution in under-
situation is similar in diploid cells. Many of the haploid-
standing plant development (Hake and Meyerowitz,
specific genes encode products that allow the cells to
1998) here. A celebration of pioneers such as Theodor
mate.
Boveri, Hans Driesch, Oscar Hertwig, Wilhelm Roux,
Remarkable changes in phenotype can therefore in-
Hans Spemann, Walter Sutton, August Weismann, Ed-
volve a reasonably small fraction of the genes. As more
mund Wilson, and others is in Horder et al. (1985). experiments are done, we will learn how many genes
do not change their transcription rate at all. In the yeast
Transcribing Different Genes experiments, 2147/6164 gene transcription levels dif-
The central mystery of development was simply stated fered by less than 10% between a and ␣. Sixteen hun-
by August Weismann in 1883: “How is a single germ dred eighty-eight genes varied between haploid and
cell capable of reproducing the entire body with all its diploid ␣ cells. During yeast sporulation, approximately
detail?” Morgan connected this basic question with ge- 1000 of the approximately 6200 genes changed their
netics in his Nobel lecture of 1935: “If as is generally levels of transcription (Chu et al., 1998). Many genes
implied in genetic work (although not often explicitly may be active only under certain nutritional, lifecycle,
stated), all of the genes are active all of the time, and if or temperature conditions. Indeed the genes that are
the characters of the individual are determined by the active regardless of conditions may be small. Gene
genes, then why are not all the cells of the body exactly arrays applied to one particular cell culture change give
alike? . . . Every cell comes to contain the same kind of a hint of what may be expected during development.
genes. Why then is it that some cells become muscle When a fibroblast in culture is exposed to serum, more
Review
29

than 500 genes of 8613 examined substantially change Temporal control of gene expression is implicit in the
their transcription level (Iyer et al., 1999). The types of time it takes to produce a functional gap protein or pair-
genes that become active suggest that exposure to se- rule protein; downstream genes will become active only
rum signals the fibroblast to engage in wound repair, as after activating transcription factors are produced. The
it would in response to contact with serum in a wounded cell cycle is controlled by oscillating levels of functional
human. cyclin/CDC complexes, controlled in turn by rates of
phosphorylation and protein degradation (see review by
Spatial and Temporal Control of Gene Expression Nurse, 2000 [this issue of Cell]). Any one clock, like
Another revelation came with the invention of in situ the cell cycle oscillator, has the potential to affect and
hybridization for detecting RNA fixed in tissues. Spatial therefore schedule other events in development. The
differences in patterns of transcription, largely con- transcription of certain genes is linked to circadian
cealed when solution hybridization or RNA blots were rhythm, including c-hairy1, whose transcription cycles
used, suddenly became visible. Fields of apparently ho- in time with the formation of somites (Pourquie, 1998).
mogeneous cells took on great character as stripes and Other types of timers rely on the dilution or exhaustion
spots of RNA expression came to light. of factors, as in the embryonic initiation of zygotic tran-
The Drosophila segmentation genes (Nusslein-Vol- scription in flies or frogs that occurs as the population
hard and Wieschaus, 1980) have become a favorite ex- of nuclei becomes sufficiently large. Patterns of cell divi-
ample of patterned gene expression. At the stage when sion are controlled in time by heterochronic genes, as
about 6000 cells on the embryo’s surface enclose the is clear from fascinating work on C. elegans. Mutations
yolk, and most of the cells look the same, beautiful in heterochronic genes lead to premature or postponed
patterns of striped gene expression are detected for sets of cell divisions. One of the heterochronic genes is
some of the gap, pair-rule, and segment-polarity genes. related to circadian rhythm genes found first in flies
Most of the gap and pair-rule genes encode transcrip- (Jeon et al., 1999), suggesting a link between seemingly
tion factors that participate in a cascade of regulators different types of timing mechanisms.
that refines the stripes until they are just one cell wide.
Gap proteins are produced in broad stripes under the DNA Rearrangement to Change Cell Type
control of localized regulators provided maternally. Gap Yeast cells can change mating-type every generation,
proteins regulate pair-rule (and Hox) transcription, and in thus having the advantages of spore formation in starva-
turn pair-rule proteins regulate striped segment-polarity tion conditions and of recombination. The differences
gene transcription. As in yeast mating–type, combina-
between the two types of haploid yeast cells are due to
tions of enhancer-binding proteins govern transcription,
a locus called MAT that has unusual properties (Haber,
in this case often combinations of different gap or pair-
1998). Two alleles, a and ␣, contain alternative versions
rule proteins (Rivera-Pomar and Jackle, 1996). Because
of a 700 bp sequence. The locus is changed from one
gap gene expression domains occur in broad peaks,
version to the other by a programmed DNA sequence
graded toward anterior and posterior, and because they
change that transfers a sequence from either of two
overlap, each location along the anterior–posterior (A–P)
donor sites to the MAT locus, inserting different “cas-
axis has particular amounts and combinations of tran-
settes.” The genes at the donor loci, HML and HMR, are
scription factors. Those combinations govern where
kept silent by a chromatin protein repression system.
pair-rule genes become active, and also affect gap gene
A yeast cell buds to reproduce; a mother produces a
transcription in feedback loops. Some proteins repress,
daughter cell that is readily distinguishable morphologi-
some activate, and some may do both. Ubiquitous co-
cally. After such a cell division, the daughter cell will
factors assist in the regulation, as they do in yeast.
The key to integrating the upstream regulators lies in divide again without switching cell types, but the mother
enhancer sequences, where the arrangement of bound cell will divide to produce two cells, both of which have
proteins determines the outcome. A pair-rule gene may switched their mating-type. The resulting four cells will
have several enhancers, each one governing one or constitute two mating pairs, which will often form two
more stripes. The gene may be “on” in response to diploid cells. Switching ability, an aspect of differentia-
any of several different combinations of regulators. For tion itself, is precisely controlled so that it occurs only
example, typically such a “successful” combination of in certain cell cycles, coordinated with cell lineage.
activating regulators occurs within each of seven stripes Switching occurs only during the G1 phase of the cell
of cells along the A–P axis. If an activating combination cycle, which ensures that both cells formed at the next
of proteins resides on any one of the enhancers, the division will have the same genotype. Switching is initi-
gene will be on, while repressing combinations of pro- ated by production of the HO nuclease only in cells that
teins may be present on other enhancers. At some posi- will switch; the nuclease creates a DNA break to start
tions along the A–P axis, no enhancer will host an acti- the recombination event.
vating protein combination. How a group of proteins While the MAT locus has special properties, echoes
bound to an integrating enhancer battle it out and then of those properties are heard in governance of immuno-
send a consensus signal to the promoter remains an globulin gene expression and switching (Cedar and
active area of research (Mannervik et al., 1999). Some Bergman, 1999; Oettinger, 1999). Again silent loci are
repressors act by sterically hindering binding of activa- rearranged to form an active gene, and the unused al-
tors, while others actively interfere with bound activa- leles are silent or deleted (Stavnezer, 1996). Another
tors, perhaps by competing for contacts with the “basal” DNA change occurs in differentiation of egg follicle cells
transcription apparatus. One yearns for a microscopic in Drosophila. The chorion genes must produce stag-
picture of gene expression in action. gering amounts of eggshell protein in a short period of
Cell
30

time. They manage it by undergoing a cell type–specific chromatin complex constitutes a form of cellular mem-
overreplication of the genes (Calvi and Spradling, 1999). ory. The still mysterious workings of the Pc-G genes
Trypanosomes have the challenge of tricking the im- have immense relevance to plans for modifying gene
mune system of the host. They have in their genome a activities to promote healing, because stimulating re-
large assortment of alternative versions of a surface generation processes will very likely require derepres-
protein, and they periodically rearrange their genes to sion of genes normally active only during embryonic
express a different version (Cross, 1996). development. Chromatin protein complexes that op-
Gene rearrangement, in which part of the genome is pose repression, such as the Trithorax group (Trx-G)
excised, appears to be limited to special occasions of proteins first found in Drosophila, may be useful in
when, for example, a large repertoire of related genes is manipulating genes for reactivation and healing. One of
created by combinatorial reassortment of gene pieces. the members of the Trx-G is brahma, which encodes
Evidence for retention of a complete genome in differen- an ATPase subunit of a large complex necessary for
tiated cells is based in part on experiments in which proper homeotic gene transcription. The Brahma com-
somatic nuclei are transplanted into eggs or embryos plex is related to the Swi/Snf complex as well as to other
and their ability to grow into animals is assessed. Some activating chromatin complexes found in yeast. The HO
notable successes—in growing complex animals—have locus, encoding the endonuclease required for mating-
shown that somatic nuclei can do a lot (Gurdon and type switching, is among the genes activated by Swi/
Uehlinger, 1966). The difficulty of defining the “differenti- Snf. A plethora of large chromatin protein complexes
ated” character of any particular nucleus leaves the sta- battles for control of genes; how they interact with se-
bility of the genome question incompletely resolved, but quence-specific DNA-binding proteins is a research
it is clear that adult cells exist that can be “cloned” into area of great importance (Mannervik et al., 1999).
animals (Wakayama and Yanagimachi, 1999). An additional hurdle for gene activation, not present
in Drosophila or C. elegans, is the clamping off of gene
Silent Genes expression resulting from methylation of DNA, an inter-
Needham (1942, p. 101) noted that “one of the most esting process that is also linked to cellular memory.
fundamental processes in development consists in the The imprinting of genes, so that they are selectively
closing of doors, i.e. in determination, in the progressive accessible for activation depending on whether they are
restriction of the possible fates.” In most cell types, only transmitted from the mother or father, is among the most
a fraction of genes is active at detectable levels. The dramatic kinds of regulation by methylation (Reik and
mechanisms for keeping genes quiescent must be heri- Walter, 1998).
table, yet reversible in at least some cells such as the
germline. Yeast MAT regulation has been a useful source
Localized Intracellular mRNA in Differentiation
of information about how genes are kept off, because
The regulation of the HO nuclease during yeast cell type
the HML and HMR loci in fact contain complete genes
switching has been traced to a localized mRNA that
that can become active under the mischievous control
codes for a regulator of HO. HO transcription is regulated
of a biologist. Normally the information at HML or HMR
by, among other things, a protein called Swi5p. Swi5p
is expressed only after it is copied into the MAT locus.
is found in mother cell nuclei, due to the regulation of
The HML and HMR genes are flanked by “I” and “E”
SWI5 transcription by the Ash1p transcription factor,
sequences that silence the gene in cis. Proper repres-
which may directly repress SWI5 in daughter cells. In
sion requires histones, the DNA origin replication com-
this way the restriction of HO function to precise cells
plex, acetylases and deacetylases, four Silent Informa-
in the lineage has been reduced to understanding how
tion Repressor (SIR) proteins, and chromatin assembly
complexes (Haber, 1998). By analogy to the visibly com- Ash1p is restricted to daughter cells. ASH1 mRNA is
pact, relatively silent chromatin of other eukaryotes, the localized to one region of cytoplasm in mother cells prior
3 kb of DNA that is silenced by this entourage of proteins to cell division (Bobola et al., 1996; Sil and Herskowitz,
is described as heterochromatin. This 3 kb is transcrip- 1996). The localization is controlled by actin and the
tionally silent, resistant to endonuclease, and is associ- myosin-like Myo4p. Presumably ASH1 RNA localization
ated with hypoacetylated histones. SIR proteins also is one aspect of the many structural differences between
affect mitotic chromatin structure and rDNA recombina- mother cell and bud.
tion, and are necessary for DNA ligation. These findings Localized mRNAs are absolutely critical for the devel-
emphasize the links between chromatin structure and opment of many organisms, most notably in the mater-
both transcription and DNA modification. nal dowry provided to oocytes (Bashirullah et al., 1998).
In multicellular developmental contexts, gene silenc- Dramatic examples include bicoid RNA localized in head
ing also depends on chromatin protein complexes. The primordia to polarize the A–P axis of the Drosophila egg,
complexes may activate or repress genes, and the regu- and nanos RNA, which is a posterior determinant. The
lation can be reversible. Genes of the Polycomb group encoded proteins are often transcription factors, RNA-
(Pc-G) are required to repress Hox and other genes in binding proteins, or both. Some RNA molecules are
Drosophila (Pirrotta, 1998). Related mammalian genes moved to specific locations within cells, including those
are implicated in Hox control, skeletal development, and coding for many cytoskeletal proteins. The relevant con-
hematopoiesis (Gould, 1997). Proteins encoded by Pc-G trol elements, usually in the 3⬘ untranslated regions,
genes form complexes that maintain genes in repressed work in multiple species, suggesting ancient origins for
states. In the fly embryo, the initial repression of genes the elements. Asymmetric cell divisions of the sort that
by transient spatial regulators is replaced by Pc-G main- occur in yeast also occur frequently in neurogenesis,
tenance systems. As more cells are produced, the Pc-G and again localized mRNAs or proteins such as prospero
Review
31

are prominent in controlling the different dowries of the type switching, promoters and enhancers of genes acti-
two daughter cells (Hawkins and Garriga, 1998). vated by myogenic proteins are assembly sites for pro-
tein complexes (Firulli and Olson, 1997). Once the right
Regulation of Batteries of Genes genes are activated, the cells can start to have fun,
by Transcription Factors mating or differentiating.
DNA rearrangement, though found in diverse organisms,
is still viewed as the exception to having identical Wild Genes and Mating Habits
genomes in every cell. Transcription appears to be a The romance between haploid yeast cells of opposite
much more prevalent level of regulation than gene re- mating-type begins with the differential gene expression
arrangement. described above. Yeast cells of opposite mating-type
The transcription factors encoded by MAT are well must find each other to combine their haploid genomes
understood, but new information is providing a more to form diploids. The process occurs through a system
complete picture of what they do (Johnson, 1998). MAT␣ of signals. The a cells secrete a protein signal called a
is active in ␣ cells and codes for two proteins, MAT␣1p factor that binds to the Ste2p receptor on ␣ cells. When
and MAT␣2p. MAT␣1p is a transcription factor that acti- the signal is received, the receptor, a transmembrane
vates the transcription of ␣-specific genes. MAT␣2p, a protein, triggers a signal transduction process that cul-
repressor protein that contains a homeodomain, re- minates in changes in cell shape and formation of sur-
presses a-specific genes. In a cells, a-specific genes face structures suitable for mating. Concomitantly, ␣
are activated by STE12p transcription factor, which is factor protein secreted by ␣ cells is received by the
made by a and ␣ cells. The combined actions of Ste12p Ste3p receptor on a cells, and triggers the appropriate
and Mcm1p, a protein that is made in all three cell types, differentiation events for mating in those cells. The two
turns on a-specific genes. a-specific genes are not ac- signals and their two receptors are transcribed in the
tive in ␣ cells despite the presence of Ste12p and proper haploid cell types under the control of MAT al-
Mcm1p, due to the MAT␣2p repressor. leles. In ␣ cells, MAT␣1p, in combination with the non–
Yeast MAT products work together with other pro- cell type specific transcription factor Mcm1p, activates
teins. For example, repression by MAT␣2p in a cell re- the ␣-specific genes’ ␣ factor and STE2, while MAT␣2p
quires Mcm1p and a repressor composed of Tup1p and (working with Mcm1p, Tup1p, and Ssn6p) represses
Ssn6p, all proteins that are produced in a, ␣, and a/␣ the a-specific genes’ a factor and Ste3p. In a cells, the
cells. The other possible resident at the MAT locus, a-specific genes are constitutive and the ␣-specific genes
MATa, is active in a and a/␣ cells and codes for MATa1p. are silent because MAT␣1 is repressed by MATa1p.
In diploids MATa1p cooperates with MAT␣2p and the Mating behavior in animals involves evolutionary
Ssn6/Tup1 repressor to repress haploid-specific genes flourishes ranging from the huge antlers of Irish elk to
and MAT␣1. The complex binds to a target sequence that the elaborate nests of bowerbirds. Genes controlling
is distinct from the sequence recognized by MAT␣2p sex differentiation also exhibit rapid evolution and a
alone. Thus, Mcm1p is particularly interesting, since remarkable range of mechanisms. Intensive studies of
it acts as an activator in a cells and as a repressor in sexual differentiation in worms and flies have provided
the other two cell types. When neither MAT allele is exceptionally well-understood pathways (Cline and
active, the default state is a, since the relevant target Meyer, 1996; Marin and Baker, 1998) and major progress
genes are constitutive without repressor around. In this has been made with mammals (Swain and Lovell-Badge,
situation ␣-specific genes are silent due to the absence 1999). In flies and worms, the X chromosome to au-
of MAT␣1p. tosome ratio is measured with known counting ele-
From the MAT studies, some generalities emerge ments, setting in motion a cascade of RNA splicing and
(Johnson, 1998): transcription factors are fairly ineffec- transcription regulatory events that has been traced all
tive alone but powerful in complexes, the DNA acts as the way to the production of differentiation products
a nucleation site for assemblies of protein complexes, such as yolk protein. Although most of the molecules
and proteins (like scientists) can have positive or nega- involved in controlling sexual differentiation and dosage
tive effects depending on their collaborators. compensation differ between worms, flies, and mam-
With the advent of recombinant DNA, specific genes mals, some of the genetic logic is similar. Critical ele-
could be assessed in differentiated cell types. The myo- ments for sex determination, in particular Sry, have been
genic transcription factors are a notable case of coordi- identified on the mouse and human Y chromosome, and
nate gene regulation. In muscle cells transcripts for all now will come the filling in of the pathway between this
the structural proteins appear with similar kinetics upon most upstream regulatory element and sexual differenti-
stimulation by activating genes such as myogenin and ation. Elaborate dosage compensation systems are nec-
MyoD in susceptible cell types. We now recognize two essary to provide the developing animal with the proper
main families of proteins that stimulate muscle develop- ratio of X chromosome products and autosome-derived
ment: the bHLH family that includes myogenin and products (Lyon, 1999). The mechanisms are quite differ-
MyoD, and the myocyte-enhancing factor (MEF) family ent in mammals, flies, and worms, with one X chromo-
(Yun and Wold, 1996). Members of the two families co- some inactivated in mammalian females, transcription
operate, in both vertebrates and invertebrates (Baylies heightened on one X chromosome in male flies, and
et al., 1998), to promote muscle differentiation by acti- lowered transcription from one X chromosome in worm
vating muscle-specific genes. The regulation of muscle hermaphrodites. Different chromatin regulators have
differentiation has parallels with regulation in the growth been adapted, different ones by different organisms, in
of the peripheral nervous system. As in yeast mating– order to regulate sex-specific levels of gene expression.
Cell
32

Sex determination is often noted as an example of repeatedly. Early in Drosophila embryogenesis, wg is

rapid evolution, and indeed the organization of hetero- activated by pair-rule transcription factors in transverse
typic chromosomes and the regulators of sexual differ- stripes that are located near the center of each segment
entiation appear to change quickly (Marin and Baker, primordium. Maintenance of wg transcription requires
1998). However, some regulators are conserved, and a Hedgehog signal originating from stripes of cells just
more parallels will probably come to light. The fly dou- posterior to each wg stripe. wg function is necessary
blesex gene encodes a transcription factor that controls for formation of segmental divisions, for keeping cells
sex-specific gene products. Alternative splicing of dsx alive, and for formation of the proper segmental pattern
RNA causes production of one protein form in males of denticles (bristles) on the surface of the cuticle. Wg
and a different protein form in females. In worms the protein carries a signal to the underlying visceral meso-
mab-3 gene, which controls sex determination, encodes derm cells to subdivide the mesoderm into repeating
a Dsx-related protein (Raymond et al., 1998). units. Wg is used again in the formation of appendages.
The small clusters of cells set aside during embryogene-
Animal Communication Systems sis to form legs and wings are determined in their fates,
The yeast signaling proteins have characteristics similar and spatially localized, by the intersection of transverse
to signaling proteins involved in development of multi- stripes of Wg with longitudinal stripes of the TGF␤ mole-
cellular animals. Indeed, gonadotrophin releasing hor- cule Dpp. Homeotic genes, which govern segment-spe-
mone (GnRH) is similar in structure to yeast ␣ factor cific appendage pattern, modulate the effect of the Wg/
(Loumaye et al., 1982). A fascinating area of develop- Dpp combination. In another phase of its function, wg
mental biology is the exploration of the origins of higher is required after early segmentation events for the heart
eukaryotic signals as revealed by tracing related mole- precursors to develop. The name “wingless” derives
cules in a wide variety of organisms. from the original partial loss-of-function mutation that
The first evidence for chemical factors that could alter abolishes wing development. Wg actually has multiple
development was the production of galls in plants under roles in wing development, first subdividing the thoracic
the influence of chemicals from insect salivary gland primordium into body wall and wing, then distinguishing
extracts. In 1894–1895, C. Herbst set out to identify dorsal and ventral wing surfaces, then stimulating pro-
external stimuli that alter development, including cal- duction of distinctive bristles along the wing margin. Wg
cium, light, gravity, salts, and temperature (Needham, regulates cell division in the wing imaginal disc. Without
1942). Hints of chemical influence also came from exo- wg function, half the fly’s brain does not develop. Wg is
gastrulation caused by lithium treatment of amphibian needed to pattern sensory organs on the body surface,
embryos. Herbst found that “one stimulus might have eyes, and appendages. Proper dorsal–ventral (D–V) pat-
quite different responses if it acted on different tissues.” terning in the eye requires wg. The Wingless saga is
Hormone signals drive both sexual differentiation and typical of many important regulatory molecules; they are
the equally astonishing process of metamorphosis, a used repeatedly during development but with different
process central to some of the most remarkable natural outcomes. The specific actions of a regulator depend
history of animals and genes. In Darwin’s words, “Many on what other regulators are acting in concert, and on
insects, and especially certain crustaceans, show us the competence of cells to respond.
what wonderful changes of structure can be effected Wg is one of the few proteins for which good evidence
during development. Such changes, however, reach has been obtained for concentration-dependent effects
their acme in the so-called alternate generations of some on cell fate determination. Morphogens are defined as
of the lower animals. It is, for instance, an astonishing substances that direct different cell fates depending on
fact that a delicate branching coralline, studded with their abundance, and although candidate morphogens
polypi and attached to a submarine rock, should pro- have been endlessly proposed and discussed, it is in fact
duce, first by budding and then by transverse division, challenging to show that under normal developmental
a host of huge floating jelly-fishes; and that these should circumstances, different concentrations of a protein di-
produce eggs, from which are hatched swimming ani- rect different cell fates (Neumann and Cohen, 1997). The
malcules, which attach themselves to rocks and become difficulty is partly in making quantitative measurements
developed into branching corallines; and so on in an in vivo, and partly in distinguishing the effects of multiple
endless cycle.” Metamorphic transformations are espe- factors. Nonetheless, several regulators, including ac-
cially significant among human disease parasites. Ad- tivin and hedgehog in vertebrates as well as bicoid,
vances in the developmental biology of these organisms dorsal, decapentaplegic, and wingless in flies, seem to
will be significant in disease control as well as interesting have morphogen properties under some circumstances.
in their own right. It is worth noting that most signal transduction sys-
The most remarkable finding from decades of work tems are similar in animals separated by vast evolution-
on developmental signaling is that few new types of ary distances. Similar does not mean identical. Distinc-
signals have been found. The routine approach to the tive components and distinctive regulatory relationships
development of any tissue is now to check for the have been found. Increasingly, linear pathway diagrams
involvement of FGF, TGF␤, Hedgehog, Wnt signals, a are being replaced by networks of arrows representing
few others, and their transduction machinery. Not all a virtual ecosystem of interacting proteins. Often the
these signals are involved in every process, but often social groupings have considerable complexity. We are
several of them are relevant. in great need of better ways to monitor and represent
The natural history of the Drosophila wingless (wg) the flow of information, changes in protein modifications
gene, a Wnt family member, shows how signals are used and activities, and changes in subcellular localization
Review
33

that happen rapidly and concurrently as signals are pro- made to human knowledge, is the law that all animals
cessed. and plants, however infinitely various their form may
seem to be, are, in reality, constructed upon a very few
Competence plans; in other words, that the parts of animals and
At one moment a cell will respond to a signal, at the plants are associated and arranged according to certain
next it will not. At one time production of a transcription fixed laws.” This comment was about morphology, and
factor will send a cell into a differentiation pathway, at the rational basis for taxonomy, but applies equally well
another time it will not. The difference is called compe- to the gene systems that control morphology. Huxley
tence, a word first used in the context of development would have enjoyed the conceptual simplification that
to describe timing-dependent susceptibilities of tissues comes from knowing the similar crystal structures of a
to inductive influences (Waddington, 1932). Needham homeodomain, ␭ repressor, and yeast MAT␣2p.
(1942, p. 119) put competence into molecular terms, The remarkable similarity of the genetic regulation of
noting “that an inductor may be a substrate, and that development in distant organisms has heralded a new
during the unavoidable reaction which will follow its conception of evolution. It was a big surprise when evo-
entry into the competent cell, the physical conformation lutionary conservation of the Krebs cycle, the genetic
of the enzyme protein may itself be changed.” An animal code, and classes of structural proteins was extended
will respond only to signals it recognizes, and gene re- to regulation of development. The diversity of organisms
sponses have similar restrictions: (1) Sensory abilities: had fooled everyone into thinking that the evolution of
Signals will be sensed only if receptors are available. completely different regulatory processes, or at least
Yeast a cells do not respond to the secreted a factor completely different uses of the same genes, was likely
because they do not have the right receptor. (2) The to be responsible for evolutionary change. Embryos of
need for specific social interactions: If a transcription different vertebrate species are more similar to each
factor requires a partner to work, the time when the other than are the corresponding adults, a useful hint of
partner is present determines competence. Most gene relatedness. Darwin complained in 1860: “Embryology is
activation events during development require multiple to me by far the strongest single class of facts in favour
transcription factors, as in yeast differentiation, so com- of change of forms and not one, I think, of my reviewers
petence depends on the presence of all the necessary has alluded to this” (Oppenheimer, 1967, p. 222). Reten-
factors. Nuclear receptor proteins often act in combina- tion of working regulatory systems for animal develop-
tion, as do segmentation proteins. (3) Interfering signals: ment would seem to be much more likely than invention
Some genes are inactivated, for example, by methyla- of dramatically new systems.
tion, during differentiation. In some cases only part of We can tally up some of the outstanding cases of
a gene, such as a particular enhancer, need be blocked. evolutionary conservation of gene function. First came
A compact inaccessible chromatin state will make a cell the Hox genes, clustered genes that are differentially
incompetent for anything requiring one of the repressed activated along the head to tail axis of the embryo
genes. (4) Tribal conflict: Modifying agents, such as pro- (Lewis, 1978; McGinnis and Krumlauf, 1992). The combi-
teases or phosphatases that destroy or modify signals nation of Hox genes active in a certain part of the animal
governs pattern formation there. For example, the shape
or other proteins, can make a cell incompetent to re-
of vertebrae is altered in mice carrying Hox gene muta-
spond. (5) Environmental impacts: A cell may sense a
tions, and Hox genes govern the type of appendages
regulator but be unable to carry out a differentiation
formed on insects. Hox genes are necessary for regional
program due to nutritional or other environmental defi-
neuronal differentiation in the Drosophila brain and in
cits. A variation of this concept may account for temper-
the mammalian hindbrain (Reichert and Simeone, 1999).
ature-dependent regulation of sex in reptiles.
Repeating units formed by the action of segmentation
Limb development provides nice examples of the inte-
genes (Figure 1, top) become different (Figure 1, bottom)
gration of multiple signals, all of them acting on compe-
due to Hox gene action. Hox gene action is not limited
tent cells but not others (Johnson and Tabin, 1997). In
to distinguishing repeating body units. Complex over-
limbs and other tissues, far more progress has been lapping patterns of Hox gene expression contribute to
made in identifying signals and regulators than in defin- limb patterning and Hox genes are active in blood cells.
ing competence. For example, the application of FGF Hox genes encode homeodomain transcription factors
molecules to the flank of a chick embryo can induce similar in structure to the yeast MAT␣2 protein.
formation of an extra leg (Cohn et al., 1995), but we do Tinman homeodomain protein is another case of strik-
not understand why those cells can do it while cells in ing evolutionary conservation. Tinman is required for
other places or at other times in development cannot. heart and visceral muscle development in flies; the re-
When we fully understand the answer, limb regeneration lated gene Nkx 2.5 is necessary for proper heart devel-
might become possible in humans. opment in mice and humans (Evans, 1999). Heart devel-
opment has a central astounding aspect: the need for
Evolutionary Conservation of Gene Function circulation early in the embryogenesis of many species
In addition to the similarity of yeast ␣ factor to GnRH, means that the heart must begin functioning when it is
many yeast proteins are related to well-known verte- tiny and continue functioning as it grows. The phenome-
brate regulators, such as ras. In 1857, two years before non is akin to keeping a rowboat’s outboard motor run-
The Origin of Species was published, Thomas Huxley ning while changing it into an engine for a cruise ship.
wrote, “The most important of all the generalizations of Another example is the Pax6 homeodomain protein that
natural history, and, indeed, one of the most brilliant is required for eye development in humans, mice, and
additions which the progress of modern science has flies. In flies Pax6 works with the sine oculis and eyes
Cell
34

Figure 2. Example of Conserved Genes Affecting Dorsal–Ventral

Cell Fates in Insect and Mammalian Nervous Systems
In situ hybridizations from serial sections are superimposed at the
left and diagrammed at the right. Corresponding genes are shown
in the same color. The order of expression of the genes is preserved
from mice (left) to flies (right). Modified from Weiss et al. (1998).

form the neural tube does the flipping. Other types of

regulation in neural development, such as the determi-
nation of neuronal subtypes by bHLH proteins, also
seem similar in Drosophila and mammals (Chan and
Jan, 1999).
What exactly does the dedication of particular tran-
scription factors to particular tissues or patterning
events mean? Let us take Tinman as an example. Pre-
sumably, more than half a billion years ago, Tinman
became irreversibly associated with one or more target
genes involved in building some sort of pump, or per-
haps even earlier to define a particular type of meso-
derm. An ancestral Nk-class homeobox gene would acti-
vate some generally useful target genes in many cell
types. If Tinman arose as a duplicate of another NK
Figure 1. Segmentation and Homeosis; the Same Two Houses at
gene, it might have been free to change either its spatial
Different Times regulation or its binding specificity for DNA or cofactor
After segmentation produces repeating structural elements (top), proteins. Since modern day Tinman can bind in vitro to
action of Hox genes in specific segments gives them individual sequences that other NK proteins bind, either the bind-
properties (bottom). Photographs by Robert S. Brantley (Brand, ing differences are subtle or they are irrelevant. So, let
1995). us suppose that the original distinguishing change in the
tinman gene was due to the acquisition of a regulatory
absent genes, and at least some of their homologs are element (responsive to Hox genes or TGF␤ inducers?)
required for fish and mouse eye development (Gehring to make tinman active in only a subset of the mesoderm
and Ikeo, 1999). cells. This might turn up transcription of a Tinman target
Dorsal-ventral patterning in the early vertebrate and gene in that subset of cells, resulting in changed mor-
fly embryo requires localized expression and action of phology, electrical conduction, or whatever. Once any
signaling proteins of the TGF␤ class, such as Dpp in useful change along these lines occurred, Tinman’s as-
flies and BMP2 and 4 in vertebrates. The proteins direct sociation with the particular subset of cells would be
polarization of the embryo and are restricted in their selectable. Now other genes could fall under Tinman’s
actions by antagonist proteins of the Sog/Chordin class influence in those cells simply by acquisition of the short
working from the opposite pole. In flies Dpp acts in DNA enhancer sequences necessary for Tinman regula-
dorsal cells and Sog in ventral cells, whereas in verte- tion. Point mutations might do it, as would transposition
brates BMPs are ventral and Chordin dorsal. This and of a piece of DNA containing Tinman target enhancer
other evidence has suggested that the axes may have sequences. There would be strong selection for animals
been flipped during evolution (Holley et al., 1995). Fur- where Tinman had acquired targets that enhanced heart
ther support for this view comes from conserved expres- or visceral mesoderm function.
sion patterns of three homeobox genes in early neural Whatever the original course of events, the dedication
development (Chan and Jan, 1999). In flies the order of of a transcription factor to a particular organogenesis
gene expression is ventral-vnd-ind-msh-dorsal, and in event implies conservation of at least some target
the vertebrate neural tube, the order for the genes genes, which we could call the primeval target genes.
most closely related in sequence is ventral-Nkx2.2(vnd)- They may be but a small subset of the current array
Gsh1,2(ind)-Msx1(msh)-dorsal (Figure 2). The apparent of targets, and distinguishing them from subsequently
discrepancy—the axis does not look flipped—is re- acquired targets may unveil the process of evolutionary
solved because the invagination of the neural plate to recruitment of genes for a task.
Review
35

Some signals also appear to have conserved their more complex than parts of insects. Some of the in-
dedication to a particular developmental process, creased complexity is illusory; “simple” organisms often
though this is the exception rather than the rule. The are not.
rule is that the same signal is used for many events that What is the most important change: gene regulation,
are not obviously linked in different organisms. The use with members of families expressed in different pat-
of a particular signal, FGF, in controlling branching mor- terns, or protein diversification within a family? Few tests
phogenesis appears to have been conserved (Metz- have been done to rigorously assess the equivalence
ger and Krasnow, 1999). FGF signals are critical for of different members of a gene family. The coding region
branching of the trachea in flies and for branching of of the mouse engrailed1 gene has been replaced by that
the lung buds in vertebrates. In both cases respiratory of engrailed2, with few detrimental effects (Hanks et al.,
requirements interact with genetically programmed 1995). On the other hand, fetal and adult hemoglobin
events to ensure proper access by all cells to oxygen. genes are expressed at different times for a clear reason:
FGF actions must be driven by local low oxygen ten- the proteins provide appropriate and distinct oxygen
sion. FGF molecules are used for many purposes, but affinities. In cases where the proteins really are equiva-
branching morphogenesis is one that is recognizably lent, one can imagine a different history in which a single
common to animals separated by hundreds of millions coding region gradually acquired a plethora of cis regu-
of years of evolution. latory sequences, and this seems to have happened in
By late in his life, Linnaeus fully appreciated the value numerous cases. One can therefore view gene families
of examining different stages of development as a guide as essential for protein diversification but not neces-
to classification, since relationships may become evi- sarily for regulatory diversification. Families could be
dent with one criterion but not another. Similarly, a gene essential for regulatory diversification if incompatible
discovered for one of its roles may in fact have originally enhancers and silencers must control the same protein-
been dedicated to another, more evolutionarily con- coding sequence. It seems possible that the existence
served, purpose. Linnaeus was stern in his view of of gene families is to some extent a smokescreen and
sloppy taxonomists who did not pay attention to multiple that most of the important changes during evolution
criteria: “Paying too little regard to Nature, they disunited happened with gene families present but do not require
natural genera, on account of the most trifling distinc- that there be gene families.
tions. This made their continuance in the science of very Much of the current emphasis on similarities between
short duration; our business here is not to suppose but vertebrate and invertebrate regulators of development
to examine what nature will allow of, and what she will is driven by amazement; much less similarity was ex-
not. Knowledge of this sort, built on opinion only, will pected. There are tremendous differences as well, and
not stand. We are therefore to look into the science with exploring them and their origins will unveil a more com-
great accuracy; and the larva of the insect, its manner plete picture of how all Linnaeus’ animals came to exist.
of changing, and other things of moment, are to be Quite a few rapidly evolving genes are known. For exam-
known, before we presume to form a new genus, as ple, the bicoid protein is required as a concentration-
men of experience will readily admit. Daily experience dependent regulator, a morphogen, of anterior–posterior
in botany teaches us that none are more apt to form polarity in fly embryos, but this function may have arisen
new genera, than those least qualified for it.” only during the evolution of Diptera. We need to under-
A good example of the importance of paying sufficient stand rapid evolution as well as conservation.
regard to the natural history of genes comes from the
Toll transmembrane protein. Toll was identified as a key
Origins of Developmental Regulators
component of the regulatory system that polarizes the
Drosophila oocyte along the D–V axis. Proteins related A billion years ago or so, differentiation was accom-
to Toll have now been identified in many organisms plished using components that worked so well that the
including mammals. D–V patterning is not the evolution- same system is still used. It is striking that homeodomain
arily conserved function. By examining adult as well as proteins, like MAT␣2p, are used to control cell differenti-
larval gene functions, observing the genes at multiple ation in yeast just as they are in humans. The origins
stages of their life cycle, Toll-related proteins were found of many other regulators is far less clear. Hedgehog
to share a role in immunity to bacterial and fungal infec- proteins are like other classes of signaling proteins in
tion. That role is common to humans, flies, and tobacco that no sign of them can be found in bacteria or yeast,
(Meister et al., 1997). Toll is accompanied in its con- and it is not clear where they first arose. However, yeast
served role by the rest of its ecosystem. For example, cells have a gene related to Niemann-Pick C1 (NPC1),
NF␬B proteins are transcription factors whose entry into a human disease syndrome gene essential for proper
the nucleus is regulated by extracellular signals, coordi- transport of cholesterol and other lipids in mammalian
nating the inflammation response to infection. Dorsal, cells. The amino acid sequence of the Hedgehog recep-
a protein related to NF␬B, transduces the Toll signal tor, Patched, is closely related to NPC1. During Hedge-
and is also linked to the fly’s immune response (Lemaitre hog synthesis cholesterol is covalently joined to the
et al., 1996). signaling portion of the Hedgehog protein. Also, certain
The genome projects will complete documentation of cholesterol analogs are powerful inhibitors of the recep-
a pattern that is now familiar: many genes present as tion of the Hedgehog signal. These and other data point
single copies in worms or flies are present as families at the possibility that some parts of the Hedgehog sig-
in vertebrates (Miklos and Rubin, 1996). It is difficult to naling pathway are derived from proteins needed for
assess whether proliferation of genes into families was intracellular lipid trafficking (Beachy et al., 1997). The
essential for evolving the parts of vertebrates that are recruitment of Toll to D–V patterning may have been a
Cell
36

relatively recent theft (and duplication) of an immunolog-

ical function. Other genes have probably been coopted
from basic metabolic processes.
One of the most interesting questions is whether there
is any logic to which types of regulators are used in
which processes. Why is a zinc finger protein used here
and a homeodomain protein there? Is the present sce-
nario a historical accident, or does it reflect features
of the proteins that make them especially suitable for
certain tasks? The origins of the machinery that regu-
lates development remain mostly unknown.

Death
As young biologists who assisted the explosive ad-
vances in developmental biology have become middle-
aged biologists, the developmental biology community
has become fascinated with death. This at first took the
mild form of an interest in cell death (Horvitz, 1999),
which was comfortably separate from real death. Cell
death is an extremely important mechanism during de-
Figure 3. High Precision in Spatial Gene Regulation and Signaling
velopment, for example in shaping digits or allowing
Spatial regulation of a Hedgehog target gene. Mouse embryo with
only functional neurons or the right lymphocytes to sur-
patched1 expression shown. Hedgehog signals induce expression
vive, and in disease, for example by regulating (or failing of patched1 and other target genes in precisely controlled spatial
to regulate) inappropriate growth. Now the trend is a and temporal patterns, as is shown here using a lacZ gene inserted
greater interest in avoiding death altogether; the field into the patched1 locus. The regulatory relationships in Hedgehog
of aging research is flourishing as never before. It is signaling are largely conserved in all animals where they have been
fascinating that both cell death and aging appear to be tested. From Ljiljana Milenkovic.
programmed genetically just as the construction of the
organism is programmed (Kenyon, 1996; Defossez et normal developmental roles, APC in the Wnt signaling
al., 1998; Guarente et al., 1998; Lin et al., 1998). Individual pathway and Rb in eye development. The presenilin
cells age too. The earliest known event associated with proteases that are crucial in familial Alzheimer’s disease
the aging of yeast cells is genetic instability in the cluster have been linked to roles in processing Notch signaling
of ribosomal DNA genes (Defossez et al., 1998). Non- proteins that are active in many developmental events
transformed eukaryotic cells in culture also have a life- (Levitan and Greenwald, 1998). An insulin receptor-like
time timer running, quite possibly linked to telomerase gene, daf-2 of C. elegans, was discovered due to its
function and chromosome shortening. Reactivation of role in inhibiting dauer formation (a developmental form
telomerase allows cells to continue dividing in culture of the worm adapted for diapause) but has also been
beyond the time they would otherwise enter crisis and implicated in affecting longevity (Kimura et al., 1997;
die. In accordance with this view, most human tumor Tissenbaum and Ruvkun, 1998). In worms insulin signal-
cells contain active telomerase, whereas most somatic ing may be required to steer metabolism away from fat
cells do not (Hodes, 1999). The rapid advances in aging formation and is also involved in embryonic develop-
research demand frequent review; I will review them- ment. Dauer formation also requires a worm homolog
again in 2050 and 2100. of the PTEN gene, which is a tumor suppressor gene in
humans (Rouault et al., 1999). Genes required to deter-
Developmental Biology, in Sickness and in Health mine neuronal cell type in nematodes are closely related
Genes discovered for their roles in development are also to genes involved in human polycystic kidney disease;
critical in human disease. Linnaeus was interested in the worm’s genetic pathway including these genes pro-
nosology, the classification of diseases. Continuing dis- vides ideas about what other proteins are relevant (Em-
coveries of the roles of developmental genes in disease mons and Somlo, 1999). A gene related to the Drosophila
will allow more rigorous and unambiguous diagnosis. epithelial polarity gene crumbs is the cause of a form
Excitement has come from finding genes that guide cell of retinitis that affects 1.5 million people (den Hollander
differentiation and pattern formation and that are linked et al., 1999). In each of these cases, the excitement
to human disease. For example, mutations in ␤-catenin comes from connecting a protein involved in human
contribute to human colon and other cancers (Morin et disease to a whole pathway of interacting components.
al., 1997). Mutations in human Sonic hedgehog result in The elucidation of the ras pathway, so important in hu-
holoprosencephaly; mutations in the hedgehog receptor man cancer and development, has benefited dramati-
Patched (Figure 3) lead to polydactyly and spina bifida cally from its connections to developmental events in
in Gorlin’s syndrome and to basal cell carcinoma, medul- flies and worms (Kayne and Sternberg, 1995; Wassar-
loblastoma, and rhabdomyosarcoma (Goodrich and man et al., 1995).
Scott, 1998). Mutations in the tinman-related human Developmental biology has great potential for impact
gene Nkx2.5 cause heart disease (Schott et al., 1998). on the clinic. Already, treatments involving skin grafts
Cell cycle regulators such as APC and Rb are critical in and growth hormones have helped many people. Isola-
human cancer and are now viewed in the context of their tion of stem cells for a variety of tissues and better
Review
37

manipulations of stem cells based on newfound knowl- limits to child design or selection, which many people
edge of growth and differentiation controls will surely find offensive, will almost certainly become a consider-
make regeneration more useful for healing and tissue able social problem. The present controversies over
replacement (see reviews by Fuchs and Segre, 2000 and modification of tomato ripening properties, bovine
Weissman, 2000 [both in this issue of Cell]). We see growth hormone produced in bacteria, and pesticides
progress in identifying signals for producing neurons genetically introduced into plants are forerunners of de-
that are lost in Parkinson’s disease, in inducing growth of bates about the proper limits of intervention in medicine.
hair, in following pancreas differentiation to learn about The current tremendous inequities in access to medical
diabetes, in stimulating growth of lung buds in culture, care may or may not be improved by new types of
and in growing arteries in culture. The biggest obstacle developmental medicine.
is likely to be finding out how to create cells with the
ability to respond rather than to find the best cocktail What New Tools Are Needed?
of signals. Our present picture of gene activation during develop-
The developmental biology of parasites also holds ment is largely dependent on static views of embryos.
enormous promise for new approaches to dread dis- Major limitations include the following: (1) Detection of
eases (Teixiera, 1998). Trypanosomes, leishmania, and markers like fluorescent proteins is slow because the
malaria parasites together account for hundreds of mil- time needed for the protein to fold delays detection for
lions of infected people. The lifecycles of parasites are significant periods of development. (2) At present we
fascinating for their evasions of the immune response cannot watch more than four proteins, or gene expres-
and for the adaptability of many parasites to multiple sion patterns, at a time. In the seething cytoplasm, thou-
host organisms, as well as for unusual molecular fea- sands of changes occur, while we watch a few events
tures such as the polycistronic transcription and intron- at a time. We need new ways to see, and new represen-
less genome of trypanosomes. tations of what we see. (3) It is difficult to quantitate
the concentration of any of the relevant proteins as a
What Can We Build? function of time, let alone their activity states. (4) Modifi-
One measure of progress in developmental biology will cations of proteins, such as phosphorylation or glycosyl-
be whether we can direct the growth of a useful tissue. ation, are hard to monitor or manipulate. (5) The chroma-
Here is a test of whether we really know how things tin is preset to allow a gene to respond or not, and this
work: take cells growing in culture and manipulate them cannot readily be viewed, particularly not at the single
to make either of two alternative organs by manipulating cell level.
signals and gene activities. Organ development means With in situ hybridization to RNA in tissues, and detec-
not just activating differentiation markers but making a tion of proteins with antibody stains, we can discriminate
fully functional, well-shaped tissue that works. Make the among previously indistinguishable cells. This has been
epithelium fold where a fold would be useful; cause an fantastically revealing about cell determination, as it
organizing center to appear in a certain spot; make some gives us one view of what the cell is “thinking,” long
cells delaminate and form another layer; make differenti- before morphological changes occur. This has led to a
ated cell types appear; make some cells migrate to a few markers being used to indicate “ventral-ness” or
useful new position; direct the formation of a left-right “liverish-ness.” While this may be safe, cells might acti-
symmetry and then asymmetry; and make a colorful vate a few indicator molecules without turning on the
pattern! If we could direct the same cell population into full set characteristic of a tissue type or stage of develop-
either of two completely different paths, we must have ment. The more complete views of gene activation that
learned something. have arrived with microarrays will help to clarify whether
How can such knowledge be useful? The shaping of this is a real problem.
tissues during healing and regeneration is mostly done Until recently gene expression during development
surgically now, and major limitations leave people in has been viewed by reconstructing events from different
therapy or with reduced opportunities for decades. Fa- individual animals. Progress here will come from nonin-
cilitating a patient’s natural healing processes to pro- vasive imaging techniques such as MRI applied to ani-
mote more complete healing, more rapid healing, or mals (Jacobs et al., 1999) and watching gene expression
better organized healing would help tremendous num- using proteins tagged with fluorescent markers, such
bers of people. Altering properties of cells to make them as green fluorescent protein (GFP) (Chalfie et al., 1994).
more resistant to infection or more aggressive in fighting GFP has already been extremely useful in watching the
infection will also become possible. Stimulating natural movements of proteins and cells in living embryos. Fluo-
growth processes to repair nerve damage, to grow new rescence resonance energy transfer (FRET) is a tech-
teeth, to replace blood without a marrow transplant, or nique that detects interactions between proteins based
even to regenerate limbs would be of clear benefit. on the proximity of an emitting fluorescent molecule
What are the risks? One is cancer. By stimulating and a sensing fluorescent molecule that emits at a new
growth processes necessary for repair at a time in life wavelength if sufficiently stimulated by the first emitter
when (perhaps) normal restraints are no longer op- (Periasamy and Day, 1999). GFP together with FRET,
erating, cancer might be a higher risk. A second concern and refinements and variations to come, will allow the
is misuse of the knowledge. The science fiction literature associations of proteins to be monitored in living em-
is replete with examples of body modifications chosen bryos.
to be repugnant. Many people find blood donation or Our view of development, and what matters in devel-
cosmetic “repair” after injury acceptable, whereas the opment, might very well be differently skewed if we
Cell
38

could observe activation of kinase cascades in space thank Roel Nusse and the anonymous reviewers for helpful com-
and time rather than gene transcription and protein ac- ments on the manuscript, Drs. Tim Galitski and Gerry Fink for helping
with microarray analysis, and Drs. Ljiljana Milenkovic and Joseph
cumulation. The ability to detect only activated forms Weiss for figures. I am particularly grateful to Allan Spradling and
of a kinase with specific antibodies (e.g., Gabay et al., Mary Lou Pardue for inspiration, guidance, and friendship, and I
1997) is valuable, but does not allow imaging fast thank my students and colleagues for all the excitement they have
enough to keep up with the rapid dynamics of living cell brought. The Howard Hughes Medical Institute and grants from the
metabolism. NIH, DARPA, and the Parseghian Foundation support research in
my laboratory.
There is a dearth of small molecules in our under-
standing of development. We have steroids in hormonal References
coordination of metamorphosis and growth, cAMP in
many events, nitric oxide in vascularization, and a few Ashburner, M. (1990). Puffs, genes, and hormones revisited. Cell 61,
others, yet it seems likely that small molecules are as 1–3.
ubiquitous and crucial in development as they are in Bashirullah, A., Cooperstock, R.L., and Lipshitz, H.D. (1998). RNA
regulation of bacterial metabolism. How could we under- localization in development. Annu. Rev. Biochem. 67, 335–394.
stand Trp repressor without knowing about tryptophan? Baylies, M.K., Bate, M., and Ruiz Gomez, M. (1998). Myogenesis: a
view from Drosophila. Cell 93, 921–927.
Where Did We Come From? Beachy, P.A., Cooper, M.K., Young, K.E., von Kessler, D.P., Park,
W.J., Hall, T.M., Leahy, D.J., and Porter, J.A. (1997). Multiple roles
How can developmental biology help us look into the of cholesterol in hedgehog protein biogenesis and signaling. Cold
past and understand human origins? Our own place in Spring Harb. Symp. Quant. Biol. 62, 191–204.
the animal kingdom was noted in a letter written by Beermann, W. (1952). Chromomeren Konstanz und Spezifische
Linnaeus to a friend in 1747: “You disapprove my having Modifikation der Chromosomenstruktur in der Entwicklung und Or-
located Man among the Anthropomorphi. But man gan Differenzierung von Chironomus tentans. Chromosoma 5,
knows himself . . . I demand of you, and of the whole 139–198.
world, that you show me a generic character—one that Bobola, N., Jansen, R.P., Shin, T.H., and Nasmyth, K. (1996). Asym-
is according to generally accepted principles of classifi- metric accumulation of Ash1p in postanaphase nuclei depends on
a myosin and restricts yeast mating–type switching to mother cells.
cation—by which to distinguish between Man and Ape.
Cell 84, 699–709.
I myself most assuredly know of none. I wish somebody
Brand, S. (1995). How Buildings Learn: What Happens After They’re
would indicate one to me. But, if I had called man an Built (New York: Penguin Books).
ape, or vice versa, I should have fallen under the ban Calvi, B.R., and Spradling, A.C. (1999). Chorion gene amplification
of all the ecclesiastics. It may be that as a naturalist I in Drosophila: a model for metazoan origins of DNA replication and
should have done so” (Greene, 1909). S-phase control. Methods 18, 407–417.
Now we know ourselves somewhat better and realize Cedar, H., and Bergman, Y. (1999). Developmental regulation of
that to know ourselves better still we must know all life immune system gene rearrangement. Curr. Opin. Immunol. 11,
better. At age 25 Linnaeus took an adventurous 3000 64–69.
mile journey through Lapland that had an influence on Chalfie, M., Tu, Y., Euskirchen, G., Ward, W.W., and Prasher, D.C.
his science reminiscent of the effect of the Beagle voy- (1994). Green fluorescent protein as a marker for gene expression.
Science 263, 802–805.
age on young Darwin. Linnaeus observed the natural
Chan, Y.-M., and Jan, Y.N. (1999). Conservation of neurogenic genes
world intensely despite storms, whitewater boat acci-
and mechanisms. Curr. Opin. Neurobiol. 9, 582–588.
dents, and near starvation. Along the way he encoun-
Chu, S., DeRisi, J., Eisen, M., Mulholland, J., Botstein, D., Brown,
tered frog development while helping a woman who P.O., and Herskowitz, I. (1998). The transcriptional program of sporu-
believed she had three frogs in her stomach (she heard lation in budding yeast. Science 282, 699–705. Erratum: Science
them croaking) as a result of drinking water containing 282 (5393), 1998.
frog spawn. Even without such medical interludes, we Cline, T.W., and Meyer, B.J. (1996). Vive la difference: males vs
can learn as Linnaeus did, by searching for natural sur- females in flies vs worms. Annu. Rev. Genet. 30, 637–702.
prises. Our few model organisms provide an incomplete Cohn, M.J., Izpisua-Belmonte, J.C., Abud, H., Heath, J.K., and Tickle,
view. We must first preserve what remains of the natural C. (1995). Fibroblast growth factors induce additional limb develop-
world around us and then, like Linnaeus and Darwin, ment from the flank of chick embryos. Cell 80, 739–746.
walk through and watch developments and develop- Cross, G.A. (1996). Antigenic variation in trypanosomes: secrets
surface slowly. Bioessays 18, 283–291.
ment with open eyes and minds.
Developmental biology is a celebration of natural Darwin, C. (1871). The Descent of Man and Selection in Relation to
Sex (New York: Hurst and Co.).
beauty combined with the elegance of molecular control
Defossez, P.A., Park, P.U., and Guarente, L. (1998). Vicious circles:
systems. The more we know, the more astounding the
a mechanism for yeast aging. Curr. Opin. Microbiol. 1, 707–711.
renewal of life becomes. Now the genome projects have
den Hollander, A.I., ten Brink, J.B., de Kok, Y.J., van Soest, S.,
stalked a host of completely untamed genes, never van den Born, L.I., van Driel, M.A., van de Pol, D.J., Payne, A.M.,
sighted before. As with animals, wild genes are even Bhattacharya, S.S., Kellner, U., et al. (1999). Mutations in a human
more fun than domesticated ones. To appreciate and homologue of Drosophila crumbs cause retinitis pigmentosa (RP12).
explore the new genes in their full natural splendor, fresh Nat. Genet. 23, 217–221.
from the jungle, is the delightful task facing develop- Emmons, S.W., and Somlo, S. (1999). Signal transduction. Mating,
mental biologists. channels and kidney cysts. Nature 401, 339–340.
Evans, S.M. (1999). Vertebrate tinman homologues and cardiac dif-
Acknowledgments ferentiation. Semin. Cell. Dev. Biol. 10, 73–83.
Firulli, A.B., and Olson, E.N. (1997). Modular regulation of muscle
With a subject of this scope it is impossible to credit all the people gene transcription: a mechanism for muscle cell diversity. Trends
who have made major contributions to developmental biology. I Genet. 13, 364–369.
Review
39

Fraser, S.E., and Harland, R.M. (2000). The molecular metamorpho- Kimura, K.D., Tissenbaum, H.A., Liu, Y., and Ruvkun G. (1997). daf-2,
sis of experimental embryology. Cell 100, this issue, 41–55. an insulin receptor-like gene that regulates longevity and diapause
Fuchs, E., and Segre, J.A. (2000). Stem cells: a new lease on life. in Caenorhabditis elegans. Science 277, 942–946.
Cell 100, this issue, 143–155. Lemaitre, B., Nicolas, E., Michaut, L., Reichhart, J.M., and Hoffmann,
Gabay, L., Seger, R., and Shilo, B.Z. (1997). In situ activation pattern J.A. (1996). The dorsoventral regulatory gene cassette spatzle/Toll/
of Drosophila EGF receptor pathway during development. Science cactus controls the potent antifungal response in Drosophila adults.
277, 1103–1106. Cell 86, 973–983.
Galau, G.A., Britten, R.J., and Davidson E.H. (1974). A measurement Levitan, D., and Greenwald, I. (1998). Effects of SEL-12 presenilin
of the sequence complexity of polysomal messenger RNA in sea on LIN-12 localization and function in Caenorhabditis elegans. De-
urchin embryos. Cell 2, 9–20. velopment 125, 3599–3606.
Galitski, T., Saldanha, A.J., Styles, C.A., Lander, E.S., and Fink, Lewis, E.B. (1978). A gene complex controlling segmentation in Dro-
G.R. (1999). Ploidy regulation of gene expression [see comments]. sophila. Nature 276, 565–570.
Science 285, 251–254. Lin, Y.J., Seroude, L., and Benzer, S. (1998). Extended life-span
Gehring, W.J., and Ikeo, K. (1999). Pax 6: mastering eye morphogen- and stress resistance in the Drosophila mutant methuselah [see
esis and eye evolution. Trends Genet. 15, 371–377. comments]. Science 282, 943–946.
Goerke, H. (1973). Linnaeus (Charles Scribner’s Sons). Linnaeus, C. (1735). Systema Naturae. Reprinted in 1964 by Nieuw-
Goodrich, L.V., and Scott, M.P. (1998). Hedgehog and patched in koop and B. De Graaf, Holland.
neural development and disease. Neuron 21, 1243–1257. Loumaye, E., Thorner, J., and Catt, K.J. (1982). Yeast mating phero-
Gould, A. (1997). Functions of mammalian Polycomb group and mone activates mammalian gonadotrophs: evolutionary conserva-
trithorax group related genes. Curr. Opin. Genet. Dev. 7, 488–494. tion of a reproductive hormone? Science 218, 1323–1325.
Greene, E.L. (1909). Linnaeus as an evolutionist. Proc. Washington Lyon, M.F. (1999). X-chromosome inactivation. Curr. Biol. 9, R235–
Acad. Sciences 11, 17–26. R237.
Guarente, L., Ruvkun, G., and Amasino R. (1998). Aging, life span, Mannervik, M., Nibu, Y., Zhang, H., and Levine M. (1999). Transcrip-
and senescence. Proc. Natl. Acad. Sci. USA 95, 11034–11036. tional coregulators in development. Science 284, 606–609.
Gurdon, J.B., and Uehlinger, V. (1966). “Fertile” intestine nuclei. Marin, I., and Baker, B.S. (1998). The evolutionary dynamics of sex
Nature 210, 1240–1241. determination. Science 281, 1990–1994.
Haber, J.E. (1998). Mating-type gene switching in Saccharomyces McGinnis, W., and Krumlauf, R. (1992). Homeobox genes and axial
cerevisiae. Annu. Rev. Genet. 32, 561–599. patterning. Cell 68, 283–302.
Hake, S., and Meyerowitz, E.M. (1998). Growth and development: Meister, M., Lemaitre, B., and Hoffmann, J.A. (1997). Antimicrobial
growing up green. Curr. Opin. Plant Biol. 1, 9–11. peptide defense in Drosophila. Bioessays 19, 1019–1026.
Hanks, M., Wurst, W., Anson-Cartwright, L., Auerbach, A.B., and Metzger, R.J., and Krasnow, M.A. (1999). Genetic control of
Joyner, A.L. (1995). Rescue of the En-1 mutant phenotype by re- branching morphogenesis. Science 284, 1635–1639.
placement of En-1 with En-2. Science 269, 679–682. Miklos, G.L., and Rubin, G.M. (1996). The role of the genome project
Hawkins, N., and Garriga, G. (1998). Asymmetric cell division: from in determining gene function: insights from model organisms. Cell
A to Z. Genes Dev. 12, 3625–3638. 86, 521–529.
Hendrix, R.W., Roberts, J.W., Stahl, F.W., and Weisberg R.A. (1983). Morin, P.J., Sparks, A.B., Korinek, V., Barker, N., Clevers, H., Vo-
Lambda II (Cold Spring Harbor, NY: Cold Spring Harbor Laboratory gelstein, B., and Kinzler, K.W. (1997). Activation of beta-catenin-
Press). Tcf signaling in colon cancer by mutations in beta-catenin or APC.
Science 275, 1787–1790.
Hodes, R.J. (1999). Telomere length, aging, and somatic cell turn-
over. J. Exp. Med. 190, 153–156. Needham, J. (1942). Biochemistry and Morphogenesis (Cambridge:
Cambridge University Press).
Holley, S.A., Jackson, P.D., Sasai, Y., Lu, B., De Robertis, E.M.,
Hoffmann, F.M., and Ferguson, E.L. (1995). A conserved system for Neumann, C., and Cohen, S. (1997). Morphogens and pattern forma-
dorsal–ventral patterning in insects and vertebrates involving sog tion. Bioessays 19, 721–729.
and chordin. Nature 376, 249–253. Nurse, P. (2000). The long twentieth century of the cell cycle and
Horder, T.J., Witkowski, J.A., and Wylie C.C. (1985). A History of beyond. Cell 100, this issue, 71–78.
Embryology (Cambridge: Cambridge University Press). Nusslein-Volhard, C., and Wieschaus, E. (1980). Mutations affecting
Horvitz, H.R. (1999). Genetic control of programmed cell death in the segment number and polarity in Drosophila. Nature 287, 795–801.
nematode Caenorhabditis elegans. Cancer Res. 59, 1701s-1706s. Oettinger, M.A. (1999). V(D)J recombination: on the cutting edge.
Hough, B.R., Smith, M.J., Britten, R.J., and Davidson E.H. (1975). Curr. Opin. Cell. Biol. 11, 325–329.
Sequence complexity of heterogeneous nuclear RNA in sea urchin Oppenheimer, J.M. (1967). Essays in the History of Embryology and
embryos. Cell 5, 291–299. Biology (Cambridge, MA: MIT Press).
Iyer, V.R., Eisen, M.B., Ross, D.T., Schuler, G., Moore, T., Lee, J.C.F., Periasamy, A., and Day, R.N. (1999). Visualizing protein interactions
Trent, J.M., Staudt, L.M., Hudson, J., Jr., et al. (1999). The transcrip- in living cells using digitized GFP imaging and FRET microscopy.
tional program in the response of human fibroblasts to serum. Sci- Methods Cell Biol. 58, 293–314.
ence 283, 83–87. Pirrotta, V. (1998). Polycombing the genome: PcG, trxG, and chro-
Jacobs, R.E., Ahrens, E.T., Meade, T.J., and Fraser, S.E. (1999). matin silencing. Cell 93, 333–336.
Looking deeper into vertebrate development. Trends Cell. Biol. 9, Pourquie, O. (1998). Clocks regulating developmental processes.
73–76. Curr. Opin. Neurobiol. 8, 665–670.
Jeon, M., Gardner, H.F., Miller, E.A., Deshler, J., and Rougvie, A.E. Raymond, C.S., Shamu, C.E., Shen, M.M., Seifert, K.J., Hirsch, B.,
(1999). Similarity of the C. elegans developmental timing protein Hodgkin, J., and Zarkower, D. (1998). Evidence for evolutionary con-
LIN-42 to circadian rhythm proteins. Science 286, 1141–1146. servation of sex-determining genes. Nature 391, 691–695.
Johnson, A.D. (1998). Molecular mechanisms of cell-type determina- Reichert, H., and Simeone, A. (1999). Conserved usage of gap and
tion in yeast. Curr. Opin. Genet. Dev. 5, 552–558. homeotic genes in patterning the CNS. Curr. Opin. Neurobiol. 9,
Johnson, R.L., and Tabin, C.J. (1997). Molecular models for verte- 589–595.
brate limb development. Cell 90, 979–990. Reik, W., and Walter, J. (1998). Imprinting mechanisms in mammals.
Kayne, P.S., and Sternberg, P.W. (1995). Ras pathways in Caeno- Curr. Opin. Genet. Dev. 8, 154–164.
rhabditis elegans. Curr. Opin. Genet. Dev. 5, 38–43. Rivera-Pomar, R., and Jackle, H. (1996). From gradients to stripes
Kenyon, C. (1996). Ponce d’elegans: genetic quest for the fountain in Drosophila embryogenesis: filling in the gaps. Trends Genet. 12,
of youth. Cell 84, 501–504. 478–483.
Cell
40

Rouault, J.P., Kuwabara, P.E., Sinilnikova, O.M., Duret, L., Thierry-

Mieg, D., and Billaud, M. (1999). Regulation of dauer larva develop-
ment in Caenorhabditis elegans by daf-18, a homologue of the tu-
mour suppressor PTEN. Curr. Biol. 9, 329–332.
Sander, K. (1985). The role of genes in ontogenesis-evolving con-
cepts from 1883–1983 as perceived by an insect embryologist. In
A History of Embryology, T.J. Horder, J.A. Witkowski, and C.C. Wylie,
eds. (Cambridge: Cambridge University Press), pp. 363–395.
Schott, J.J., Benson, D.W., Basson, C.T., Pease, W., Silberbach,
G.M., Moak, J.P., Maron, B.J., Seidman, C.E., and Seidman, J.G.
(1998). Congenital heart disease caused by mutations in the tran-
scription factor NKX2–5. Science 281, 108–111.
Sil, A., and Herskowitz, I. (1996). Identification of asymmetrically
localized determinant, Ash1p, required for lineage-specific tran-
scription of the yeast HO gene. Cell 84, 711–722.
Stavnezer, J. (1996). Immunoglobulin class switching. Curr. Opin.
Immunol. 8, 199–205.
Swain, A., and Lovell-Badge, R. (1999). Mammalian sex determina-
tion: a molecular drama. Genes Dev. 13, 755–767.
Teixiera, S.M.R. (1998). Control of gene expression in trypanoso-
matidae. J. Med. Biol. Res. (Brazil) 31, 1503–1516.
Tissenbaum, H.A., and Ruvkun, G. (1998). An insulin-like signaling
pathway affects both longevity and reproduction in Caenorhabditis
elegans. Genetics 148, 703–717.
Waddington, C.R. (1932). III. Experiments on the development of
chick and duck embryos, cultivated in vitro. Philos. Transactions
Royal Soc. B. 221, 179–230.
Wakayama, T., and Yanagimachi, R. (1999). Cloning the laboratory
mouse. Semin. Cell. Dev. Biol. 10, 253–258.
Wassarman, D.A., Therrien, M., and Rubin, G.M. (1995). The Ras
signaling pathway in Drosophila. Curr. Opin. Genet. Dev. 5, 44–50.
Weissman, I.L. (2000). Stem cells: units of development, units of regen-
eration, and units of evolution. Cell 100, this issue, 157–168.
Weiss, J.B., von Ohlen, T., Mellerick, D., Dressler, G., Doe, C.Q.,
and Scott, M.P. (1998). Dorsal-ventral patterning in the Drosophila
central nervous system: the intermediate neuroblast defective ho-
meobox gene specifies intermediate column identity. Genes Dev.
12, 3591–3602.
Yun, K., and Wold, B. (1996). Skeletal muscle determination and
differentiation: story of a core regulatory network and its context.
Curr. Opin. Cell. Biol. 8, 877–889.