Principles of experimental embryology...Environmental Developmental Biology The developing embryo is...

DESCRIPTIVE EMBRYOLOGY AND EVOLUTIONARY EMBRYOLOGY both had their rootsin anatomy. At the end of the nineteenth century, however, the new biologi-cal science of physiology made inroads into embryological research. The

questions of “what?” became questions of “how?” A new generation of embryologistsfelt that embryology should not merely be a guide to the study of anatomy and evo-lution, but should answer the question, “How does an egg become an adult?” Embry-ologists were urged to study the mechanisms of organ formation (morphogenesis)and differentiation. This new program was called Entwicklungsmechanik, often trans-lated as “causal embryology,” “physiological embryology,” or “developmental me-chanics.” Its goals were to find the molecules and processes that caused the visiblechanges in embryos. Experimentation was to supplement observation in the study ofembryos, and embryologists were expected to discover the properties of the embryoby seeing how the embryonic cells responded to perturbations and disruptions. Wil-helm Roux (1894), one of the founders of this branch of embryology, saw it as agrand undertaking:

We must not hide from ourselves the fact that the causal investigation of organ-isms is one of the most difficult, if not the most difficult, problem which thehuman intellect has attempted to solve … since every new cause ascertainedonly gives rise to fresh questions regarding the cause of this cause.

In this chapter, we will discuss three of the major research programs in experimentalembryology. The first concerns how forces outside the embryo influence its develop-ment. The second concerns how forces within the embryo cause the differentiationof its cells. The third looks at how the cells order themselves into tissues and organs.

WEBSITE 3.1 Establishing experimental embryology. The foundations ofEntwicklungsmechanik were laid by a group of young investigators who desired amore physiological approach to embryology. These scientists disagreed with one an-other concerning the mechanisms of development, but they cooperated to secureplaces to perform and publish their research.

Environmental Developmental Biology

The developing embryo is not isolated from its environment. In numerous instances,environmental cues are a fundamental part of the organism’s life cycle. Moreover, re-moving or altering these environmental parameters can alter development.

51

It is possible, I think, by means of exper-imentation alone, to determine how farand in what sense we can pursue the in-vestigation of the causes of form.

THOMAS HUNT MORGAN (1898)

The behaviour of a cell in an embryo de-pends on the extent to which it listens toits mother or its neighbourhood. The sizeand the nature of the noise, the way inwhich it is heard and the response areunpredictable and can only be discov-ered by experimentation.

JONATHAN BARD (1997)

Principles ofexperimental embryology

c h a p t e r3

2ND PASS PAGE PROOFS

52 Chapter 3

Environmental sexdetermination

SEX DETERMINATION IN AN ECHIUROID WORM: BONELLIA.

When the field of developmental mechanics was first formu-lated, some of the obvious variables to manipulate were thetemperature and media in which embryos were developing.These early studies initiated several experimental programson the effects of the environment on development. For in-stance, Baltzer (1914) showed that the sex of the echiuroidworm Bonellia viridis depended on where the larva settled.The female Bonellia worm is a marine, rock-dwelling animal,with a body about 10 cm long (Figure 3.1). She has a proboscisthat can extend over a meter in length. The male Bonellia,however, is only 1–3 mm long and resides within the uterus ofthe female, fertilizing her eggs. Baltzer showed that if aBonellia larva settles on the seafloor, it becomes a female.However, should a larva land on a female’s proboscis (whichapparently emits chemical signals that attract larvae), it entersthe female’s mouth, migrates into her uterus, and differenti-ates into a male. Thus, if a larva lands on the seafloor, it be-comes female; if it settles on a proboscis, it becomes male.Baltzer (1914) and Leutert (1974) were able to duplicate thisphenomenon in the laboratory, incubating larvae in either theabsence or presence of adult females (Figure 3.2).

SEX DETERMINATION IN A VERTEBRATE: ALLIGATOR. The ef-fects of the environment on development can have importantconsequences. Recent research has shown that the sex of alli-gators, crocodiles, and many other reptiles depends not onchromosomes, but on temperature. After studying the sex de-

termination of the Mississippi alligator both in the laboratoryand in the field, Ferguson and Joanen (1982) concluded thatsex is determined by the temperature of the egg during thesecond and third weeks of incubation. Eggs incubated at 30°Cor below during this time period produce female alligators,whereas those eggs incubated at 34°C or above produce males.(At 32°C, 87% of the hatchlings were female.) Moreover,whereas nests built in wet marshes (close to 30°C) produce fe-males, nests constructed on levees (close to 34°C) give rise tomales. These findings are obviously important to wildlifemanagers and farmers who wish to breed this species. Theyalso raise questions of environmental policy, since the shadeof buildings or the heat of thermal effluents can have dra-matic effects on sex ratios among reptiles. We will discuss themechanisms of temperature-dependent sex determinationfurther in Chapter 17.

WEBSITE 3.2 The hazards of environmental sex deter-mination. Ferguson and Joanen (1982) speculate that tem-perature-dependent sex determination may have been responsible for the extinction of the dinosaurs. The depen-dence on temperature for sex determination may also bedangerous for reptilian species in our present era of climatechange.

Adaptation of embryos and larvae to their environments

PHENOTYPIC PLASTICITY. Another program of environmen-tal developmental biology concerns how the embryo adapts toits particular environment. August Weismann (1875) pio-neered the study of larval adaptations, and recent research inthis area has provided some fascinating insights into how anorganism’s development is keyed to its environment.Weismann noted that butterflies that hatched during different

Figure 3.1Sexual dimorphism in Bonellia viridis. The body of the mature fe-male is about 10 cm in length, but the proboscis can extend up to ameter. The body of the symbiotic male is a minute 1–3 mm inlength. While the body of the adult female is buried in the oceansediments, her proboscis extends out of the sediments, where it canbe used for feeding or attracting larvae.

10 cm

Male Bonellia (lives symbiotically insidethe reproductiveorgans of the female)3 mm

Female Bonellia

Proboscis (mayextend 1 meter)

Male Female

100

80

60

40

20

0

Per

cen

t

Pure seawater

Indifferent

Seawater and proboscis fragments

Figure 3.2In vitro analysis of Bonellia sex determination. Larval Bonellia wereplaced either in normal seawater or in seawater containing frag-ments of the female proboscis. A majority of the animals cultured inthe presence of the proboscis fragments became males, whereas intheir absence, most became females. (After Leutert 1974.)


seasons were colored differently, and that this season-depen-dent coloration could be mimicked by incubating larvae atdifferent temperatures. Phenotypic variants that result fromenvironmental differences are often called morphs. One ex-ample of such seasonal variation is the European map butter-fly, Araschnia levana, which has two seasonal phenotypes sodifferent that Linnaeus classified them as two different species(van der Weele 1995). The spring morph is bright orange withblack spots, while the summer morph is mostly black with awhite band (Figure 3.3). The shift from spring to summermorph is controlled by changes in both day length and tem-perature during the larval period. When researchers experi-mentally mimic spring conditions, summer caterpillars cangive rise to “spring” butterflies (Koch and Buchmann 1987;Nijhout 1991).

Another dramatic example of seasonal change in devel-opment occurs in the moth Nemoria arizonaria. This mothhas a fairly typical insect life cycle. Eggs hatch in the spring,and the caterpillars feed on young oak flowers (catkins).These larvae metamorphose in the late spring, mate in thesummer, and lay eggs on the oak trees, producing anotherbrood of caterpillars. These caterpillars eat the oak leaves,metamorphose, and mate. Their eggs overwinter to start thecycle over again next spring. What is remarkable is that the

caterpillars that hatch in the spring look nothing like theirprogeny that hatch in the summer (Figure 3.4). The caterpil-lars that hatch in the spring and eat oak catkins (flowers) areyellow-brown, rugose, and beaded, resembling nothing elsebut an oak catkin. They are magnificently camouflagedagainst predation. But what of the caterpillars that hatch inthe summer, after all the catkins are gone? They, too, are wellcamouflaged, resembling year-old oak twigs. What controlsthis difference? By doing reciprocal feeding experiments,Greene (1989) was able to convert spring caterpillars intosummer morphs by feeding them oak leaves. The reciprocalexperiment did not turn the summer morphs into catkin-likecaterpillars. Thus, it appears that the catkin form is the “de-fault state” and that something induces the twiglike morphol-ogy. That something is probably a tannin that is concentratedin oak leaves as they mature.

Embryologists have emphasized that what gets inheritedis not a deterministic genotype, but rather a genotype that en-codes a potential range of phenotypes. The environment is

Principles of experimental embryology 532ND PASS PAGE PROOFS

Figure 3.3Two morphs of Araschnia levana, the European map butterfly. Thesummer morph is represented at the top, the spring morph at thebottom. In this species, the phenotypic differences are elicited bydifferences in day length and temperature during the larval period.(Photographs courtesy of H. F. Nijhout.)

Figure 3.4Two morphs of Nemoria arizonaria. (A) Caterpillars that hatch inthe spring eat oak catkins and develop a cuticle that resembles theseflowers. (B) Caterpillars that hatch in the summer (after the catkinsare gone) eat oak leaves. These caterpillars develop a cuticle that re-sembles young oak twigs. (Photographs courtesy of E. Greene.)

(A)

(B)

often able to select the phenotype that is adaptive forthat season or habitat. The continuous range of phe-notypes expressed by a single genotype across arange of environmental conditions is called the reac-tion norm (Woltereck 1909; Schmalhausen 1949;Stearns et al. 1991; Schlichting and Pigliucci 1998).

PROTECTING THE EGG FROM UV RADIATION.

Survival in their environments poses daunting chal-lenges for embryos. Indeed, as Darwin clearly noted,most eggs and embryos fail to survive. A sea urchinmay broadcast tens of thousands of eggs into the sea-water, but only one or two of the resulting embryoswill become adult urchins. Most become food forother organisms. Moreover, if the environmentchanges, embryonic survival may be increased or de-creased dramatically. For instance, many eggs andearly embryos lie in direct sunlight for long periods.If we lie in the sun for hours without sunscreen, weget a burn from the sun’s ultraviolet rays of the sun;this UV radiation is harmful to our DNA. So how doeggs survive all those hours of constant exposure tothe sun (often on the same beaches where we sunourselves)?

First, it seems that many eggs have evolved nat-ural sunscreens. The eggs of many marine organismscontain high concentrations of mycosporine-likeamino acid pigments, which absorb ultraviolet radi-ation (UV-B). Moreover, just like our melanin pig-ment, these pigments can be induced by exposure toUV-B radiation (Jokiel and York 1982; Siebeck 1988). Theeggs of tunicates are very resistant to UV-B radiation, andmuch of this resistance comes from extracellular coats that areenriched with mycosporine compounds (Mead and Epel1995). Adams and Shick (1996; 2001) experimentally manip-ulated the amount of mycosporine-like amino acids in seaurchin eggs and found that embryos from eggs with more ofthese compounds were better protected from UV damagethan embryos with less. Moreover, when mycosporine-defi-cient eggs were exposed to ultraviolet radiation, significantdevelopmental anomalies were seen (Figure 3.5). Thus, thesemycosporine-like amino acids appear to play an importantrole in protecting the developing sea urchin embryo againstultraviolet radiation.

VADE MECUM Sea urchins and UV radiation. This seg-ment presents data documenting the protection of seaurchin embryos by mycosporine-like amino acids. Thistype of research is linking developmental biology with ecol-ogy and conservation biology.[Click on Sea Urchin-UV]

It is possible that increased UV-B exposure could be animportant factor in the decline in amphibian populationsseen throughout the world during the past two decades. Pop-

ulations of amphibians in widely scattered locations havebeen drastically reduced in the past two decades, and some ofthese species (such as the golden toad of Costa Rica) have re-cently become extinct (Phillips 1994). Blaustein and his col-leagues (1994) have looked at levels of photolyase, an enzymethat repairs UV damage to DNA by excising and replacingdamaged thymidine residues, in amphibian eggs and oocytes.Levels of photolyase varied 80-fold among the tested species,and were correlated with the site of egg laying. Eggs more ex-posed to the sun had higher levels of photolyase (Table 3.1).These levels also correlated with whether or not the specieswas suffering population decline. The highest photolyase lev-els were found in those species (such as the Pacific tree frog,Hyla regilla) whose populations were not in decline. The low-

54 Chapter 32ND PASS PAGE PROOFS

UV-filtered lightUV-filtered light +UV-A and UV-B

(A) (D)

(B) (E)

(C) (F)

Earlyblastula

Gastrula

Pluteuslarva

Figure 3.5The effect of ultraviolet (UV) radiation on embryos of the seaurchin Strongylocentrotus droebachiensis. Eggs were fertilized andplaced in seawater lacking sources of mycosporine-like amino acids.The first column (A–C) represents embryos grown in light lackingultraviolet radiation. The second column (D–F) shows the samestage embryos grown in the presence of such filtered light, but withultraviolet radiation added. The “s” shows isolated skeletal spicules.(From Adams and Shick 2001; photographs courtesy of the authors.)

est levels were seen in those species (such as the Western toad,Bufo boreas, and the Cascades frog, Rana cascadae) whosepopulations had declined dramatically.

Blaustein and his colleagues tested whether or not UV-Bcould be a factor in lowering the hatching rate of amphibianeggs. At two field sites, they divided the eggs of each of threeamphibian species into three groups (Figure 3.6). The firstgroup developed without any sun filter. The second group de-veloped under a filter that allowed UV-B to pass through. Thethird group developed under a filter that blocked UV-B fromreaching the eggs. For Hyla regilla, the filters had no effect,and hatching success was excellent under all three conditions.For Rana cascadea and Bufo boreas, however, the UV-B block-ing filter raised the percentage of eggs hatched from about60% to close to 80%.

The effects of UV-B radiation in mediating amphibianpopulation declines appears to be complex, involving climate

change and fungal pathogens. Using long-term observationaldata and by manipulating the depth of pond water in whichfrogs laid their eggs, Kiesecker and colleagues (2001) showedthat climate-induced reductions in pond water depth increasethe exposure of eggs and embryos to UV-B radiation and,consequently, increase their vulnerability to fungal infection.In these studies, water depth and UV-B exposure in the PacificNorthwest of the United States was strongly linked to El Niñocycles. Moreover, elevated sea-surface temperatures in thesouthern Pacific since the mid-1970s, which have affected theclimate over much of the world, could produce conditions forthe pathogen-mediated amphibian declines in many regions.Therefore, UV-B radiation may be the factor that linkschanges in climate with changes in amphibian mortality.

The environmental programs of experimental embryol-ogy were a major part of the discipline when Entwick-lungsmechanik was first established. However, it soon became


TABLE 3.1 Photolyase activity correlated with exposure of eggs to ultraviolet radiation in 10 amphibian species

Species Photolyase activitya Mode of egg-laying Exposure to sunlight

Plethodon dunni <0.1 Eggs hidden None

Xenopus laevis 0.1 Eggs laid in laboratoryb Limited

Triturus granulosa 0.2 Eggs hidden Limited

Rana variegatus 0.3 Eggs hidden None

Plethodon vehiculum 0.5 Eggs hidden None

Ambystoma macrodactylum 0.8 Eggs often laid in open water Some

Ambystoma gracile 1.0 Eggs often laid in open water Some

Bufo boreas 1.3 Eggs laid in open, often in shallow water High

Rana cascadae 2.4 Eggs laid in open shallow water High

Hyla regilla 7.5 Eggs laid in open shallow water High

Source: After Blaustein et al. 1994.aSpecific activity of photolyase, 1011 thymidine dimers separated per hour per mg. The values are averages of 6–8 assays.

bIn nature, Xenopus laevis eggs are laid under vegetation with limited exposure to sunlight.

P

P

P

O

DNA backbone

Thymidinemonomers

Photolyase

UV (200–300 nm)

O

CH3H

C

CN O

H

O

O

H

H

N

C C

O

O

O

DNA backbone

Thymidinedimer

P

P

TC N

T

O

CH3H

C

CN O

H

N

C C

T

CH3H

C

CN O

C O

N

C

CH3HC

C

C

T

P

N

Normal UV-damaged

Base spacing distorted

Thymidinedimer bonds

obvious that experimental variables couldbe better controlled in the laboratory thanin the field, and that a scientist could domany more experiments in the laboratory.Thus, field experimentation in embryologydwindled in the first decades of the twenti-eth century (see Nyhart 1995). However,with our increasing concern about the en-vironment, this area of developmental biol-ogy has become increasingly important.Other recent work in this field will be de-tailed in Chapter 22.

The Developmental Dynamics of Cell Specification

An embryo’s environment may be a tide pool, a pond, or auterus. As we saw above, the embryo interacts with its envi-ronment, and its developmental trajectory can be guided byinformation from its surroundings. On a smaller scale, the en-vironment of an embryonic cell consists of the surroundingtissues within the embryo, and the fate of that cell (for in-stance, whether it becomes part of the skin or part of the lens)often depends upon its interactions with other components ofits immediate “ecosystem.”

Thus, a second research program of experimental embry-ology studies how interactions between embryonic cells gen-erate the embryo. The development of specialized cell types iscalled differentiation (Table 3.2). These overt changes in cel-lular biochemistry and function are preceded by a process re-sulting in the commitment of the cell to a certain fate. At thispoint, even though the cell or tissue does not differ phenotyp-ically from its uncommitted state, its developmental fate hasbecome restricted.

The process of commitment can be divided into twostages (Harrison 1933; Slack 1991). The first stage is a labilephase called specification. The fate of a cell or a tissue is saidto be specified when it is capable of differentiating au-tonomously when placed in a neutral environment, such as apetri dish or test tube. (The environment is neutral with re-spect to the developmental pathway.) At this stage, the com-mitment is still capable of being reversed. The second stage of

commitment is determination. A cell or tissue is said to be de-termined when it is capable of differentiating autonomouslyeven when placed into another region of the embryo. If it isable to differentiate according to its original fate even underthese circumstances, it is assumed that the commitment is irre-versible.*

Autonomous specification

Three basic modes of commitment have been described(Table 3.3; Davidson 1991). The first is called autonomousspecification. In this case, if a particular blastomere is re-moved from an embryo early in its development, that isolatedblastomere will produce the same types of cells that it wouldhave made if it were still part of the embryo (Figure 3.7).Moreover, the embryo from which that blastomere is takenwill lack those cells (and only those cells) that would havebeen produced by the missing blastomere. Autonomous speci-fication gives rise to a pattern of embryogenesis referred to asmosaic development, since the embryo appears to be con-structed like a tile mosaic of independent, self-differentiatingparts. Invertebrate embryos, especially those of molluscs, an-nelids, and tunicates, often use autonomous specification todetermine the fates of their cells. In these embryos, morpho-genetic determinants (certain proteins or messenger RNAs)


1009080706050

Site 1Hyla regilla

Rana cascadae

Bufo boreas

Site 2

Site 3

Site 2

Site 2

Site 4

Percent surviving to hatching

UV-B blocking filter

UV-B transmitting filter

No filter

Figure 3.6Hatching success rates in three amphibian species in thefield. At each of two sites, eggs were placed in enclosuresthat were unshielded, shielded with an acetate screen thatadmitted UV-B radiation, or shielded with a Mylar screenthat blocked UV-B radiation. Eggs of the tree frog Hylaregilla hatched successfully under all three conditions. Eggsof the frog Rana cascadae and the toad Bufo boreas hatchedsignificantly better when protected from UV-B radiation.(After Blaustein et al. 1994.)

*Recall the discussion of specification of spore and stalk cells in the slimemold Dictyostelium in the previous chapter. This irreversibility of determina-tion and differentiation is only with respect to normal development. As Dollyand other cloned animals have shown, the nucleus of a differentiated cell canbe reprogrammed experimentally to give rise to any cell type in the body. Wewill discuss this phenomenon in detail in the next chapter.

are placed in different regions of the eggcytoplasm and are apportioned to thedifferent cells as the embryo divides.These morphogenetic determinantsspecify the cell type.

Autonomous specification was firstdemonstrated in 1887 by a French med-ical student, Laurent Chabry. Chabry de-sired to know the causes of birth defects,and he reasoned that such malformationsmight be caused by the lack of certaincells. He decided to perform experimentson tunicate embryos, since they have rel-atively large cells and were abundant in anearby bay. This was a fortunate choice,because tunicate embryos develop rap-idly into larvae with relatively few cellsand cell types (Chabry 1887; Fischer1991). Chabry set out to produce specificmalformations by isolating or lancingspecific blastomeres of the cleaving tuni-cate embryo. He discovered that eachblastomere was responsible for produc-ing a particular set of larval tissues (Fig-ure 3.8). In the absence of particularblastomeres, the larva lacked just thosestructures normally formed by thosecells. Moreover, he observed that whenparticular cells were isolated from the


Cell type Differentiatedof cell cell product Specialized function

Keratinocyte Keratin Protection against(epidermal cell) abrasion, desiccation

Erythrocyte Hemoglobin Transport of oxygen(red blood cell)

Lens cell Crystallins Transmission of light

B lymphocyte Immunoglobulins Antibody synthesis

T lymphocyte Cytokines Destruction of foreigncells; regulation ofimmune response

Melanocyte Melanin Pigment production

Pancreatic islet cell Insulin Regulation of carbohydratemetabolism

Leydig cell (ç) Testosterone Male sexual characteristics

Chondrocyte Chondroitin sulfate; Tendons and ligaments(cartilage cell) type II collagen

Osteoblast Bone matrix Skeletal support(bone-forming cell)

Myocyte Muscle actin Contraction(muscle cell) and myosin

Hepatocyte Serum albumin; Production of serum(liver cell) numerous proteins and numerous

enzymes enzymatic functions

Neurons Neurotransmitters Transmission of(acetylcholine, electric impulsesepinephrine, etc.)

Tubule cell (å) of Ovalbumin Egg white proteins for hen oviduct nutrition and protection

of embryo

Follicle cell (å) of Chorion proteins Eggshell proteins forinsect ovary protection of embryo

TABLE 3.2 Some differentiated cell types and their major products

Normal development of Patella

Presumptivetrochoblast

(A)

(D) (E) (F) (G)

(B) (C)

Isolated trochoblast development

Figure 3.7Autonomous specification (mosaic develop-ment). (A–C) Differentiation of trochoblast(ciliated) cells of the mollusc Patella. (A) 16-cell stage seen from the side; the presump-tive trochoblast cells are shaded. (B) 48-cellstage. (C) Ciliated larval stage, seen from theanimal pole. (D–G) Differentiation of aPatella trochoblast cell isolated from the 16-cell stage and cultured in vitro. (E, F) Resultsof the first and second divisions in culture.(G) Ciliated products of (F). Even in isolatedculture, these cells divide and become ciliat-ed at the correct time. (After Wilson 1904.)

*This was not the answer Chabry expected, nor theone he had hoped to find. In nineteenth-centuryFrance, conservatives favored preformationistviews, which were interpreted to support hereditaryinequalities between members of a human commu-nity. What you were was determined by your lin-eage. Liberals, especially Socialists, favored epige-netic views, which were interpreted to indicate thateveryone started off with an equal hereditary en-dowment, and that no one had a “right” to a higherposition than any other person. Chabry, a Socialistwho hated the inherited rights of the aristocrats,took pains not to extrapolate his data to anythingbeyond tunicate embryos (see Fischer 1991).

rest of the embryo, they formed their characteristic structuresapart from the context of the other cells. Thus, each of the tu-nicate cells appeared to be developing autonomously.*

Recent studies have confirmed that when particular cells ofthe 8-cell tunicate embryo are removed, the embryo lacks thosestructures normally produced by the missing cells, and the iso-lated cells produce these structures away from the embryo. J. R.Whittaker provided dramatic biochemical confirmation of thecytoplasmic segregation of the morphogenetic determinants re-sponsible for this pattern. Whittaker (1973) stained blastomeresfor the presence of the enzyme acetylcholinesterase. This en-zyme is found only in muscle tissue and is involved in enablinglarval muscles to respond to repeated nerve impulses. From thecell lineage studies of Conklin and others (see Chapter 1), it wasknown that only one pair of blastomeres (the posterior vegetalpair, B4.1) in the 8-cell tunicate embryo is capable of producingtail muscle tissue. (As discussed in Chapter 1, the B4.1 blas-tomere pair contains the yellow crescent cytoplasm that corre-lates with muscle determination.) When Whittaker removedthese two cells and placed them in isolation, they producedmuscle tissue that stained positively for the presence of acetyl-

cholinesterase (Figure 3.9). When hetransferred some of the yellow crescentcytoplasm of the B4.1 (muscle-forming)blastomere into the b4.2 (ectoderm-forming) blastomere of an 8-cell tunicateembryo, the ectoderm-forming blas-tomere generated muscle cells as well asits normal ectodermal progeny (Figure3.10; Whittaker 1982). The mechanismsof autonomous specification will be de-tailed in Chapter 8.

Conditional specification

THE PHENOMENON OF CONDITIONAL SPEC-

IFICATION. A second mode of commit-ment involves interactions among neigh-boring cells. In this type of specification,each cell originally has the ability to be-come any of many different cell types.However, interactions of the cell withother cells restrict the fate of one or moreof the participants. This mode of com-mitment is sometimes called conditional


TABLE 3.3 Modes of cell type specification and their characteristics

I. Autonomous specificationCharacteristic of most invertebrates.

Specification by differential acquisition of certain cytoplasmic molecules present in the egg.

Invariant cleavages produce the same lineages in each embryo of the species. Blasto-mere fates are generally invariant.

Cell type specification precedes any large-scale embryonic cell migration.

Produces “mosaic” development: cells cannot change fate if a blastomere is lost.

II. Conditional specificationCharacteristic of all vertebrates and few invertebrates.

Specification by interactions between cells. Relative positions are important.

Variable cleavages produce no invariant fate assignments to cells.

Massive cell rearrangements and migrations precede or accompany specification.

Capacity for “regulative” development: allows cells to acquire different functions.

III. Syncytial specificationCharacteristic of most insect classes.

Specification of body regions by interactions between cytoplasmic regions prior to cellularization of the blastoderm.

Variable cleavage produces no rigid cell fates for particular nuclei.

After cellularization, conditional specification is most often seen.

Source: After Davidson 1991.

Anterior Posterior

Vegetal pole

Animal pole

Muscle

MesenchymeEndoderm

Notochord

Ectoderm

Nervoussystem b4.2

B4.1

a4.2

A4.1

Ectoderm

b4.2

B4.1

a4.2

A4.1

Ectoderm

Endoderm Endoderm

Muscle

Mesenchyme

Notochord

Figure 3.8Autonomous specification in the early tunicate embryo. When thefour blastomere pairs of the 8-cell embryo are dissociated, eachforms the structures it would have formed had it remained in theembryo. (The fate map of the tunicate shows that the left and rightsides produce identical cell lineages.) (After Reverberi and Minganti1946.)

specification because the fate of a cell depends upon the condi-tions in which the cell finds itself. If a blastomere is removedfrom an early embryo that uses conditional specification, theremaining embryonic cells alter their fates so that the roles ofthe missing cells can be taken over. This ability of embryoniccells to change their fates to compensate for the missing partsis called regulation (Figure 3.11). The isolated blastomere canalso give rise to a wide variety of cell types (and sometimesgenerates cell types that the cell would normally not havemade if it were still part of the embryo). Thus, conditionalspecification gives rise to a pattern of embryogenesis calledregulative development.* Regulative development is seen inmost vertebrate embryos, and it is obviously critical in the de-velopment of identical twins. In the formation of such twins,the cleavage-stage cells of a single embryo divide into twogroups, and each group of cells produces a fully developed in-dividual (Figure 3.12).

The research leading to the discovery of conditional spec-ification began with the testing of a hypothesis claiming thatthere was no such thing. In 1893, August Weismann proposedthe first testable model of cell specification, the germ plasmtheory. Based on the scant knowledge of fertilization availableat that time, Weismann boldly proposed that the sperm andegg provided equal chromosomal contributions, both quanti-tatively and qualitatively, to the new organism. Moreover, hepostulated that the chromosomes carried the inherited poten-


(A)

A4.1

b4.2a4.2

B4.1

Muscle-forming cytoplasm(“yellow crescent”)

Blastomere pair B4.1separated by glass needle

(B) (C)

(D)

Figure 3.9Acetylcholinesterase in the progeny of the muscle lineage blas-tomeres (B4.1) isolated from a tunicate embryo at the 8-cell stage.(A) Diagram of the isolation procedure. (B) Localization of acetyl-cholinesterase in the tail muscles of an intact tunicate larva. Thepresence of the enzyme is demonstrated by the dark staining. Thesame dark staining is seen in the progeny of the B4.1 blastomerepair (C), but not in the remaining 6/8 of the embryo (D) when in-cubated for the length of time it normally takes to form a larva.(From Whittaker 1977; photographs courtesy of J. R. Whittaker.)

Vegetal poleMuscle-formingregion

Needle pushes muscle-formingcytoplasm into animal cells

Animal pole

B4.1

b4.2 b4.2

B4.1

b4.2

B4.1

b4.2

B4.1

b4.2

B4.1

b4.2 b4.2Tissue includingmuscle cells

Tissue includingmuscle cells

b4.2

B4.1B4.1 B4.1

Figure 3.10Microsurgery on tunicate eggs forces some of the yellow crescent cytoplasm of the muscle-forming B4.1 blastomeres to enter the b4.2 (epidermis- and nerve-producing) blastomere pair.Pressing the B4.1 blastomeres with a glass needle causes the regression of the cleavage furrow.The furrow will re-form at a more vegetal position where the cells are cut with a needle. Thenew furrow will thereby separate the cells in such a way that the b4.2 blastomeres receive someof the muscle-forming (“yellow crescent”) B4.1 cytoplasm. These modified b4.2 cells producemuscle cells as well as their normal ectodermal progeny. (After Whittaker 1982.)

*Sydney Brenner (quoted in Wilkins 1993) has remarked that animal devel-opment can proceed according to either the American or the European plan.Under the European plan (autonomous specification), you are what yourprogenitors were. Lineage is important. Under the American plan (condition-al specification), the cells start off undetermined, but with certain biases.There is a great deal of mixing, lineages are not critical, and one tends to be-come what one’s neighbors are.

†Embryologists were thinking in these terms some 15 years before the redis-covery of Mendel’s work. Weismann (1892, 1893) also speculated that thesenuclear determinants of inheritance functioned by elaborating substancesthat became active in the cytoplasm!

tials of this new organism.† However, not all the determinantson the chromosomes were thought to enter every cell of theembryo. Instead of dividing equally, the chromosomes werehypothesized to divide in such a way that different chromoso-mal determinants entered different cells. Whereas the fertil-ized egg would carry the full complement of determinants,certain somatic cells would retain the “blood-forming” deter-minants while others would retain the “muscle-forming” de-


(A)

(B)

Blastula

Cells formback tissue

Cells formbelly tissue

Normaldevelopment

Glass needleremoves cells

Notransplantation(normal development)

Normalbackcells

Transplant normalback cells tobelly region

Figure 3.11Conditional specification. (A)What a cell becomes dependsupon its position in the embryo.Its fate is determined by interac-tions with neighboring cells. (B) Ifcells are removed from the em-bryo, the remaining cells can regu-late and compensate for the miss-ing part.

Figure 3.12In the early developmental stages of many vertebrates, the separa-tion of the embryonic cells into two parts can create twins. Thisphenomenon occurs sporadically in humans. However, in the nine-banded armadillo, Dasypus novemcinctus, the original embryo al-ways splits into four separate groups of cells, each of which forms itsown embryo. (Photograph courtesy of K. Benirschke.)

Neu

ral d

eter

minants

Blood determinants

Muscle

determinants

All determinants

Germcell

Germcell

Germcell

Continuity of germ cells

Differentiationof somaticcells

Figure 3.13Weismann’s theory of inheritance. The germ cell gives rise to thedifferentiating somatic cells of the body (indicated in color), as wellas to new germ cells (blue). Weismann hypothesized that only thegerm cells contained all the inherited determinants. The somaticcells were each thought to contain a subset of the determinants. Thetypes of determinants found in the nucleus would determine the celltype. (After Wilson 1896.)

terminants, and so forth (Figure 3.13). Only in the nuclei ofthose cells destined to become gametes (the germ cells) wereall types of determinants thought to be retained. The nuclei ofall other cells would have only a subset of the original deter-minant types.

In postulating this model, Weismann had proposed a hy-pothesis of development that could be tested immediately.Based on the fate map of the frog embryo, Weismann claimedthat when the first cleavage division separated the future righthalf of the embryo from the future left half, there would be aseparation of “right” determinants from “left” determinants inthe resulting blastomeres. The testing of this hypothesis pio-neered three of the four major techniques involved in experi-mental embryology:

• The defect experiment, wherein one destroys a portionof the embryo and then observes the development of theimpaired embryo.

• The isolation experiment, wherein one removes a por-tion of the embryo and then observes the development ofthe partial embryo and the isolated part.

• The recombination experiment, wherein one observesthe development of the embryo after replacing an origi-nal part with a part from a different region of the em-bryo.

• The transplantation experiment, wherein one portionof the embryo is replaced by a portion from a differentembryo. This fourth technique was used by some of thesame scientists when they first constructed fate maps ofearly embryos (see Chapter 1).

One of the first scientists to test Weismann’s hypothesiswas Wilhelm Roux, a young German embryologist. In 1888,Roux published the results of a series of defect experiments inwhich he took 2- and 4-cell frog embryos and destroyed someof the cells of each embryo with a hot needle. Weismann’s hy-pothesis predicted the formation of right or left half-embryos.Roux obtained half-blastulae, just as Weismann had predicted(Figure 3.14). These developed into half-neurulae having acomplete right or left side, with one neural fold, one ear pit,and so on. He therefore concluded that the frog embryo was a

mosaic of self-differentiating parts, and that each cell proba-bly received a specific set of determinants and differentiatedaccordingly.

Nobody appreciated Roux’s work and the experimentalapproach to embryology more than Hans Driesch. Driesch’sgoal was to explain development in terms of the laws ofphysics and mathematics. His initial investigations were simi-lar to those of Roux. However, while Roux’s studies were de-fect experiments that answered the question of how the re-maining blastomeres of an embryo would develop when asubset of blastomeres was destroyed, Driesch (1892) sought toextend this research by performing isolation experiments. Heseparated sea urchin blastomeres from each other by vigorousshaking (or, later, by placing them in calcium-free seawater).To Driesch’s surprise, each of the blastomeres from a 2-cellembryo developed into a complete larva. Similarly, when Dri-esch separated the blastomeres of 4- and 8-cell embryos, someof the isolated cells produced entire pluteus larvae (Figure3.15). Here was a result drastically different from the predic-tions of Weismann or Roux. Rather than self-differentiatinginto its future embryonic part, each isolated blastomere regu-lated its development so as to produce a complete organism.Moreover, these experiments provided the first experimentallyobservable instance of regulative development.

Driesch confirmed regulative development in sea urchinembryos by performing an intricate recombination experi-ment. In sea urchin eggs, the first two cleavage planes are nor-mally meridional, passing through both the animal and vege-tal poles, whereas the third division is equatorial, dividing theembryo into four upper and four lower cells. Driesch (1893)changed the direction of the third cleavage by gently com-pressing early embryos between two glass plates, thus causingthe third division to be meridional like the preceding two.After he released the pressure, the fourth division was equato-rial. This procedure reshuffled the nuclei, causing a nucleusthat normally would be in the region destined to form endo-


Hot needle

Cleavage

Fertilizedfrog egg

2-Cell stage Blastula Neurula stage

Destroyed half (dead tissue)

Deadtissue

Livingtissue

Half-embryo

Figure 3.14Roux’s attempt to demonstrate mosaic development. Destroying(but not removing) one cell of a 2-cell frog embryo results in thedevelopment of only one-half of the embryo.

derm to now be in the presumptive ectoderm re-gion. Some nuclei that would normally have pro-duced dorsal structures were now found in theventral cells (Figure 3.16). If segregation of nucleardeterminants had occurred (as had been proposedby Weismann and Roux), the resulting embryoshould have been strangely disordered. However,Driesch obtained normal larvae from these em-bryos. He concluded, “The relative position of ablastomere within the whole will probably in ageneral way determine what shall come from it.”

The consequences of these experiments weremomentous, both for embryology and for Drieschpersonally. First, Driesch had demonstrated that the prospec-tive potency of an isolated blastomere (those cell types it waspossible for it to form) is greater than its prospective fate(those cell types it would normally give rise to over the unal-tered course of its development). According to Weismann andRoux, the prospective potency and the prospective fate of ablastomere should have been identical. Second, Driesch con-cluded that the sea urchin embryo is a “harmonious equipo-tential system” because all of its potentially independent partsfunctioned together to form a single organism. Third, he con-cluded that the fate of a nucleus depended solely on its loca-tion in the embryo. Driesch (1894) hypothesized a series ofevents wherein development proceeded by the interactions ofthe nucleus and cytoplasm:

Insofar as it contains a nucleus, every cell, during devel-opment, carries the totality of all primordia; insofar asit contains a specific cytoplasmic cell body, it is specifi-cally enabled by this to respond to specific effects only.…When nuclear material is activated, then, under itsguidance, the cytoplasm of its cell that had first influ-enced the nucleus is in turn changed, and thus the basisis established for a new elementary process, which itselfis not only the result but also a cause.

This strikingly modern concept of nuclear-cytoplasmic inter-action and nuclear equivalence eventually caused Driesch toabandon science. Because the embryo could be subdividedinto parts that were each capable of re-forming the entire or-

ganism, he could no longer envision it as a physical machine.In other words, Driesch had come to believe that developmentcould not be explained by physical forces. Harking back toAristotle, he invoked a vital force, entelechy (“internal goal-di-rected force”), to explain how development proceeds. Essen-tially, he believed that the embryo was imbued with an inter-nal psyche and the wisdom to accomplish its goals despite theobstacles embryologists placed in its path. Unable to explainhis results in terms of the physics of his day, Driesch re-nounced the study of developmental physiology and became aphilosophy professor, proclaiming vitalism (the doctrine thatliving things cannot be explained by physical forces alone)


Normal pluteus larva

Plutei developed from single cells of 4-cell embryo

Removefertilizationenvelope

Separate into 4 cells

(B)

(A) Fertilization envelopeFigure 3.15Driesch’s demonstration of regulative development.(A) An intact 4-cell sea urchin embryo generates anormal pluteus larva. (B) When one removes the 4-cell embryo from its fertilization envelope and iso-lates each of the four cells, each cell can form asmaller, but normal, pluteus larva. (All larvae aredrawn to the same scale.) Note that the four larvaederived in this way are not identical, despite theirability to generate all the necessary cell types. Suchvariations are also seen in adult sea urchins formedin this way (Marcus 1979). (Photograph courtesy ofG. Watchmaker.)

*Driesch also became an outspoken opponent of the Nazis, and was one of thefirst non-Jewish professors to be forcibly retired when Hitler came to power(Harrington 1996). Hertwig used Driesch’s experiments and some of his ownto strengthen within embryology a type of materialist philosophy calledwholist organicism. This philosophy embraces the views that (1) the proper-ties of the whole cannot be predicted solely from the properties of the com-ponent parts, and (2) the properties of the parts are informed by their rela-tionship to the whole. As an analogy, the meaning of a sentence obviouslydepends on the meanings of its component parts, words. However, the mean-ing of each word depends on the entire sentence. In the sentence, “The partyleaders were split on the platform,” the possible meanings of each noun andverb are limited by the meaning of the entire sentence and by their relation-ships to other words within the sentence. Similarly, the phenotype of a cell inthe embryo depends on its interactions within the entire embryo. The oppo-site materialist view is reductionism, which maintains that the properties ofthe whole can be known if all the properties of the parts are known.Embryology has traditionally espoused wholist organicism as its ontology(model of reality) while maintaining a reductionist methodology (experimen-tal procedures) (Needham 1943; Haraway 1976; Hamburger 1988; Gilbert andFaber 1996).

until his death in 1941. However, others, especially Oscar Her-twig (1894), were able to incorporate Driesch’s experimentsinto a more sophisticated experimental embryology.*

VADE MECUM Sea urchin development. Roux’s andDreisch’s experiments manipulated normal development.Normal sea urchin development is seen here in video andlabeled photographs.[Click on Sea Urchin]

The differences between Roux’s experiments and those ofDriesch are summarized in Table 3.4. The difference betweenisolation and defect experiments and the importance of theinteractions among blastomeres were highlighted in 1910,when J. F. McClendon showed that isolated frog blastomeres

behave just like separated sea urchin cells. Therefore, the mo-saic-like development of the first two frog blastomeres inRoux’s study was an artifact of the defect experiment. Some-thing in or on the dead blastomere still informed the live cellsthat it existed. Therefore, even though Weismann and Rouxpioneered the study of developmental physiology, theirproposition that differentiation is caused by the segregation ofnuclear determinants was soon shown to be incorrect.

MORPHOGEN GRADIENTS. Cell fates may be specified byneighboring cells, but cell fates can also be specified by specif-ic amounts of soluble molecules secreted at a distance from thetarget cells. Such a soluble molecule is called a morphogen,and a morphogen may specify more than one cell type byforming a concentration gradient.

The concept of morphogen gradients had been used tomodel another phenomenon of regulative development: re-generation. It had been known since the 1700s that when hy-dras and planarian flatworms were cut in half, the head halfwould regenerate a tail from the wound site, while the tail halfwould regenerate a head. Allman (1864) had called attention


TABLE 3.4 Experimental procedures and results of Roux and Dreisch

Type of Interpretation concerning Investigator Organism experiment Conclusion potency and fate

Roux (1888) Frog Defect Mosaic development Prospective potency(Rana fusca) (autonomous specification) equals prospective fate

Driesch (1892) Sea urchin Isolation Regulative development Prospective potency(Echinus (conditional specification) is greater thanmicrotuberculatus) prospective fate

Dreisch (1893) Sea urchin Recombination Regulative development Prospective potency(Echinus and (conditional specification) is greater thanParacentrotus) prospective fate

8-Cell

Topview

Sideview

88B

8A 2B

6B 4A

7A7A 1B5B 3A

1A

5A 3B

2A

6A 4B

7

7

1

5

5

3

3

86

4

2

6 4

8-Cell16-Cell 16-Cell

(A) NORMAL CLEAVAGE (B) CLEAVAGE UNDER PRESSURE

3A

4A

Glass plate7B

7A5B 5A

6B 6A

3B

88A 7A 6A 5A 4A

8B 7B 6B 5B 4B7 6

45

18 1B8B

4B5B

8A7B 2B

6B 3B

1A

5A 4A

7A 2A

6A 3A36

27

5 48A 4B

8B

Figure 3.16Driesch’s pressure-plate experiment for altering the distribution ofnuclei. (A) Normal cleavage in 8- to 16-cell sea urchin embryos,seen from the animal pole (upper sequence) and from the side(lower sequence). (B) Abnormal cleavage planes formed under pres-sure, as seen from the animal pole and from the side. (After Huxleyand de Beer 1934.)

to the fact that this phenomenon indicated a polarity in theorganization of the hydra. It was not until 1905, however, thatThomas Hunt Morgan (1905, 1906) realized that such polar-ity indicated an important principle in development. Hepointed out that if the head and tail were both cut off a flat-worm, leaving only the medial segment, that segment wouldregenerate a head from the former anterior end and a tailfrom the former posterior end— never the reverse (Figure3.17A,B). Moreover, if the medial segment were sufficientlysmall, the regenerating portions would be abnormal (Figure3.17C). Morgan postulated a gradient of anterior-producingmaterials concentrated in the head region. The middle seg-ment would be told what to regenerate at both ends by the

concentration gradient of these materials. If the piece weretoo small, however, the gradient would not be sensed withinthe segment. (It is possible that there are actually two gradi-ents in the flatworm, one to direct the formation of a headand one to direct the production of a tail. Regeneration willbe discussed in more detail in Chapter 18.)

VADE MECUM Flatworm regeneration. You should see itfor yourself. Flatworms are easy to obtain, and cutting theanimal in half does nothing more than what the animaldoes to itself. Here are videos and easy instructions for ex-perimenting with these fascinating animals.[Click on Flatworm]

In the 1930s through the 1950s, gradient models wereused to explain conditional cell specification in sea urchinand amphibian embryos (Hörstadius and Wolsky 1936;Hörstadius 1939; Toivonen and Saxén 1955). In the 1960s,these gradient models were extended to explain how cellsmight be told their position along an embryonic axis(Lawrence 1966; Stumpf 1966; Wolpert 1968, 1969). Accord-ing to such models, a soluble substance—the morphogen—diffuses from its site of synthesis (source) to its site of degra-dation (sink). Wolpert (1968) illustrated such a gradient ofpositional information using “the French flag analogy.” Imag-ine a row of “flag cells,” each of which is capable of differenti-ating into a red, white, or blue cell. Then imagine a mor-phogen whose source is on the left-hand edge of the bluestripe, and whose sink is at the other end of the flag, on theright-hand edge of the red stripe. A concentration gradient isthus formed, being highest at one end of the “flag tissue” andlowest at the other. The specification of the multipotentialcells in this tissue is accomplished by the concentration of themorphogen. Cells sensing a high concentration of the mor-phogen become blue. Then there is a threshold of mor-phogen concentration below which cells become white. Asthe declining concentration of morphogen falls below an-other threshold, the cells become red (Figure 3.18A).

Different tissues may use the same gradient system, butrespond to the gradient in a different way. If cells that wouldnormally become the middle segment of a Drosophila leg areremoved from the leg-forming area of the larva and placed inthe region that will become the tip of the fly’s antenna, theydifferentiate into claws. These cells retain their committed sta-tus as leg cells, but respond to the positional information of


(A)

(B)

(C)

(D)

BisectRegeneration

Regeneration

Regeneration

Regeneration

Regeneration

Regeneration

Removehead first

Next, removetail

Figure 3.17Flatworm regeneration and its limits. (A) If a flatworm is cut in two, the anterior portionof the lower half regenerates a head, while the posterior of the upper half regenerates atail. The same tissue can generate a head (if it is at the anterior portion of the tail piece)or a tail (if it at the posterior portion of the head piece). (B) If a flatworm is cut into threepieces, the middle piece will regenerate a head from its anterior end and a tail from itsposterior end. (C) However, if the middle piece is too thin, there is no morphogen gradi-ent within it, and regeneration is abnormal. (D) If the second cut is delayed, however, anequally thin middle section forms a normal worm, since the time lag has allowed an ante-rior–posterior gradient to become established. (After Gosse 1969.)

their environment. Thereby, they became leg tip cells—claws.This phenomenon, said Wolpert, is analogous to reciprocallytransplanting portions of the American and French flags intoeach other. The segments will retain their identity (French orAmerican), but will be positionally specified (develop colors)appropriate to their new positions (Figure 3.18B).

WEBSITE 3.3 Receptor gradients. In addition to a gra-dient of morphogen, there can also be a gradient of thosemolecules that recognize the morphogen. The interplay ofmorphogen gradients and the gradients of molecules thatinterpret them can give rise to interesting developmentalpatterns.

The molecules involved in establishing morphogen gradi-ents are beginning to be identified. For a diffusible moleculeto be considered a morphogen, it must be demonstrated thatcells respond directly to that molecule and that the differenti-ation of those cells depends upon the concentration of thatmolecule. One such system currently being analyzed concernsthe ability of different concentrations of the protein activin tospecify different fates in the frog Xenopus. In the Xenopus blas-tula, the cells in the middle of the embryo become mesoder-mal by responding to activin (or an activin-like compound)produced in the vegetal hemisphere. Makoto Asashima andhis colleagues (Fukui and Asashima 1994; Ariizumi andAsashima 1994) have shown that the animal cap of the Xeno-pus embryo (which normally becomes ectoderm, but whichcan be induced to form mesoderm if transplanted into otherregions within the embryo) responds differently to differentconcentrations of activin. If left untreated in saline solution,animal cap blastomeres form an epidermis-like mass of cells.However, if exposed to small amounts of activin, they formventral mesodermal tissue—blood and connective tissue. Pro-gressively higher concentrations of activin will cause the ani-mal cap cells to develop into other types of mesodermal cells:muscles, notochord cells, and heart cells (Figure 3.19).

John Gurdon’s laboratory has shown that these animal capcells respond to activin by changing their expression of partic-ular genes (Figure 3.20; Gurdon et al. 1994, 1995). Gurdon andhis colleagues placed activin-releasing beads or control beadsinto “sandwiches” of Xenopus animal cap cells. They found thatcells exposed to little or no activin failed to express any of thegenes associated with mesodermal tissues; these cells differen-tiated into ectoderm. Higher concentrations of activin turnedon genes such as Brachyury, which are responsible for instruct-ing cells to become mesoderm. Still higher concentrations ofactivin caused the cells to express genes such as goosecoid,which are associated with the most dorsal mesodermal struc-ture, the notochord. The expression of the Brachyury andgoosecoid genes has been correlated with the number of activinreceptors on each cell that are bound by activin. Each cell hasabout 500 activin receptors. If about 100 of them are bound,Brachyury expression is activated and the tissue becomes ven-trolateral mesoderm, such as blood and connective tissue cells.


Source

Thresholdblue

Thresholdwhite

Cell color

(A)

(B)

Blue White Red

Sink

Gradient

Reciprocal transplants developaccording to their final positions

in the “donor” flag

Figure 3.18The French flag analogy for the operation of a gradient of positionalinformation. (A) In this model, positional information is deliveredby a gradient of a diffusible morphogen extending from a source toa sink. The thresholds indicated on the left are cellular propertiesthat enable the gradient to be interpreted. For example, cells becomeblue at one concentration of the morphogen, but as the concentra-tion declines below a certain threshold, cells become white. Wherethe concentration falls below another threshold, cells become red.The result is a pattern of three colors. (B) An important feature ofthis model is that a piece of tissue transplanted from one region ofan embryo to another retains its identity (as to its origin), but dif-ferentiates according to its new positional instructions. This phe-nomenon is indicated schematically by reciprocal “grafts” betweenthe flag of the United States of America and the French flag. (AfterWolpert 1978.)


Animalhemisphere

Presumptive ectoderm

Blastula

Saline (with or without activin)

Activin~1 ng/ml

Activin~10 ng/ml

Activin~100 ng/ml

Muscle cells

Notochordcells

Heartcells

Bloodlike cells, coelomicepithelium,mesenchyme

Atypicalepidermis

Vegetalhemisphere

Activin~0.1 ng/ml

Control(no treatment)

Figure 3.19Activin (or a closely related protein, such asNodal) is thought to be responsible for con-verting animal hemisphere cells into meso-derm. When animal cap cells were removedfrom Xenopus blastulae and placed in salinesolutions containing activin, the activin con-ferred different fates on the cells at differentconcentrations. (After Fukui and Asashima1994.)

(A) Beads containingno activin

Animalcap cells

Region of Brachyuryexpression

(B) Beads containing1 nM activin(low concentration)

(C) Beads containing4 nM activin(high concentration)

Region of goosecoid expression

300Number of occupied receptors

goosecoidthreshold

Activin gradient

Brachyurythreshold

100

High LowActivin concentration(distance from source)

Activin beads

Very low expression (no mesodermal gene activated)

Region of goosecoid gene expression (high concentration)

Region of Brachyury gene expression (low concentration)

Figure 3.20A gradient of activin causes different gene expression in Xenopus an-imal cap cells. The mRNAs from the Brachyury and goosecoid genescan be monitored by hybridization techniques like those discussedin the next chapter. The cells containing these mRNAs appear darkerthan the cells not expressing them. Beads containing activin inducethe expression (transcription of mRNA) of the Brachyury gene atdistances removed from the beads. (A) Beads containing no activindid not elicit Brachyury gene expression. (B) Beads containing 1 nMactivin elicited Brachyury expression near the beads. (C) Beads con-taining 4 nM activin elicited Brachyury expression several cell diam-eters away from the beads. However, goosecoid expression was seenwhere the concentration of activin was higher, near the source.Thus, it appears that Brachyury gene expression is induced at partic-ular concentrations of activin, and that goosecoid is induced at high-er concentrations of activin. (D) Interpretation of activin gradientby Xenopus animal cap cells. High concentrations of activin activatethe goosecoid gene, while lower concentrations activate theBrachyury gene. This pattern correlates with the number of activinreceptors occupied on the individual cells. A threshold value appearsto exist that determines whether a cell will express goosecoid,Brachyury, or neither gene. (A–C after Gurdon et al. 1994, 1995; Dafter Gurdon et al. 1998.)

If about 300 of these receptors are occupied, the cell turns onits goosecoid gene and differentiates into a more dorsal meso-dermal cell type, such as notochord (Figure 3.20D; Dyson andGurdon 1998; Shimizu and Gurdon 1999).

MORPHOGENETIC FIELDS. One of the most interesting ideasto come from experimental embryology has been that of themorphogenetic field. A morphogenetic field can be describedas a group of cells whose position and fate are specified withrespect to the same set of boundaries (Weiss 1939; Wolpert

1977). The general fate of a morphogenetic field is deter-mined; thus, a particular field of cells will give rise to its par-ticular organ (forelimb, eye, heart, etc.) even when trans-planted to a different part of the embryo. However, theindividual cells within the field are not committed, and thecells of the field can regulate their fates to make up for cellsmissing from the field (Huxley and de Beer 1934; Opitz 1985;De Robertis et al. 1991). Moreover, as described earlier (i.e.,the case of presumptive Drosophila leg cells transposed to thetip region of the presumptive antennal field), if cells from one


Science is both about concluding whatsomething is and about concludingwhat something is not. A piece of sci-

ence is only as good as the controls it usesto tell what something is not, and the dif-ference between science and non-science isusually a matter of controls. Just because aparticular compound causes cells in thesame population to differentiate into par-ticular cell types does not mean that it is amorphogen.

In The Adventure of Black Peter Sher-lock Holmes further noted, “One should al-ways look for a possible alternative andprovide against it.” What other possibilitiescould there be besides the morphogen hy-pothesis? A morphogen is expected to be adiffusible molecule that acts through a con-centration gradient. However, there are atleast two other possibilities besides gradi-ents to explain the phenomenon wherebycells appear to differentiate with respect totheir proximity to a source of activin. First,there could be a cascade of inductions.Activin might only induce nearby cells athigh concentrations and do nothing atlower concentrations. Instead, the inducedcells might put forth a second signal that in-duces the next set of cells, and these cellsmight put forth a third local signal, and soon. Such a sequence of events is sometimescalled a relay amplification cascade.Second, the cells near the source of activinmight respond not only by differentiating,but by migrating away to a distant positionwithin the responding tissue (or by produc-ing daughter cells that migrate). Thestronger the signal, the farther away the in-duced cells or their daughters migrate.

Gurdon and his colleagues (1994) con-trolled for these alternative possibilities.First, to distinguish whether the effects ofactivin are due to the passive diffusion ofactivin or to a relay amplification cascade,

Gurdon and colleagues placed nonrespond-ing cells (that is, cells that are not compe-tent to respond to activin) between thesource of activin and the responsive animalcap cells. If activin acted through a relayamplification cascade, the animal cap cellsshould not express Brachyury, since the cas-cade would have been broken by the inter-vening cells. However, if activin producedits effects through a concentration gradi-ent, the animal cap cells should respondbased on their distance from the source ofactivin, even if the cells between them andthe source could not respond. In this exper-iment, the animal cap cells were found toexpress Brachyury, even when the cells clos-est to the source of activin could not re-spond to it.

Second, reserachers controlled for cellmovement by injecting animal cap cells(the responding cells) with green dye andplacing these green cells between the veg-etal cells (which secrete activin) and rho-damine (red dye)- injected animal cap cells.If there were migrations of the respondingcells or their progeny, then green cellsshould be seen within the red cells. Nonewere found. Moreover, cell division andmovement were shown not to be criticalfactors. This was done by placing vegetalcell/animal cap conjugates into a mediumcontaining cytochalasin, an inhibitor of cellmovement and cell division. Expression ofthe Brachyury gene (the gene expressed atlow concentrations of activin, but not athigh concentrations) was seen several di-ameters away from the activin source, eventhough cell movement and cell divisionwere suppressed.

These controls are negative controls be-cause they tell the researcher what is notthe right answer. Any scientific investiga-tion has to have such controls; when theyare not done, the investigation is suspect.

For instance, a group of investigators pub-lished an article claiming that a particularwormlike structure was found in the uter-uses of women with a particular disease.However, no controls were mentioned.When other scientists read the originalpaper, they noted that the obvious con-trol—to look for the wormlike structure inthe uteruses of women without the dis-ease—was not reported. It turned out thatthe structure is a common entity in mostuteruses and was not associated with thedisease.

Similarly, when scientists inject a specif-ic antibody into an embryo to see if it in-hibits a particular phenomenon, they willinject unrelated antibodies into other em-bryos as negative controls. This practiceshows that the inhibition of the event isnot a result of the solvent being injectedalong with the antibody, the presence ofnonspecific antibody protein, or the fact ofinjection.

In addition to negative controls, experi-ments should also incorporate positive con-trols. Positive controls tell scientists thattheir techniques work and that they aremeasuring what they think they are mea-suring. Positive controls for the activin con-centration gradient experiment would beto expose the responding tissue to knownamounts of activin and see a change ingene expression as a result of increased ac-tivin concentrations; this would tell the re-searchers that their techniques work andthat their cells can respond to activin. Thismeans that negative results are not due toimperfections in the methods. These exper-iments have been done (Green and Smith1990; McDowell et al. 1997), and their re-sults support the conclusion that the cellsare indeed responding to decreasingamounts of activin the farther away theyare from its source.

Rules of Evidence II

Sidelights Speculations

field are placed within another field, they can use the posi-tional cues of their new location, even if they retain theirorgan-specific commitment.

The morphogenetic field has been referred to as a “fieldof organization” (Spemann 1921) and as a “cellular ecosys-tem” (Weiss 1923, 1939). The ecosystem metaphor is quite ap-propriate in that recent studies have shown that there arewebs of interactions among the cells in different regions of amorphogenetic field.

WEBSITE 3.5 “Rediscovery” of the morphogeneticfield. The morphogenetic field was one of the most impor-tant concepts of embryology during the early twentiethcentury. This concept was eclipsed by research on the rolesof genes in development, but it is being “rediscovered” as aconsequence of those developmental genetic studies.

Stem cells and commitment

One of the important principles derived from conditionalspecification is the notion of stem cells (National Institutes ofHealth 2001). Stem cells are cells that have the capacity to di-vide indefinitely and which can give rise to more specializedcells. When they divide, stem cells produce a more specializedtype of cell and also generate more stem cells (Figure 3.21A).

Some single stem cells are capable of generating all thestructures of the embryo. These cells, called pluripotent stem

cells,* can generate ectoderm, endoderm, mesoderm, andgerm cells. As well as giving rise to more pluripotent stem cells,these cells also generate committed stem cells. Committedstem cells can give rise to a smaller population of cells. For in-stance, one type of committed stem cell (the hemangioblast)gives rise to all the blood vessels, blood cells, and lymphocytes.Another type of committed stem cell, the mesenchymal stemcell, can give rise to all the different connective tissues (carti-lage, muscle, fat, etc). Committed stem cells can give rise tomore specifically committed stem cells (such as a stem cell thatgenerates only blood cells and lymphocytes, not blood vessels)or it can generate what are called progenitor cells (Figure3.21B).

Progenitor cells, also called precursor cells, are no longerstem cells, since their divisions do not create another similarprogenitor cell. Rather, the progenitor cell divides to form oneor a few related types of cells, depending on the cellular envi-ronment it is in. For example, there is a blood cell precursor(the myeloid progenitor cell) that can generate all the differenttypes of blood cells. Usually, the progenitor cell shows some


Pluripotentialstem cell

(uncommitted)

Other stemcells

Hemangioblast(committed

stem cell)

Lymphocytestem cells

Multipotentialstem cell

(committed)

Myeloidprogenitor cell

Eosinophil

Neutrophil

Basophil

Monocyte

Macrophage

Platelets

Red bloodcells

(A)

(B)fpo

Pluripotentstem cell

Committedstem cell

Progenitorcell

Differentiatedcells

Differentiated cells

Figure 3.21The stem cell concept. (A) Generalized view of the cascade from pluripotentstem cell through committed stem cell, through progenitor cell, to differentiat-ed cell. When stem cells divide they form a more committed cell and anotherstem cell. (B) Example of blood cell formation based on the above scheme.(After National Institutes of Health 2001.)

*There are also stem cells called totipotent stem cells, and they will be discussedin Chapters 11 and 21. Totipotent stem cells are those very early mammaliancells that can form both the entire embryo and the fetal placenta (trophoblast)around it. The pluripotent stem cells of mammals can form the embryo, butnot its surrounding tissues.

evidence of differentiation, but the process is not completeuntil the differentiated cell has been formed.

The restriction on the potency of stem cells is gradual,and the potencies of these cells are determined by their sur-roundings. Once committed, though, they usually do notswitch commitment. When placed in a new environment, theywill not change the type of cells they can generate. Stem cellsare critical for maintaining cell populations that last for longperiods of time and so must be renewed. Thus, stem cells areimportant for our continued production of blood, hair, epi-dermis, and intestinal epithelial cells.

Syncytial specification

Many insects also use a third means, known as syncitial speci-fication, to commit cells to their fates. Here, interactionsoccur not between cells, but between parts of one cell. In earlyembryos of these insects, cell division is not complete. Rather,the nuclei divide within the egg cytoplasm, creating many nu-clei within one large egg cell. A cytoplasm that contains manynuclei is called a syncytium. The egg cytoplasm, however, isnot uniform. Rather, the anterior of the egg cytoplasm ismarkedly different from the posterior.

In the fruit fly, for instance, the anteriormost portion ofthe egg contains an mRNA that encodes a protein called Bi-coid. The posteriormost portion of the egg contains anmRNA that encodes a protein called Nanos. When the egg islaid and fertilized, these two mRNAs are translated into theirrespective proteins. The concentration of Bicoid protein ishighest in the anterior and declines toward the posterior; thatof Nanos protein is highest in the posterior and declines as itdiffuses anteriorly. Thus, the long axis of the Drosophila egg isspanned by two opposing morphogen gradients—one of Bi-coid protein coming from the anterior, and one of Nanos pro-tein coming from the posterior. The Bicoid and Nanos pro-teins form a coordinate system based on their ratios, such thateach region of the embryo is distinguished by a different ratioof the two proteins. As the nuclei divide and enter different re-gions of the egg cytoplasm, they will be instructed by these ra-tios as to their position along the anterior-posterior axis.Those nuclei in regions containing high amounts of Bicoidand little Nanos will be instructed to activate those genes nec-essary for producing the head. Those nuclei in regions withslightly less Bicoid but with a small amount of Nanos will beinstructed to activate those genes that generate the thorax.Those nuclei in regions that have little or no Bicoid and plentyof Nanos will be instructed to form the abdominal structures(Figure 3.22; Nüsslein-Volhard et al. 1987). The mechanismsof syncytial specification will be detailed in Chapter 9.

No embryo uses only autonomous, conditional, or syncy-tial mechanisms to specify its cells. One finds autonomousspecification even in a “regulative embryo” such as the seaurchin, and the nervous system and some musculature of the“autonomously developing” tunicate have been shown to

come from regulative interactions between its cells. Insectssuch as Drosophila use all three modes of specification tocommit their cells to particular fates. Later chapters will detailthe mechanisms by which cell fates are committed in thesespecies.

Morphogenesis and Cell AdhesionA body is more than a collection of randomly distributed cell


Egg layingFertilizationNuclear division

bicoid mRNA

Nucleus

Mature egg

Cellularization

nanos mRNA

Gradient ofBicoid protein

Gradient ofNanos protein

Acron (head cap)- forming region

Telson (tail)- forming region

Pole cells(germ line)

Head-formingregion

Abdomen-formingregion

Thorax-formingregion

Micropyle

Figure 3.22Syncytial specification in the fruit fly Drosophila melanogaster. An-terior-posterior specification originates from morphogen gradientswithin the egg cytoplasm. Bicoid mRNA is stabilized in the most an-terior portion of the egg, while Nanos mRNA is tethered to the poste-rior end. (The anterior can be recognized by the micropyle on theshell; this structure permits sperm to enter.) When the egg is laid andfertilized, these two mRNAs are translated into proteins. The Bicoidprotein forms a gradient that is highest at the anterior end, and theNanos protein forms a gradient that is highest at the posterior end.These two proteins form a coordinate system based on their ratios.Each position along the axis is thus distinguished from any other po-sition. When nuclear division occurs, each nucleus is given its posi-tional information by the ratio of these proteins. The proteins form-ing these gradients activate the transcription of the genes specifyingthe segmental identities of the larva and the adult fly.

types. Development involves not only the differentiation ofcells, but also their organization into multicellular arrange-ments such as tissues and organs. When we observe the de-tailed anatomy of a tissue such as the neural retina of the eye,we see an intricate and precise arrangement of many types ofcells. How can matter organize itself so as to create a complexstructure such as a limb or an eye?

There are five major questions for embryologists whostudy morphogenesis:

1. How are tissues formed from populations of cells? Forexample, how do neural retina cells stick to other neuralretina cells rather than becoming integrated into the pig-mented retina or iris cells next to them? How are the vari-ous cell types within the retina (the three distinct layersof photoreceptors, bipolar neurons, and ganglion cells)arranged so that the retina is functional?

2. How are organs constructed from tissues? The retina ofthe eye forms at a precise distance behind the cornea andthe lens. The retina would be useless if it developed be-hind a bone or in the middle of the kidney. Moreover,neurons from the retina must enter the brain to inner-vate the regions of the brain cortex that analyze visualinformation. All these connections must be preciselyordered.

3. How do organs form in particular locations, and howdo migrating cells reach their destinations? Eyes de-velop only in the head and nowhere else. What stops aneye from forming in some other area of the body? Somecells—for instance, the precursors of our pigment cells,germ cells, and blood cells—must travel long distances toreach their final destinations. How are cells instructed totravel along certain routes in our embryonic bodies, andhow are they told to stop once they have reached their ap-propriate destinations?

4. How do organs and their cells grow, and how is theirgrowth coordinated throughout development? The cellsof all the tissues in the eye must grow in a coordinatedfashion if one is to see. Some cells, including most neu-rons, do not divide after birth. In contrast, the intestine isconstantly shedding cells, and new intestinal cells are re-generated each day. The mitotic rate of each tissue mustbe carefully regulated. If the intestine generated morecells than it sloughed off, it could produce tumorous out-growths. If it produced fewer cells than it sloughed off, itwould soon become nonfunctional. What controls therate of mitosis in the intestine?

5. How do organs achieve polarity? If one were to look at across section of the fingers, one would see a certain orga-nized collection of tissues—bone, cartilage, muscle, fat,dermis, epidermis, blood, and neurons. Looking at a crosssection of the forearm, one would find the same collec-

tion of tissues. But they are arranged very differently indifferent parts of the arm. How is it that the same celltypes can be arranged in different ways in different partsof the same structure?

All these questions concern aspects of cell behavior. There aretwo major types of cell arrangements in the embryo: epithe-lial cells, which are tightly connected to one another in sheetsor tubes, and mesenchymal cells, which are unconnected toone another and operate as independent units. Morphogene-sis is brought about through a limited repertoire of variationsin cellular processes within these two types of arrangements:(1) the direction and number of cell divisions; (2) cell shapechanges; (3) cell movement; (4) cell growth; (5) cell death;and (6) changes in the composition of the cell membrane orsecreted products. We will discuss the last of these considera-tions here.

WEBSITE 3.6 How morphogenetic behaviors work.Although the repertoire of morphogenetic behaviors issmall, cells can do a great deal with this limited set of in-structions. This website illustrates the epithelial and mes-enchymal changes that effect development.

Differential cell affinity

Many of the answers to our questions about morphogenesisinvolve the properties of the cell surface. The cell surface lookspretty much the same in all cell types, and many early investi-gators thought that the cell surface was not even a living partof the cell. We now know that each type of cell has a differentset of proteins at its surfaces, and that some of these differ-ences are responsible for forming the structure of the tissuesand organs during development. Observations of fertilizationand early embryonic development made by E. E. Just (1939)suggested that the cell membrane differed among cell types,but the modern analysis of morphogenesis began with the ex-periments of Townes and Holtfreter in 1955. Taking advan-tage of the discovery that amphibian tissues become dissoci-ated into single cells when placed in alkaline solutions, theyprepared single-cell suspensions from each of the three germlayers of amphibian embryos soon after the neural tube hadformed. Two or more of these single-cell suspensions could becombined in various ways. When the pH of the solution wasnormalized, the cells adhered to one another, forming aggre-gates on agar-coated petri dishes. By using embryos fromspecies having cells of different sizes and colors, Townes andHoltfreter were able to follow the behavior of the recombinedcells (Figure 3.23).

The results of their experiments were striking. First, theyfound that reaggregated cells become spatially segregated.That is, instead of the two cell types remaining mixed, eachcell type sorts out into its own region. Thus, when epidermal(ectodermal) and mesodermal cells are brought together toform a mixed aggregate, the epidermal cells move to the pe-


riphery of the aggregate and the mesodermal cells move to theinside. In no case do the recombined cells remain randomlymixed, and in most cases, one tissue type completely envelopsthe other.

Second, the researchers found that the final positions ofthe reaggregated cells reflect their embryonic positions. Themesoderm migrates centrally with respect to the epidermis,adhering to the inner epidermal surface (Figure 3.24A). Themesoderm also migrates centrally with respect to the gut orendoderm (Figure 3.24B). However, when the three germ lay-ers are mixed together, the endoderm separates from the ecto-derm and mesoderm and is then enveloped by them (Figure3.24C). In the final configuration, the ectoderm is on the pe-riphery, the endoderm is internal, and the mesoderm lies inthe region between them. Holtfreter interpreted this findingin terms of selective affinity. The inner surface of the ecto-derm has a positive affinity for mesodermal cells and a nega-tive affinity for the endoderm, while the mesoderm has posi-tive affinities for both ectodermal and endodermal cells.Mimicry of normal embryonic structure by cell aggregates isalso seen in the recombination of epidermis and neural platecells (Figures 3.23 and 3.24D). The presumptive epidermalcells migrate to the periphery as before; the neural plate cellsmigrate inward, forming a structure reminiscent of the neuraltube. When axial mesoderm (notochord) cells are added to asuspension of presumptive epidermal and presumptive neuralcells, cell segregation results in an external epidermal layer, acentrally located neural tissue, and a layer of mesodermal tis-sue between them (Figure 3.24E). Somehow, the cells are ableto sort out into their proper embryonic positions.

Such selective affinities were also noted by Boucaut

(1974), who injected individual cells from specific germ layersinto the body cavity of amphibian gastrulae. He found thatthese cells migrated back to their appropriate germ layer. En-dodermal cells found positions in the host endoderm, whereasectodermal cells were found only in host ectoderm. Thus, se-lective affinity appears to be important for imparting posi-tional information to embryonic cells.

The third conclusion of Holtfreter and his colleagues wasthat selective affinities change during development. Suchchanges should be expected, because embryonic cells do notretain a single stable relationship with other cell types. For de-velopment to occur, cells must interact differently with othercell populations at specific times. Such changes in cell affinityare extremely important in the processes of morphogenesis.

The experimental reconstruction of aggregates from cellsof later embryos of birds and mammals was accomplished bythe use of the protease trypsin to dissociate the cells from oneanother (Moscona 1952). When the resulting single cells weremixed together in a flask and swirled so that the shear forcewould break any nonspecific adhesions, the cells sorted them-selves out according to their cell type. In so doing, they recon-structed the organization of the original tissue (Moscona1961; Giudice and Just 1962). Figure 3.25 shows the “recon-struction” of skin tissue from a 15-day embryonic mouse. Theepidermal cells are separated by proteolytic enzymes and thenaggregated in a rotary culture. The epidermal cells of each ag-gregate migrate to the periphery, and the dermal cells migratetoward the center. In 72 hours, the epidermis has been recon-stituted, a keratin layer has formed, and interactions betweenthese tissues form hair follicles in the dermal region. Such re-construction of complex tissues from individual cells is called


Presumptiveepidermal cells

Dissociationof cells

Neural platecells

Segregationof cell types

Cross section

Outside

Epidermal cells

Spontaneousreaggregation

Neural cells

Figure 3.23Reaggregation of cells from amphibian neurulae. Presumptive epidermal cells from pigment-ed embryos and neural plate cells from unpigmented embryos are dissociated and mixed to-gether. The cells reaggregate so that one type (here, the presumptive epidermis) covers theother. (After Townes and Holtfreter 1955.)

histotypic aggregation.

The thermodynamic model of cell interactions

Cells, then, do not sort randomly, but can actively move tocreate tissue organization. What forces direct cell movementduring morphogenesis? In 1964, Malcolm Steinberg proposedthe differential adhesion hypothesis, a model that sought toexplain patterns of cell sorting based on thermodynamic prin-ciples. Using cells derived from trypsinized embryonic tissues,Steinberg showed that certain cell types always migrate cen-trally when combined with some cell types, but migrate pe-ripherally when combined with others. Figure 3.26 illustratesthe interactions between pigmented retina cells and neuralretina cells. When single-cell suspensions of these two celltypes are mixed together, they form aggregates of randomly

arranged cells. However, after several hours, no pigmentedretina cells are seen on the periphery of the aggregates, andafter 2 days, two distinct layers are seen, with the pigmentedretina cells lying internal to the neural retina cells. Moreover,such interactions form a hierarchy (Steinberg 1970). If thefinal position of one cell type, A, is internal to a second celltype, B, and the final position of B is internal to a third celltype, C, then the final position of A will always be internal toC. For example, pigmented retina cells migrate internally toneural retina cells, and heart cells migrate internally to pig-mented retina cells. Therefore, heart cells migrate internally toneural retina cells.

This observation led Steinberg to propose that cells inter-act so as to form an aggregate with the smallest interfacial freeenergy. In other words, the cells rearrange themselves into themost thermodynamically stable pattern. If cell types A and Bhave different strengths of adhesion, and if the strength of A-A connections is greater than the strength of A-B or B-B con-nections, sorting will occur, with the A cells becoming central.


Epidermis Epidermis EpidermisEndoderm Mesoderm

Mesoderm

(A) (B) (C) (D) (E)

Mesoderm Endoderm Epidermis

Neural plate

Neural plate

Epidermis+

mesoderm

Mesoderm+

endoderm

Neural plate+

epidermis

Epidermis+

mesoderm+

endoderm

Neural plate+

axial mesoderm+

epidermis

Neural plate

Neural plate

Mesoderm

Figure 3.24Sorting out and reconstruction of spatial relationships in aggregatesof embryonic amphibian cells. (After Townes and Holtfreter 1955.)

On the other hand, if the strength of A-A connections is lessthan or equal to the strength of A-B connections, then the ag-gregate will remain as a random mix of cells. Finally, if thestrength of A-A connections is far greater than the strength ofA-B connections—in other words, if A and B cells show essen-tially no adhesivity toward one another—then A cells and B


(A)

(B)

(C)

(D)

(E) Dermis

Hair follicles

Dermis

Epidermis DermisPrimaryhair follicle

Epidermis Keratinized layer

Figure 3.25Reconstruction of skin from a suspension of skin cells from a 15-day embryonicmouse. (A) Section through intact embryonic skin, showing epidermis, dermis, andprimary hair follicle. (B) Suspension of single skin cells from both the dermis andthe epidermis. (C) Aggregates after 24 hours. (D) Section through an aggregate,showing migration of epidermal cells to the periphery. (E) Further differentiation ofaggregates after 72 hours, showing reconstituted epidermis and dermis, completewith hair follicles and keratinized layer. (From Monroy and Moscona 1979; pho-tographs courtesy of A. Moscona.)

Figure 3.26Aggregates formed by mixing 7-day chick embryo neural retina (un-pigmented) cells with pigmented retina cells. (A) Five hours afterthe single-cell suspensions are mixed, aggregates of randomly dis-tributed cells are seen. (B) At 19 hours, the pigmented retina cellsare no longer seen on the periphery. (C) At 2 days, a great majorityof the pigmented retina cells are located in a central internal mass,surrounded by the neural retina cells. (The scattered pigmented cellsare probably dead cells.) (From Armstrong 1989; photographs cour-tesy of P. B. Armstrong.)

(A)

(C)

(B)

cells will form separate aggregates. According to this hypothe-sis, the early embryo can be viewed as existing in an equilib-rium state until some change in gene activity changes the cellsurface molecules. The movements that result seek to restorethe cells to a new equilibrium configuration. All that is neededfor sorting to occur is that cell types differ in the strengths oftheir adhesion.

In 1996, Foty and his colleagues in Steinberg’s laboratorydemonstrated that this was indeed the case: the cell types thathad greater surface cohesion sorted within those cells that hadless surface tension (Figure 3.27; Foty et al. 1996). In the sim-plest form of this model, all cells could have the same type of“glue” on the cell surface. The amount of this cell surfaceproduct, or the cellular architecture that allows the substanceto be differentially distributed across the surface, could causea difference in the number of stable contacts made betweencell types. In a more specific version of this model, the ther-modynamic differences could be caused by different types ofadhesion molecules (see Moscona 1974). When Holtfreter’sstudies were revisited using modern techniques, Davis andcolleagues (1997) found that the tissue surface tensions of theindividual germ layers were precisely those required for thesorting patterns observed both in vitro and in vivo.

WEBSITE 3.7 Demonstrating the thermodynamicmodel. The original in vivo evidence for the thermody-namic model of cell adhesion came from studies of limbregeneration. This website goes into some of the details ofthese experiments and how they are interpreted.

Cadherins and cell adhesion

Recent evidence shows that such boundaries between tissuescan indeed be created by different cell types having both dif-ferent types and different amounts of cell adhesion molecules.Several classes of molecules can mediate cell adhesion, but hemajor cell adhesion molecules appear to be the cadherins.

As their name suggests, cadherins are calcium-dependentadhesion molecules. They are critical for establishing andmaintaining intercellular connections, and they appear to becrucial to the spatial segregation of cell types and to the orga-nization of animal form (Takeichi 1987). Cadherins interactwith other cadherins on adjacent cells, and they are anchoredinto the cell by a complex of proteins called catenins (Figure

3.28). The cadherin–catenin complex forms the classic ad-herens junctions that help connect epithelial cells together.Moreover, since the catenins bind to the actin cytoskeleton ofthe cell, they integrate the epithelial cells into a mechanicalunit.

In vertebrate embryos, several major cadherin classeshave been identified:

• E-cadherin (epithelial cadherin, also called uvomorulinand L-CAM) is expressed on all early mammalian em-bryonic cells, even at the zygote stage. Later, this mole-cule is restricted to epithelial tissues of embryos andadults.


Tissue Surface tension(dyne/cm)

20.1

12.6

Equilibrium configuration

Limb bud(green)

8.5

Pigmentedepithelium(red)

4.6

Heart(yellow)

1.6

Liver(blue)

Neuralretina(orange)

Figure 3.27Hierarchy of cell sorting in order of decreasing surface tensions. Theequilibrium configuration reflects the strength of cell cohesion, withthe cell types having the greater cell cohesion segregating inside thecells with less cohesion. The images were obtained by sectioning theaggregates and assigning colors to the cell types by computer. Theblack areas represent cells whose signal was edited out in the pro-gram of image optimization. (From Foty et al. 1996; photographcourtesy of M. S. Steinberg and R. A. Foty.)

• P-cadherin (placental cadherin) appears to be expressedprimarily on the trophoblast cells (those placental cells ofthe mammalian embryo that contact the uterine wall)and on the uterine wall epithelium (Nose and Takeichi1986). It is possible that P-cadherin facilitates the con-nection of the embryo to the uterus, since P-cadherin onthe uterine cells is seen to contact P-cadherin on the tro-phoblast cells of mouse embryos (Kadokawa et al. 1989).

• N-cadherin (neural cadherin) is first seen on mesoder-mal cells in the gastrulating embryo as they lose their E-cadherin expression. It is also highly expressed on thecells of the developing central nervous system (Hatta andTakeichi 1986).

• EP-cadherin (C-cadherin) is critical for maintaining ad-hesion between the blastomeres of the Xenopus blastulaand is required for the normal movements of gastrula-

tion (Figure 3.29; Heasman et al. 1994; Lee andGumbiner 1995).

• Protocadherins are calcium-dependent adhesion pro-teins that differ from the classic cadherins in that theylack connections to the cytoskeleton through catenins.Protocadherins are very important in separating the no-tochord from the other mesodermal tissues duringXenopus gastrulation (see Chapter 10).

Cadherins join cells together by binding to the same type ofcadherin on another cell. Thus, cells with E-cadherin stickbest to other cells with E-cadherin, and they will sort out fromcells containing N-cadherin in their membranes. This adhe-sion pattern, which is called homophilic binding, may haveimportant consequences in the embryo. In the gastrula of thefrog Xenopus, for example, the neural tube expresses N-cad-herin, while the epidermis expresses E-cadherin. Normally,these two tissues separate from each other such that theneural tube is inside the body and the epidermis covers thebody. If the epidermis is experimentally manipulated to re-move its E-cadherin, the epidermal epithelium cannot holdtogether. If the epidermis is made to express N-cadherin, or ifthe neural cells are made to lose it, the neural tube will notseparate from the epidermis (Figure 3.30; Detrick et al. 1990;Fujimori et al. 1990). Thus, boundaries can be created be-tween cell populations expressing different cadherins.

The amount of a particular cadherin can also be criticalin development. Univalent (Fab) antibody fragments (Fab; seeChapter 2) against cadherins will convert a three-dimensional,histotypic aggregate of cells into a single layer of cells (Take-ichi et al. 1979). The amount of cadherin can also mediate the


EC2 EC3 EC4

Catenins

Phosphorylation sites

CellmembraneCalcium-

binding site

Adhesive recognitionsite

Actin

EC5

Cadherin– cadherinbinding

Cadherin

γβα

EC1

Figure 3.28Schematic representation of cadherin-mediated cell adhesion.Cadherins are associated with three types of catenins. The cateninscan become associated with the actin microfilament system withinthe cell. (After Takeichi 1991.)

Figure 3.29The importance of cadherins for maintaining cohesion between de-veloping cells can be demonstrated by interfering with their produc-tion. When an oocyte is injected with an antisense oligonucleotideagainst a maternally inherited cadherin mRNA (thus preventing thesynthesis of the cadherin), the inner cells of the resulting embryodisperse when the animal cap is removed (left). In control embryos(right), the inner cells remain together. (After Heasman et al. 1994;photograph courtesy of J. Heasman.)

formation of embryonic structures. This was first shown to bea possibility when Steinberg and Takeichi (1994) collaboratedon an experiment using two cell lines that were identical ex-cept that they synthesized different amounts of P-cadherin.When these two groups of cells, each expressing a differentamount of cadherin, were mixed, the cells that expressedmore cadherin had a higher surface cohesion and migratedinternally to the lower-expressing group of cells. This processalso appears to work in the embryo as well (Godt and Tepass1998; González-Reyes and St. Johnston 1998).

The timing of particular developmental events can de-pend on cadherin expression. For instance, N-cadherin ap-pears in the mesenchyme of the developing chick leg just be-fore these mesenchymal cells condense and form nodules ofcartilage (which are the precursors to the bone tissue). It isnot seen prior to condensation, nor is it seen afterwards. If thelimbs are injected just prior to mesenchyme condensationwith antibodies that block N-cadherin, the mesenchyme cellsfail to condense and cartilage fails to form (Oberlander andTuan 1994). Therefore, it appears that the signal to begin car-tilage formation in the chick limb is the appearance of N-cad-herin.

WEBSITE 3.8 Cadherins: Functional anatomy. The cad-herin molecule has several functional domains that mediateits activities, and the mechanisms of homophilic adhesionare currently being resolved.

WEBSITE 3.9 Other cell adhesion molecules. There aremore types of cell adhesion molecules than cadherins. Thiswebsite looks at some of the other cell adhesion and sub-strate adhesion molecules that have been discovered.

During development, the cadherins often work withother adhesion systems. For instance, one of the most criticaltimes in a mammal’s life is when the embryo is passingthrough the uterus. If development is to continue, the embryomust adhere to the uterus and embed itself in the uterine wall.That is why the first differentiation event in mammalian de-velopment distinguishes the trophoblast cells (the outer cellsthat bind to the uterus) from the inner cell mass (those cellsthat will generate the adult organism). This process occurs asthe embryo travels from the upper regions of the oviduct onits way to the uterus. The trophoblast cells are endowed withseveral adhesion molecules to anchor the embryo to the uter-ine wall. First, they contain both E-cadherins and P-cadherins(Kadokawa et al. 1989), and these cadherins recognize similarcadherins on the uterine cells. Second, they have receptors(integrin proteins) for the collagen and the heparan sulfateglycoproteins of the uterine wall (Farach et al. 1987; Carson etal. 1988, 1993; Cross et al. 1994). Third, the trophoblast cellsalso have a modified glycosyltransferase enzyme that extendsout from the call membrane and can bind to specific carbohy-drate residues on uterine glycoproteins (Dutt et al. 1987). Forsomething as important as the implantation of the mam-malian embryo, it is not surprising that several cell adhesionsystems appear to be working together.

The questions of morphogenesis remain some of the


Figure 3.30The importance of N-cadherin in the separation of neural and epi-dermal ectoderm. At the 4-cell stage, the blastomeres that formedthe left side of this Xenopus embryo were injected with an mRNAfor N-cadherin that lacks the extracellular region of the cadherin.This mutation blocks N-cadherin function. During neurulation, thecells with the mutant protein did not form a coherent layer distinctfrom the epidermis. (From Kintner 1993; photograph courtesy of C.Kintner.)

most fascinating of all developmental biology. Think, for ex-ample, of the thousands of specific connections made by themillions of cells within the human brain, or ponder the mech-anisms by which the heart chambers form on the correct sidesand become connected to the appropriate arteries and veins.These and other questions will be specifically addressed in

later chapters.1. Norms of reaction describe an embryo’s inherited ability

to develop a range of phenotypes. The environment canplay a role in selecting which phenotype is expressed.(Examples include temperature-dependent sex determi-nation and seasonal phenotypic changes in caterpillars

Principles of Development: Experimental Embryology

and butterflies.)

2. Developing organisms are adapted to the ecological nich-es in which they develop. (Examples include the ability offrog eggs exposed to sunlight to repair DNA damage.)

3. Before cells overtly differentiate into the many cell types ofthe body, they undergo a “covert” commitment to a cer-tain fate. This commitment is first labile (the specificationstep) but later becomes irreversible (the determinationstep).

4. In some embryos, removal of a blastomere from an em-bryo causes the absence in the embryo of those tissuesformed by that blastomere. This autonomous specifica-tion produces a mosaic pattern of development.(Examples include early snail and tunicate embryos.)

5. In autonomous specification, morphogenetic determi-nants in the egg cytoplasm are apportioned to differentblastomeres during cleavage. (An example is the yellowcrescent cytoplasm that is found in the muscle-formingcells of tunicate embryos.)

6. In some embryos, the removal of a blastomere can becompensated for by the other cells’ changing their fates.

Each cell has the potential to give rise to more cell typesthan it normally does. This conditional specification pro-duces a regulative pattern of development wherein cellfates are determined relatively late. (Examples include frogand mammalian embryos.)

7. In conditional specification, the fate of a cell often de-pends on interactions with its neighbors

8. In conditional specification, groups of cells can have theirfates determined by a concentration gradient of a mor-phogen. The cells specified by such a morphogen can con-stitute a morphogenetic field.

9. Stem cells are able to generate another stem cell as well asa more committed cell type. By generating more stemcells, the population continues.

10. In syncytial specification, the fates of cells are determinedby gradients of morphogens within the egg cytoplasm.

11. Different cell types can sort themselves into regions bymeans of cell surface molecules such as cadherins. Thesemolecules can be critical in patterning cells into tissuesand organs.

12. Negative controls tell investigators that a given agent does


Literature Cited

not cause an observed phenomenon.Such controls are critical in arguingthat some other agent does cause thephenomenon.

13. Positive controls confirm that an in-vestigator’s protocols are working, sothat if a negative result is observed, itis not merely due to faulty proce-dure.

Adams, N. L. and J. M. Shick. 1996. Myco-sporine-like amino acids provide protectionagainst ultraviolet radiation in eggs of the greensea urchin Strongylocentrotus droebachiensis.Photochem. Photobiol. 64: 149–158.

Adams, N. L. and J. M. Shick. 2001. Myco-sporine-like amino acids prevent UVB-inducedabnormalities during early development of thegreen sea urchin Strongylocentrotus droe-bachiensis. Mar. Biol. 138: 267–280.

Allman, J. A. 1864. Reproductive systems in theHydroidaea. Published in Report of the Thirty-third Meeting of the British Association, 1869.

Ariizumi, T. and M. Asashima. 1994. In vitrocontrol of the embryonic form of Xenopus laevisby activin A: Time- and dose-dependent induc-ing properties of activin-treated ectoderm. Dev.Growth Diff. 36: 499–507.

Armstrong, P. B. 1989. Cell sorting out: The self-assembly of tissues in vitro. CRC Crit. Rev.Biochem. Mol. Biol. 24: 119–149.

Baltzer, F. 1914. Die Bestimmung und derDimorphismus des Geschlechtes bei Bonellia.Sber. Phys.-Med. Ges. Würzb. 43: 1–4.

Blaustein, A. R., P. D. Hoffman, D. G. Hokit, J.M. Kiesecker, S. C. Walls and J. B. Hays. 1994.UV repair and resistance to solar UV-B in am-phibian eggs: A link to population declines?Proc. Natl. Acad. Sci. USA 91: 1791–1795.

Boucaut, J. C. 1974. Étude autoradiographiquede la distribution de cellules embryonnairesisolées, transplantées dans le blastocèle chezPleurodeles waltii Michah (Amphibien,Urodele). Ann. Embryol. Morphol. 7: 7–50.

Carson, D. D., J.-P. Tang and S. Gay. 1988.Collagens support embryo attachment and out-growth in vitro: Effects of the Arg-Gly-Asp se-quence. Dev. Biol. 127: 368–375.

Carson, D. D., J.-P. Tang and J. Julian. 1993.Heparan sulfate proteoglycan (perlecan) ex-

pression by mouse embryos during acquisitionof attachment competence. Dev. Biol. 155:97–106.

Chabry, L. M. 1887. Contribution a l’embryolo-gie normale tératologique des ascidies simples.J. Anat. Physiol. Norm. Pathol. 23: 167–321.

Cross, J. C., Z. Werb and S. J. Fisher. 1994.Implantation and the placenta: Key pieces of thedevelopment puzzle. Science 266: 1508–1518.

Davidson, E. H. 1991. Spatial mechanisms ofgene regulation in metazoan embryos. Develop-ment 113: 1–26.

Davis, G. S., H. M. Phillips and M. S. Steinberg.1997. Germ-layer surface tension and “tissueaffinities” in Rana pipiens: Quantitative mea-surement. Dev. Biol. 192: 630–644.

De Robertis, E. A., E. M. Morita and K. W. Y.Cho. 1991. Gradient fields and homeobox genes.Development 112: 669–678.

Detrick, R. J., D. Dickey and C. R. Kintner. 1990.The effects of N-cadherin misexpression onmorphogenesis in Xenopus embryos. Neuron 4:493–506.

Driesch, H. 1892. The potency of the first twocleavage cells in echinoderm development:Experimental production of partial and double

formations. In B. H. Willier and J. M.Oppenheimer (eds.), Foundations of Experi-mental Embryology. Hafner, New York, 1974.

Driesch, H. 1893. Zur Verlagerung derBlastomeren des Echinideneies. Anat. Anz. 8:348–357.

Driesch, H. 1894. Analytische Theorie de organ-ischen Entwicklung. W. Engelmann, Leipzig.

Dutt, A., J.-P. Tang and D. D. Carson. 1987.Lactosaminoglycans are involved in uterine ep-ithelial cell adhesion in vitro. Dev. Biol. 119:27–37.

Dyson, S. and J. B. Gurdon. 1998. The interpre-tation of position in a morphogen gradient asrevealed by occupancy of activin receptors. Cell93: 557–568.

Farach, M. C., J. P. Tang, G. L. Decker and D. D.Carson. 1987. Heparin/heparan sulfate is in-volved in attachment and spreading of mouseembryos in vitro. Dev. Biol. 123: 401–410.

Ferguson, M. W. J. and T. Joanen. 1982.Temperature of egg incubation determines sexin Alligator mississippiensis. Nature 296:850–853.

Fischer, J.-L. 1991. Laurent Chabry and the be-ginnings of experimental embryology in France.In S. Gilbert (ed.), A Conceptual History of Mod-ern Embryology. Plenum, New York, pp. 31–41.

Foty, R. A., C. M. Pfleger, G. Forgacs and M. S.Steinberg. 1996. Surface tensions of embryoniccells predict their mutual envelopment behav-ior. Development 122: 1611–1620.

Fujimori, T., S. Miyatani and M. Takeichi. 1990.Ectopic expression of N-cadherin perturbs his-togenesis in Xenopus embryos. Development110: 97–104.

Fukui, A. and M. Asashima. 1994. Control of celldifferentiation and morphogenesis in amphib-ian development. Int. J. Dev. Biol. 38: 257–266.

Gilbert, S. F. and M. Faber. 1996. Looking at em-bryos: The visual and conceptual aesthetics ofembryology. In A. I. Tauber (ed.), The ElusiveSynthesis: Aesthetics and Science. Kluwer,Dordrecht, pp. 125–151.

Giudice, G. and R. Just. 1962. Restitution ofwhole larvae from disaggregated cells of seaurchin embryos. Dev. Biol. 5: 402–411.

Godt, D. and U. Tepass. 1998. Drosophila oocytelocalization is mediated by differential cad-herin-based adhesion. Nature 395: 387–391.

González-Reyes, A. and D. St. Johnston. 1998.The Drosophila AP axis is polarised by the cad-herin-mediated positioning of the oocyte.Development 125: 3635–3644.

Gosse, R. J. 1969. Principles of Regeneration.Academic Press, New York.

Green, J. B. A. and J. C. Smith. 1990. Gradedchanges in dose of a Xenopus activin A homo-logue elicit stepwise transitions in embryoniccell fate. Nature 347: 391–394.

Greene, E. 1989. A diet-induced developmentalpolymorphism in a caterpillar. Science 243:643–646.

Gurdon, J. B., P. Harger, A. Mitchell and P.Lemaire. 1994. Activin signalling and responsesto a morphogen gradient. Nature 371: 487–493.

Gurdon, J. B., A. Mitchell and D. Mahony. 1995.Direct and continuous assessment by cells oftheir position in a morphogen gradient. Nature376: 520–521.

Gurdon, J. B., S. Dyson and D. St. Johnston.1998. Cells’ perception of position in a concen-tration gradient. Cell 95: 159–163.

Hamburger, V. 1988. The Heritage of Experi-mental Embryology: Hans Spemann and theOrganizer. Oxford University Press, New York,p. vii.

Haraway, D. J. 1976. Crystals, Fabrics and Fields:Metaphors of Organicism in Twentieth-CenturyBiology. Yale University Press, New Haven.

Harrington, A. 1996. Reenchanted Science:Holism in German Culture from Wilhelm II toHitler. Princeton University Press, Princeton,NJ.

Harrison, R. G. 1933. Some difficulties of thedetermination problem. Am. Nat. 67: 306–321.

Hatta, K. and M. Takeichi. 1986. Expression ofN-cadherin adhesion molecules associated withearly morphogenetic events in chick develop-ment. Nature 320: 447–449.

Heasman, J., D. Ginsberg, K. Goldstone, T. Pratt,C. Yoshidanaro and C. Wylie. 1994. A function-al test for maternally inherited cadherin inXenopus shows its importance in cell adhesionat the blastula stage. Development 120: 49–57.

Hertwig, O. 1894. Zeit- und Streitfragen derBiologie I. Präformation oder Epigenese?Grundzüge einer Entwicklungstheorie der Organ-ismen. Gustav Fischer, Jena.

Hörstadius, S. 1939. The mechanics of seaurchin development studied by operative meth-ods. Biol. Rev. 14: 132–179.

Hörstadius, S. and A. Wolsky. 1936. Studienüber die Determination der Bilateralsymmetriedes jungen Seeigelkeimes. Wilhelm Roux Arch.Entwicklungsmech. Org. 135: 69–113.

Huxley, J. and G. R. de Beer. 1934. The Elementsof Experimental Embryology. Cambridge Uni-versity Press, Cambridge.

Jokiel, P. L. and R. H. York, Jr. 1982. Solar ultra-violet photobiology of the reef coral Pocilloporadamicornis and symbiotic zooxanthellae. Bull.Mar. Sci. 32: 301–315.

Just, E. E. 1939. The Biology of the Cell Surface.Blackiston, Philadelphia.

Kadokawa, Y., I. Fuketa, A. Nose, M. Takeichiand N. Nakatsuji. 1989. Expression of E- and P-cadherin in mouse embryos and uteri duringthe periimplantation period. Dev. Growth Diff.31: 23–30.

Kiesecker, J. M., A. R. Blaustein and L. K. Belden.2001. Complex causes of amphibian populationdeclines. Nature 410: 681–684.

Kintner, C. 1993. Regulation of embryonic celladhesion by the cadherin cytoplasm domain.Cell 69: 225–236.

Koch, P. B. and D. Buchmann. 1987. Hormonalcontrol of seasonal morphs by the timing ofecdysteroid release in Araschnia levana(Nymphalidae: Lepidoptera). J. Insect Physiol.36: 159–164.

Lawrence, P. A. 1966. Gradients in the insectsegment: The orientation of hairs in the milk-weed bug Oncopeltus fasciatus. J. Exp. Zool. 44:602–620.

Lee, C.-H. and B. M. Gumbiner. 1995. Dis-ruption of gastrulation movements in Xenopusby a dominant-negative mutant for C-cadherin.Dev. Biol. 171: 363–373.

Leutert, T. R. 1974. Zur Geschlechtsbestimmungund Gametogenese von Bonellia viridisRolando. J. Embryol. Exp. Morphol. 32: 169–193.

Marcus, N. H. 1979. Developmental aberrationsassociated with twinning in laboratory-rearedsea urchins. Dev. Biol. 70: 274–277.

McClendon, J. F. 1910. The development of iso-lated blastomeres of the frog’s egg. Am. J. Anat.10: 425–430.

McDowell, N., A. M. Zorn, D. J. Crease and J. B.Gurdon. 1997. Activin has direct long-range sig-nalling activity and can form a concentrationgradient by diffusion. Curr. Biol. 7: 671–681.

Mead, K. S. and D. Epel. 1995. Beakers versusbreakers: How fertilization in the laboratory dif-fers from fertilization in nature. Zygote 3: 95–99.

Monroy, A. and A. A. Moscona. 1979.Introductory Concepts in Developmental Biology.University of Chicago Press, Chicago.

Morgan, T. H. 1905 An attempt to analyze thephenomenon of polarity in Tubularia. J. Exp.Zool. 1: 589–591.

Morgan, T. H. 1906. “Polarity” considered as aphenomenon of gradation of materials. J. Exp.Zool. 2: 495–506.

Moscona, A. A. 1952. Cell suspension fromorgan rudiments of chick embryos. Exp. CellRes. 3: 535–539.

Moscona, A. A. 1961. Rotation-mediated histo-genetic aggregation of dissociated cells: A quan-tifiable approach to cell interaction in vitro. Exp.Cell Res. 22: 455–475.

Moscona, A. A. 1974. Surface specification ofembryonic cells: Lectin receptors, cell recogni-tion, and specific cell ligands. In A. Monroy(ed.), The Cell Surface in Development. Wiley,New York, pp. 67–99.

National Institutes of Health. 2001. Stem Cells:Scientific Progress and Future ResearchDirections. http://www.nih.gov/news/stemcell/fullrptstem.pdf.


Needham, J. 1943. Time: The Refreshing River.Allen and Unwin, London.

Nijhout, H. F. 1991. The Development andEvolution of Butterfly Wing Patterns. Smith-sonian Institution Press, Washington, D.C.

Nose, A. and M. Takeichi. 1986. A novel cad-herin adhesion molecule: Its expression pat-terns associated with implantation and organo-genesis of mouse embryos. J. Cell Biol. 103:2649–2658.

Nüsslein-Volhard, C., H. G. Fröhnhofer and R.Lehmann. 1987. Determination of anteriopos-terior polarity in Drosophila. Science 238:1675–1681.

Nyhart, L. K. 1995. Biology Takes Form: AnimalMorphology and the German Universities,1800–1900. University of Chicago Press,Chicago.

Oberlender, S. A. and R. S. Tuan. 1994.Expression and functional involvement of N-cadherin in embryonic limb chondrogenesis.Development 120: 177–187.

Opitz, J. M. 1985. The developmental field con-cept. Am. J. Med. Genet. 21: 1–11.

Phillips, K. 1994. Tracking the Vanishing Frogs:An Ecological Mystery. St. Martin’s Press, NewYork.

Reverberi, G. and A. Minganti. 1946. Fenomenidi evocazione nello sviluppo dell’uovo diAscidie. Risultati dell’indagine spermentale sul-l’ouvo di Ascidiella aspersa e di Ascidia malacaallo stadio di 8 blastomeri. Pubbl. Staz. Zool.Napoli 20: 199–253. (Quoted in G. Reverberi,.Experimental Embryology of Marine and Fresh-water Invertebrates. North-Holland, Amster-dam, 1971, p. 537.)

Roux, W. 1888. Contributions to the develop-mental mechanics of the embryo. On the artifi-cial production of half-embryos by destructionof one of the first two blastomeres and the laterdevelopment (postgeneration) of the missinghalf of the body. In B. H. Willier and J. M.Oppenheimer (eds.) 1974, Foundations ofExperimental Embryology. Hafner, New York,pp. 2–37.

Roux, W. 1894. Einleitung zum Archiv fürEntwicklungsmechanik. Arch. Embryol. 1:1–142. English translation reprinted in J. M.Maienschein (ed.), 1986, Defining Biology,Harvard University Press, Cambridge, MA, pp.107–148.

Schlichting, C. D. and M. Pigliucci. 1998.Phenotypic Evolution: A Reaction Norm Per-spective. Sinauer Associates, Sunderland, MA.

Schmalhausen, I. I. 1949. Factors of Evolution:The Theory of Stabilizing Selection. University ofChicago Press, Chicago.

Shimizu, K. and J. B. Gurdon. 1999. A quantita-tive analysis of signal transduction from activinreceptor to nucleus and its relevance to mor-phogen gradient interpretation. Proc. Natl.Acad. Sci. USA 96: 6791–6796.

Siebeck, O. 1988. Experimental investigation ofUV tolerance in hermatypic corals. Mar. Ecol.Prog. Ser. 43: 95–103.

Slack, J. M. W. 1991. From Egg to Embryo:Regional Specification in Early Development.Cambridge University Press, New York.

Spemann, H. 1921. Die Erzeugung tierischerChimaeren durch heteroplastiche embryonaleTransplantation zwischen Triton cristatus u. tae-niatus. Wilhelm Roux Arch. Entwicklungsmech.Org. 48: 533–570.

Stearns, S. C., G. de Jong and R. A. Newman.1991. The effects of phenotypic plasticity on ge-netic correlations. Trends Ecol. Evol. 6: 122–126.

Steinberg, M. S. 1964. The problem of adhesiveselectivity in cellular interactions. In M. Locke(ed.), Cellular Membranes in Development.Academic Press, New York, pp. 321–434.

Steinberg, M. S. 1970. Does differential adhe-sion govern self-assembly processes in histogen-esis? Equilibrium configurations and the emer-gence of a hierarchy among populations ofembryonic cells. J. Exp. Zool. 173: 395–434.

Steinberg, M. S. and M. Takeichi. 1994.Experimental specification of cell sorting, tissuespreading and specific spatial patterning byquantitative differences in cadherin expression.Proc. Natl. Acad. Sci. USA 91: 206–209.

Stumpf, H. 1966. Mechanism by which cells es-timate their location within the body. Nature212: 430–431.

Takeichi, M. 1987. Cadherins: A molecular fam-ily essential for selective cell-cell adhesion andanimal morphogenesis. Trends Genet. 3:213–217.

Takeichi, M. 1991. Cadherin cell adhesion re-ceptors as a morphogenetic regulator. Science251: 1451–1455.

Takeichi, M., H. S. Ozaka, K. Tokunaga and T. S.Okada. 1979. Experimental manipulation of cell

surface to affect cellular recognition mecha-nisms. Dev. Biol. 70: 195–205.

Toivonen, S. and L. Saxén. 1955. The simultane-ous inducing action of liver and bone marrowof the guinea pig in implantation and explanta-tion experiments of Triturus. Exp. Cell Res.[Suppl.] 3: 346–357.

Townes, P. L. and J. Holtfreter. 1955. Directedmovements and selective adhesion of embryon-ic amphibian cells. J. Exp. Zool. 128: 53–120.

van der Weele, C. 1995. Images of Development:Environmental Causes in Ontogeny. Elinkwijk,Utrecht.

Weismann, A. 1875. Über den Saison-Dimorphismus der Schmetterlinge. In. Studienzur Descendenz-Theorie. Engelmann, Leipzig.

Weismann, A. 1892. Essays on Heredity andKindred Biological Problems. Translated by E. B.Poulton, S. Schoenland and A. E. Shipley.Clarendon, Oxford.

Weismann, A. 1893. The Germ-Plasm: A Theoryof Heredity. Translated by W. Newton Parkerand H. Ronnfeld. Walter Scott Ltd., London.

Weiss, P. 1923. Die Regeneration derUrodelenextremität als Selbst differenzierungdes Organrestes. Naturwissenschaften 11:669–677.

Weiss, P. 1939. Principles of Development. Holt,New York.

Whittaker, J. R. 1973. Segregation during ascidi-an embryogenesis of egg cytoplasmic informa-tion for tissue-specific enzyme development.Proc. Natl. Acad. Sci. USA 70: 2096–2100.

Whittaker, J. R. 1977. Segregation during cleav-age of a factor determining endodermal alkalinephosphatase development in ascidian embryos.J. Exp. Zool. 202: 139–153.

Whittaker, J. R. 1982. Muscle cell lineage cyto-plasm can change the developmental expressionin epidermal lineage cells of ascidian embryos.Dev. Biol. 93: 463–470.

Wilkins, A. S. 1993. Genetic Analysis of AnimalDevelopment, 2nd Ed. Wiley-Liss, New York.

Wilson, E. B. 1896. The Cell in Development andInheritance. Macmillan, New York.

Wilson, E. B. 1904. Experimental studies on ger-minal location. J. Exp. Zool. 1: 1–72.

Wolpert, L. 1968. The French flag problem: Acontribution to the discussion on pattern for-mation and regulation. In C. H. Waddington



(ed.), Towards a Theoretical Biology. Edinburgh University Press,Edinburgh, pp. 125–133.

Wolpert, L. 1969. Positional information and the spatial pattern of cellu-lar differentiation. J. Theor. Biol. 25: 1–47.

Wolpert, L. 1977. The Development of Pattern and Form in Animals.Carolina Biological, Burlington, NC.

Wolpert, L. 1978. Pattern formation in biological development. Sci. Am.239(4): 154–164.

Woltereck, R. 1909. Weitere experimentelle Untersuchungen überArtveränderung, speziell über das Wesen quantitativer Artunderscheidebei Daphniden. Versuch. Deutsch. Zool. Ges. 1909: 110–173.

Date post:	11-Aug-2020
Category:	Documents
Upload:	others
View:	2 times
Download:	0 times

Principles of experimental embryology...Environmental Developmental Biology The developing embryo is...

Documents