2ND PASS PAGE PROOFS

chapter Life cycles and the evolution 2 of developmental patterns

RADITIONAL WAYS OF CLASSIFYING ANIMALS catalog them according to their adult The view taken here is that the life cycle is the central unit in biology. … structure. But, as J. T. Bonner (1965) pointed out, this is a very artificial Evolution then becomes the alteration Tmethod, because what we consider an individual is usually just a brief slice of of life cycles through time, genetics the its life cycle. When we consider a dog, for instance, we usually picture an adult. But inheritance mechanisms between cycles, the dog is a “dog” from the moment of fertilization of a dog egg by a dog sperm. It and development all the changes in remains a dog even as a senescent dying hound. Therefore, the dog is actually the en- structure that take place during one life tire life cycle of the animal, from fertilization through death. cycle. The life cycle has to be adapted to its environment, which is composed of non- J. T. BONNER (1965) It’s the circle of life living objects as well as other life cycles. Take, for example, the life cycle of Clunio And it moves us all. marinus, a small fly that inhabits tidal waters along the coast of western Europe. Fe- TIM RICE (1994) males of this species live only 2–3 hours as adults, and they must mate and lay their eggs within this short time. To make matters even more precarious, they must lay their eggs on red algal mats that are exposed only during the lowest ebbing of the spring tide. Such low tides occur on four successive days shortly after the new and full moons (i.e., at about 15-day intervals). Therefore, the life cycle of these insects must be coordinated with the lunar cycle as well as the daily tidal rhythms such that the insects emerge from their pupal cases during the few days of the spring tide and at the correct hour for its ebb (Beck 1980; Neumann and Spindler 1991).

The Circle of Life: The Stages of Animal Development One of the major triumphs of descriptive embryology was the idea of a generalizable life cycle. Each animal, whether earthworm, eagle, or beagle, passes through similar stages of development. The life of a new individual is initiated by the fusion of ge- netic material from the two gametes—the sperm and the egg. This fusion, called fer- tilization, stimulates the egg to begin development. The stages of development be- tween fertilization and hatching are collectively called embryogenesis. Throughout the animal kingdom, an incredible variety of embryonic types exist, but most pat- terns of embryogenesis are variations on five themes: 1. Immediately following fertilization, cleavage occurs. Cleavage is a series of ex- tremely rapid mitotic divisions wherein the enormous volume of zygote cyto- plasm is divided into numerous smaller cells. These cells are called blastomeres, and by the end of cleavage, they generally form a sphere known as a blastula. 2. After the rate of mitotic division has slowed down, the blastomeres undergo dra- matic movements wherein they change their positions relative to one another. This series of extensive cell rearrangements is called gastrulation, and the em- bryo is said to be in the gastrula stage. As a result of gastrulation, the embryo

25 2ND PASS PAGE PROOFS 26 Chapter 2

contains three germ layers: the ectoderm, the endoderm, pond where it lives and on the temperature of the air and and the mesoderm. water. A combination of photoperiod (hours of daylight) and 3. Once the three germ layers are established, the cells inter- temperature tells the pituitary gland of the female frog that it is act with one another and rearrange themselves to pro- spring. If the female is mature, her pituitary gland secretes hor- duce tissues and organs. This process is called organo- mones that stimulate her ovary to make estrogen. Estrogen is a genesis. Many organs contain cells from more than one hormone that can instruct the liver to make and secrete yolk germ layer, and it is not unusual for the outside of an proteins such as vitellogenin, which are then transported organ to be derived from one layer and the inside from through the blood into the enlarging eggs in the ovary.* The another. For example, the outer layer of skin (epidermis) yolk is transported into the bottom portion of the egg (Figure comes from the ectoderm, while the inner layer (the der- 2.2A). The bottom half of the egg usually contains more yolk mis) comes from the mesoderm. Also during organogen- than the top half and is called the vegetal hemisphere of the esis, certain cells undergo long migrations from their egg. Conversely, the upper half of the egg usually has less yolk place of origin to their final location. These migrating and is called the animal hemisphere of the egg.† cells include the precursors of blood cells, lymph cells, Another ovarian hormone, progesterone, signals the egg pigment cells, and gametes. Most of the bones of our face to resume its meiotic division. This is necessary because the are derived from cells that have migrated ventrally from egg had been “frozen” in the metaphase of its first meiosis. the dorsal region of the head. When it has completed this first meiotic division, the egg is 4. In many species, a specialized portion of egg cytoplasm released from the ovary and can be fertilized. In many species, gives rise to cells that are the precursors of the gametes the eggs are enclosed in a jelly coat that acts to enhance their (sperm and egg). The gametes and their precursor cells size (so they won’t be as easily eaten), to protect them against are collectively called germ cells, and they are set aside bacteria, and to attract and activate sperm. for reproductive function. All the other cells of the body Sperm also occur on a seasonal basis. Male leopard frogs are called somatic cells. This separation of somatic cells make their sperm in the summer, and by the time they begin (which give rise to the individual body) and germ cells hibernation in autumn, they have produced all the sperm that (which contribute to the formation of a new generation) are to be available for the following spring’s breeding season. is often one of the first differentiations to occur during In most species of frogs, fertilization is external. The male frog animal development. The germ cells eventually migrate grabs the female’s back and fertilizes the eggs as the female to the gonads, where they differentiate into gametes. The frog releases them (Figure 2.2B). Rana pipiens usually lays development of gametes, called gametogenesis, is usually about 2500 eggs, while the bullfrog, Rana catesbiana, can lay not completed until the organism has become physically as many as 20,000. Some species lay their eggs in pond vegeta- mature. At maturity, the gametes may be released and tion, and the jelly adheres to the plants and anchors the eggs participate in fertilization to begin a new embryo. The (Figure 2.2C). Other species float their eggs into the center of adult organism eventually undergoes senescence and dies. the pond without any support. 5. In many species, the organism that hatches from the egg or Fertilization accomplishes several things. First, it allows is born into the world is not sexually mature. Indeed, in the egg to complete its second meiotic division, which pro- most animals, the young organism is a larva that may look vides the egg with a haploid pronucleus. The egg pronucleus significantly different from the adult. Larvae often consti- and the sperm pronucleus meet in the egg cytoplasm to form tute the stage of life that is used for feeding or dispersal. In the diploid zygote nucleus. Second, fertilization causes the cy- many species, the larval stage is the one that lasts the toplasm of the egg to move such that different parts of the cy- longest, and the adult is a brief stage solely for reproduc- toplasm find themselves in new locations (Figure 2.2D). tion. In the silkworm moths, for instance, the adults do not Third, fertilization activates those molecules necessary to have mouthparts and cannot feed. The larvae must eat begin cell cleavage and development (Rugh 1950). The sperm enough for the adult to survive and mate. Indeed, most fe- and egg die quickly unless fertilization occurs. male moths mate as soon as they eclose from their pupa, and they fly only once—to lay their eggs. Then they die. *As we will see in later chapters, there are numerous ways by which the synthe- sis of a new protein can be induced. Estrogen stimulates the production of vitel- logenin protein in two ways. First, it uses transcriptional regulation to make new vitellogenin mRNA. Before estrogen stimulation, no vitellogenin message can be seen in the liver cells. After stimulation, there are over 50,000 vitellogenin mRNA The Frog Life Cycle molecules in these cells. Estrogen also uses translational regulation to stabilize these particular messages, increasing their half-life from 16 hours to 3 weeks. In Figure 2.1 uses the development of the leopard frog, Rana pip- this way, more protein can be translated from each message. iens, to show a representative life cycle. Let us look at this life †The terms animal and vegetal reflect the movements of cells seen in some em- cycle in a bit more detail. bryos (such as those of frogs). The cells derived from the upper portion of the egg divide more rapidly and are actively mobile (hence, animated), while the In most frogs, gametogenesis and fertilization are seasonal yolk-filled cells of the vegetal half were seen as being immobile (hence, like events, because its life depends on the plants and insects in the plants). 2ND PASS PAGE PROOFS Life cycles and the evolution of developmental patterns 27

Sperm

Morula Blastula Oocyte Location of Sperm germ cells Blastocoel (male gamete) Germ plasm FERTILI- Oocyte CLEAVAGE ZATION (female gamete) Blastopore

GAMETOGENESIS GASTRULATION Sexually Ectoderm MATURITY mature adult Metamorphosis Mesoderm (in some species) ORGANOGENESIS Endoderm LARVAL Gonad STAGES Hatching Immature larval (birth) stages

Figure 2.1 Developmental history of the leopard frog, Rana pipiens. The stages etal hemisphere (until they are encircled by the ectoderm) be- from fertilization through hatching (birth) are known collectively as come the endoderm. Thus, at the end of gastrulation, the ec- embryogenesis. The region set aside for producing germ cells is toderm (the precursor of the epidermis and nerves) is on the shown in bright purple. Gametogenesis, which is completed in the sexually mature adult, begins at different times during development, outside of the embryo, the endoderm (the precursor of the depending on the species. (The sizes of the varicolored wedges gut lining) is on the inside of the embryo, and the mesoderm shown here are arbitrary and do not correspond to the proportion (the precursor of connective tissue, blood, skeleton, gonads, of the life cycle spent in each stage.) and kidneys) is between them. Organogenesis begins when the notochord—a rod of mesodermal cells in the most dorsal portion of the embryo— During cleavage, the volume of the frog egg stays the signals the ectodermal cells above it that they are not going to same, but it is divided into tens of thousands of cells (Figure become epidermis. Instead, these dorsal ectoderm cells form a 2.2E–H). The animal hemisphere of the egg divides faster tube and become the nervous system. At this stage, the em- than the vegetal hemisphere does, and the cells of the vegetal bryo is called a neurula. The neural precursor cells elongate, hemisphere become progressively larger the more vegetal the stretch, and fold into the embryo (Figure 2.3D–F), forming cytoplasm. A fluid-filled cavity, the blastocoel, forms in the the neural tube. The future back epidermal cells cover them. animal hemisphere (Figure 2.2I). This cavity will be impor- The cells that had connected the neural tube to the epidermis tant for allowing cell movements to occur during gastrulation. become the neural crest cells. The neural crest cells are almost Gastrulation in the frog begins at a point on the embryo like a fourth germ layer. They give rise to the pigment cells of surface roughly 180 degrees opposite the point of sperm entry the body (the melanocytes), the peripheral neurons, and the with the formation of a dimple, called the blastopore. At first, cartilage of the face. just a small slit is made. Cells migrating through this dorsal Once the neural tube has formed, it induces changes in blastage lip migrate toward the animal pole (Figure 2.3A,B). its neighbors, and organogenesis continues. The mesodermal These cells become the dorsal mesoderm. The blastopore ex- tissue adjacent to the notochord becomes segmented into pands into a circle (Figure 2.3C), and cells migrating through somites, the precursors of the frog’s back muscles, spinal ver- the lateral and ventral lips of this circle become the lateral and tebrae, and dermis (the inner portion of the skin). These ventral mesoderm. The cells remaining on the outside become somites appear as blocks of mesodermal tissue (Figure the ectoderm, and this outer layer expands vegetally to enclose 2.3F,G). The embryo develops a mouth and an anus, and it the entire embryo. The large yolky cells that remain at the veg- elongates into the typical tadpole structure (Figure 2.3H). The 2ND PASS PAGE PROOFS 28 Chapter 2

(A) Figure 2.2 Early development of the frog Xenopus laevis. (A) As the egg matures, it accumulates yolk (here stained yellow and green) in the vegetal cytoplasm. (B) Frogs mate by am- plexus, the male grasping the female around the belly and fertilizing the eggs as they are released. (C) A newly laid clutch of eggs. The brown area of each egg is the pig- mented animal cap. The white spot in the middle of the pigment is where the egg's nucleus resides. (D) Rearrangement of cytoplasm seen during first cleavage. Compare with the initial stage seen in (A). (E) A 2-cell embryo near the end of its first cleavage. (F) An 8-cell embryo. (G) Early blastula. Note that the cells get smaller, but the vol- ume of the egg remains the same. (H) Late blastula. (I) Cross section of a late blastula, showing the blastocoel (cavity). (A–H courtesy of Michael Danilchik and Kimberly Ray; I courtesy of J. Heasman.)

(B) (C)

(D) (E) (F)

(G) (H) (I) Blastocoel 2ND PASS PAGE PROOFS Life cycles and the evolution of developmental patterns 29

Figure 2.3 (A) Continued development of Xenopus laevis. (A) Gastrulation begins with an invagination, or slit, in the future dorsal (top) side of the embryo. (B) This slit, the dorsal blastopore lip, as seen from the ventral surface (bottom) of the embryo. (C) The slit becomes a cir- cle, the blastopore. Future mesoderm cells migrate into the interior of the embryo along the blastopore edges, and the ectoderm (future epidermis and nerves) migrates down the outside of the embryo. The remaining part, the yolk-filled endoderm, is eventually encir- cled. (D) Neural folds begin to form on the dorsal surface. (E) A groove can be seen where the bottom of the neural tube will be. (F) The neural folds come together at the dorsal midline, creating a neural tube. (G) Cross section of the Xenopus embryo at the neurula stage. (H) A pre-hatching tadpole, as the protrusions of the forebrain begin to in- duce eyes to form. (I) A mature tadpole, having swum away from the egg mass and feed- ing independently. (Photographs courtesy of Michael Danilchik and Kimberly Ray.)

Dorsal blastopore lip (B) Dorsal blastopore lip(C) Yolk plug Blastopore (D) Neural folds

(F) (G) (E) Dorsal (back) Notochord Neural tube Somite

Archenteron (future gut)

YolkyYolky endodermendoderm

Neural groove Open neural tube Epidermis Mesoderm (ectoderm) Ventral (H) (belly) (I)

Somites Brain

Gill area

Expansion of forebrain to touch surface ectoderm ((induces eyes to form) Stomodeum (mouth) Tailbud 2ND PASS PAGE PROOFS 30 Chapter 2

(A) (B)

(C)

(D)

(E)

Figure 2.4 Metamorphosis of the frog. (A) Huge changes are obvious when one contrasts the tadpole and the adult bullfrog. Note especially the differences in jaw structure and limbs. (B) Pre- (F) metamorphic tadpole. (C) Prometamorphic tadpole, showing hindlimb growth. (D) Onset of metamorphic climax as forelimbs emerge. (E, F) Climax stages. (Photograph copyright Patrice Ceisel/Stock, Boston.)

neurons make their connections to the muscles and to other neurons, the gills form, and the larva is ready to hatch from its egg jelly. The hatched tadpole will soon feed for itself once the WEBSITE 2.1 Immortal animals. Imagine a multicellu- yolk supply given it by its mother is exhausted (Figure 2.3I). lar animal that acquires immortality by reverting to its lar- Metamorphosis of the tadpole larva into an adult frog is val form instead of growing old. That seems to be what the one of the most striking transformations in all of biology (Fig- marine hydranth Turritopsis does. ure 2.4). These changes prepare an aquatic organism for a ter- restrial existence. In amphibians, metamorphosis is initiated by WEBSITE 2.2 The human life cycle. The human animal hormones from the tadpole’s thyroid gland. (The mechanisms provides a fascinating life cycle to study. Here are some by which thyroid hormones accomplish these changes will be websites that speculate about (A) when is an embryo or fetus “human”? (B) how might the strange way the human discussed in Chapter 18.) In anurans (frogs and toads), almost brain develops make childhood a necessity? and (C) do hu- every organ is subject to modification, and the resulting mans undergo metamorphosis? changes in form are striking and very obvious. The hindlimbs and forelimbs that the adult will use for locomotion differenti- VADE MECUM The frog life cycle. The life cycle of a frog ate and the tadpole’s paddle tail recedes. The cartilaginous tad- is illustrated in labeled photographs and time-lapse pole skull is replaced by the predominantly bony skull of the videomicroscopy. [Click on Amphibian] young frog. The horny teeth the tadpole uses to tear up pond plants disappear as the mouth and jaw take a new shape, and the fly-catching tongue muscle of the frog develops. Mean- The Evolution of Developmental Patterns while, the large intestine characteristic of herbivores shortens in Unicellular Protists to suit the more carnivorous diet of the adult frog. The gills regress and the lungs enlarge. As metamorphosis ends, the de- Every living organism develops. Development can be seen velopment of the first germ cells begins. In Rana pipiens,egg even among the unicellular organisms. Moreover, by studying development lasts 3 years. At that time, the female frog is sexu- the development of unicellular protists, we can see the sim- ally mature and can produce offspring of her own. plest forms of cell differentiation and sexual reproduction. The speed of metamorphosis is carefully keyed to envi- ronmental pressures. In temperate regions, for instance, Rana Control of developmental morphogenesis: The role metamorphosis must occur before ponds freeze in winter. An of the nucleus adult leopard frog can burrow into the mud and survive the A century ago, it had not yet been proved that the nucleus of winter; its tadpole cannot. the cell contained hereditary or developmental information. 2ND PASS PAGE PROOFS Life cycles and the evolution of developmental patterns 31

Some of the best evidence for this theory came from studies specific cap, but it takes several weeks to do so. Moreover, if the in which unicellular organisms were fragmented into nucleate nucleus is removed from an Acetabularia cell early in its devel- and anucleate pieces (reviewed in Wilson 1896). When vari- opment, before it first forms a cap, a normal cap is formed ous protists were cut into fragments, nearly all the pieces died. weeks later, even though the organism will eventually die. However, the fragments containing nuclei were able to live These studies suggest that (1) the nucleus contains informa- and to regenerate entire complex cellular structures. tion specifying the type of cap to be produced (i.e., it contains Nuclear control of cell morphogenesis and the interac- the genetic information that specifies the proteins required for tion of nucleus and cytoplasm are beautifully demonstrated in the production of a certain type of cap), and (2) material con- studies of the protist Acetabularia. This enormous single cell taining this information enters the cytoplasm long before cap (2–4 cm long) consists of three parts: a cap, a stalk, and a rhi- production occurs. This information in the cytoplasm is not zoid (Figure 2.5A; Mandoli 1998). The rhizoid is located at used for several weeks. the base of the cell and holds it to the substrate. The single One current hypothesis proposed to explain these observa- nucleus of the cell resides within the rhizoid. The size of Ac- tions is that the nucleus synthesizes a stable mRNA that lies dor- etabularia and the location of its nucleus allow investigators to mant in the cytoplasm until the time of cap formation (see Du- remove the nucleus from one cell and replace it with a nucleus mais et al. 2000). This hypothesis is supported by an observation from another cell. In the 1930s, J. Hämmerling took advantage that Hämmerling published in 1934. Hämmerling fractionated of these unique features and exchanged nuclei between two young Acetabularia into several parts (Figure 2.6). The portion morphologically distinct species, A. mediterranea* and A. with the nucleus eventually formed a new cap, as expected; so crenulata. As Figure 2.5A shows, these two species have very did the apical tip of the stalk. However, the intermediate portion different cap structures. Hämmerling found that when he of the stalk did not form a transferred the nucleus from one species into the stalk of an- cap. Thus, Hämmerling pos- (B) other species, the newly formed cap eventually assumed the tulated (nearly 30 years be- form associated with the donor nucleus (Figure 2.5B). Thus, fore the existence of mRNA the nucleus was seen to control Acetabularia development. was known) that the in- The formation of a cap is a complex morphogenic event structions for cap formation involving the synthesis of numerous proteins, which must be originated in the nucleus accumulated in a certain portion of the cell and then assem- and were somehow stored in bled into complex, species-specific structures. The trans- a dormant form near the tip planted nucleus does indeed direct the synthesis of its species- of the stalk. Many years later, researchers established that *After a recent formal name change, this species is now called Acetabularia ac- nucleus-derived mRNA does etabulum. For the sake of simplicity, however, we will use Hämmerling’s des- accumulate in the tip of the ignations here.

A. crenulata A. mediterranea Nuclei transplanted (A)

Nucleus Nucleus

Rhizoid

Figure 2.5 (A) Acetabularia crenulata (left) Cap structure and A. mediterranea (right). Each is that of individual is a single cell. The donor nucleus rhizoid contains the nucleus. (B) Effect of exchanging nuclei between two species of Acetabu- laria. Nuclei were transplanted into enucleated rhizoid frag- ments. A. crenulata structures are darker, A. mediterranea struc- tures lighter green. (Photographs courtesy of S. Berger.) 2ND PASS PAGE PROOFS 32 Chapter 2

sential role in cap formation. The mRNAs are not translated for weeks, even though they are present in the cytoplasm. Cap and stalk Something in the cytoplasm controls when the message is uti- regenerated lized. Hence, the expression of the cap is controlled not only Apical tip by nuclear transcription, but also by the translation of the cy- of stalk toplasmic RNA. In this unicellular organism, “development” is controlled at both the transcriptional and translational levels.

WEBSITE 2.3 Protist differentiation. Three of the most remarkable areas of protist development concern the con- Central portion No regeneration trol of sex type in fission yeast, the transformation of of stalk Naegleria amoebae into streamlined, flagellated cells, and the cortical inheritance of the cell surface in paramecia.

Unicellular protists and the origins Rhizoid of sexual reproduction and nucleus Sexual reproduction is another invention of the protists that has had a profound effect on more complex organisms. It should be noted that sex and reproduction are two distinct Total regeneration and separable processes. Reproduction involves the creation of new individuals. Sex involves the combining of genes from Figure 2.6 two different individuals into new arrangements. Reproduc- Regenerative ability of different fragments of tion in the absence of sex is characteristic of organisms that A. mediterranea. reproduce by fission (i.e., splitting into two); there is no sort- ing of genes when an amoeba divides or when a hydra buds off cells to form a new colony. Sex without reproduction is also common among unicel- stalk, and that the destruction of this mRNA or the inhibition lular organisms. Bacteria are able to transmit genes from one of protein synthesis in this region prevents cap formation individual to another by means of sex pili. This transmission is (Kloppstech and Schweiger 1975; Garcia and Dazy 1986; separate from reproduction. Protists are also able to reassort Serikawa et al. 2001). genes without reproduction. Paramecia, for instance, repro- It is clear from the preceding discussion that nuclear duce by fission, but sex is accomplished by conjugation.When transcription plays an important role in the formation of the two paramecia join together, they link their oral apparatuses Acetabularia cap. But note that the cytoplasm also plays an es- and form a cytoplasmic connection through which they can

Stationary Migratory Cytoplasmic bridge Meiotic micronucleus micronucleus Micronuclei spindle

Macronucleus Micronuclei undergo All but one of Remaining micro- Migratory micronuclei meiosis, forming each partner’s nucleus divides to cross cytoplasmic bridge Two paramecia form 8 haploid nuclei micronuclei form a stationary and fertilize partners’ cytoplasmic bridge per cell; macronuclei degenerate and a migratory stationary micronuclei degenerate micronucleus

Figure 2.7 Diploid nucleus forms and Conjugation across a cytoplasmic bridge in paramecia. Two paramecia can undergoes mitotic divisions to exchange genetic material, each ending up with genes that differ from those generate a new macronucleus with which they started. (After Strickberger 1985.) as paramecia separate 2ND PASS PAGE PROOFS Life cycles and the evolution of developmental patterns 33 exchange genetic material (Figure 2.7). The macronucleus of and minus. When a plus and a minus meet, they join their cy- each individual (which controls the metabolism of the organ- toplasms, and their nuclei fuse to form a diploid zygote. This ism) degenerates, while each micronucleus undergoes meiosis zygote is the only diploid cell in the life cycle, and it eventually to produce eight haploid micronuclei, of which all but one de- undergoes meiosis to form four new Chlamydomonas cells. generate. The remaining micronucleus divides once more to This is true sexual reproduction, for chromosomes are reas- form a stationary micronucleus and a migratory micronucleus. sorted during the meiotic divisions and more individuals are Each migratory micronucleus crosses the cytoplasmic bridge formed. Note that in this protist type of sexual reproduction, and fuses with (“fertilizes”) the partner’s stationary micronu- the gametes are morphologically identical; the distinction be- cleus, thereby creating a new diploid nucleus in each cell. This tween sperm and egg has not yet been made. diploid nucleus then divides mitotically to give rise to a new In evolving sexual reproduction, two important advances micronucleus and a new macronucleus as the two partners dis- had to be achieved. The first was the mechanism of meiosis engage. Therefore, no reproduction has occurred, only sex. (Figure 2.9), whereby the diploid complement of chromo- The union of these two distinct processes, sex and repro- somes is reduced to the haploid state (discussed in detail in duction, into sexual reproduction is seen in other unicellular Chapter 19). The second was a mechanism whereby the two eukaryotes. Figure 2.8 shows the life cycle of Chlamydomonas. different mating types could recognize each other. In Chlamy- This organism is usually haploid, having just one copy of each domonas, recognition occurs first on the flagellar membranes chromosome (like a mammalian gamete). The individuals of (Figure 2.10; Bergman et al. 1975; Wilson et al. 1997; Pan and each species, however, are divided into two mating types:plus Snell 2000). The flagella of two individuals twist around each other, enabling specific regions of the cell membranes to come together. These specialized regions contain mating type-spe- cific components that enable the cytoplasms to fuse. Follow- Asexual (mitotic) reproduction ing flagellar agglutination, the plus individual initiates fusion Plus mating type Minus mating type by extending a fertilization tube. This tube contacts and fuses (haploid) (haploid) with a specific site on the minus individual. Interestingly, the mechanism used to extend this tube—the polymerization of the protein actin to form microfilaments—is also used to ex- tend the processes of sea urchin eggs and sperm. In Chapter 7, we will see that the recognition and fusion of sperm and egg Sexual reproduction occur in an amazingly similar manner. Unicellular eukaryotes appear to possess the basic ele- ments of the developmental processes that characterize more Mating complex organisms: protein synthesis is controlled such that certain proteins are made only at certain times and in certain places; the structures of individual genes and chromosomes Cytoplasms merge are as they will be throughout eukaryotic evolution; mitosis and meiosis have been perfected; and sexual reproduction ex- ists, involving cooperation between individual cells. Such in- Zygote (diploid) tercellular cooperation becomes even more important with the evolution of multicellular organisms.

Maturation (meiosis) Multicellularity: The Evolution of Differentiation One of evolution’s most important experiments was the cre- Germination ation of multicellular organisms. There appear to be several paths by which single cells evolved multicellular arrange- Two plus and two minus mating types ments; we will discuss only two of them here (see Chapter 22 for a fuller discussion). The first path involves the orderly di- Figure 2.8 vision of the reproductive cell and the subsequent differentia- Sexual reproduction in Chlamydomonas. Two mating types, both tion of its progeny into different cell types. This path to mul- haploid, can reproduce asexually when separate. Under certain con- ditions, the two mating types can unite to produce a diploid cell ticellularity can be seen in a remarkable series of multicellular that can undergo meiosis to form four new haploid organisms. organisms collectively referred to as the family Volvocaceae, or (After Strickberger 1985.) the volvocaceans (Kirk 1999, 2000). 2ND PASS PAGE PROOFS 34 Chapter 2

Meiosis I: Separation of homologous chromosomes

Nuclear Homologous Homologous envelope Chromatin chromosomes chromatids Nucleus

Interphase Early prophase I Mid prophase I Late prophase I Metaphase I DNA replicates The nuclear envelope breaks down and homologous chromosomes (each chromosome being double, with the chromatids joined at the kinetochore) align in pairs. Chromosomal rearrangements can occur between the four homologous chromatids at this time

Figure 2.9 Summary of meiosis. The DNA and its associated proteins replicate The Volvocaceans during interphase. During prophase, the nuclear envelope breaks down and homologous chromosomes (each chromosome is double, The simpler organisms among the volvocaceans are ordered with the chromatids joined at the kinetochore) align in pairs. assemblies of numerous cells, each resembling the unicellular Chromosomal rearrangements between the four homologous chro- protist Chlamydomonas, to which they are related (Figure matids can occur at this time. After the first metaphase, the two 2.11A). A single organism of the volvocacean genus Gonium original homologous chromosomes are segregated into different (Figure 2.11B), for example, consists of a flat plate of 4 to 16 cells. During the second meiotic division, the kinetochore splits and the sister chromatids separate, leaving each new cell with one copy cells, each with its own flagellum. In a related genus, Pando- of each chromosome. rina, the 16 cells form a sphere (Figure 2.11C); and in Eudo- rina, the sphere contains 32 or 64 cells arranged in a regular pattern (Figure 2.11D). In these organisms, then, a very impor- tant developmental principle has been worked (A) (B) out: the ordered division of one cell to generate a number of cells that are organized in a pre- dictable fashion. Like cleavage in most animal embryos, the cell divisions by which a single volvocacean cell produces an organism of 4 to 64 cells occur in very rapid sequence and in the absence of cell growth. The next two genera of the volvocacean series exhibit another important principle of develop- ment: the differentiation of cell types within an individual organism. In these organisms, the re- productive cells become differentiated from the Microfilaments somatic cells. In all the genera mentioned earlier, every cell can, and normally does, produce a complete new organism by mitosis. In the genera Pleodorina and Volvox, however, relatively few cells can reproduce. In Pleodorina californica (Fig- ure 2.11E), the cells in the anterior region are re- stricted to a somatic function; only those cells on Figure 2.10 the posterior side can reproduce. In P. californica, Two-step recognition in mating Chlamydomonas. (A) Scanning electron micro- a colony usually has 128 or 64 cells, and the ratio graph (7000×) of mating pair. The interacting flagella twist around each other, ad- × of the number of somatic cells to the number of hering at the tips (arrows). (B) Transmission electron micrograph (20,000 ) of a reproductive cells is usually 3:5. Thus, a 128-cell cytoplasmic bridge connecting the two organisms. The actin microfilaments extend from the donor (lower) cell to the recipient (upper) cell. (From Goodenough and colony typically has 48 somatic cells, and a 64-cell Weiss 1975 and Bergman et al. 1975; photographs courtesy of U. Goodenough.) colony has 24. 2ND PASS PAGE PROOFS Life cycles and the evolution of developmental patterns 35

Meiosis II: Separation of the chromatids

Anaphase I Telophase I Metaphase II Anaphase II Telophase II The two original homo- The kinetochore splits Each new cell has logous chromosomes one copy of each are segregated into chromosome different cells

In Volvox, almost all the cells are somatic, and very few of pendent existence and of perpetuating the species), in V. car- the cells are able to produce new individuals. In some species teri we have a truly multicellular organism with two distinct of Volvox, reproductive cells, as in Pleodorina, are derived from and interdependent cell types (somatic and reproductive), cells that originally look and function like somatic cells before both of which are required for perpetuation of the species they enlarge and divide to form new progeny. However, in (Figure 2.11F). Although not all animals set aside the repro- other members of the genus, such as V. carteri, there is a com- ductive cells from the somatic cells (and plants hardly ever plete division of labor: the reproductive cells that will create do), this separation of germ cells from somatic cells early in the next generation are set aside during the division of the development is characteristic of many animal phyla and will original cell that is forming a new individual. The reproduc- be discussed in more detail in Chapter 19. tive cells never develop functional flagella and never con- tribute to motility or other somatic functions of the individ- WEBSITE 2.4 Volvox cell differentiation. The pathways ual; they are entirely specialized for reproduction. leading to germ cells or somatic cells are controlled by genes that cause cells to follow one or the other fate. Mutations Thus, although the simpler volvocaceans may be thought can prevent the formation of one of these lineages. of as colonial organisms (because each cell is capable of inde-

(A) (B) (C) Figure 2.11 Representatives of the order Volvocales. All but Chlamydomonas are members of the family Volvocaceae. (A) The unicel- lular protist Chlamydomonas reinhardtii. (B) Gonium pectorale, with 8 Chlamy- domonas-like cells in a convex disc. (C) Pandorina morum. (D) Eudorina el- egans. (E) Pleodorina californica.Here, all 64 cells are originally similar, but the posterior ones dedifferentiate and redif- ferentiate as asexual reproductive cells (D)(E) (F) called gonidia, while the anterior cells remain small and biflagellate, like Chlamydomonas. (F) Volvox carteri. Here, cells destined to become gonidia are set aside early in development and never have somatic characteristics. The smaller somatic cells resemble Chlamy- domonas. Complexity increases from the single-celled Chlamydomonas to the multicellular Volvox. (Photographs courtesy of D. Kirk.) 2ND PASS PAGE PROOFS 36 Chapter 2

Sidelights Speculations Sex and Individuality in Volvox

imple as it is, Volvox shares many fea- gellated somatic cells along its periphery bryo everts through this hole and then tures that characterize the life cycles and about 16 large, asexual reproductive closes it up. About a day after this is done, Sand developmental histories of much cells, called gonidia, toward one end of the the juvenile Volvox are enzymatically re- more complex organisms, including our- interior. When mature, each gonidium di- leased from the parent and swim away. selves. As already mentioned, Volvox is vides rapidly 11 or 12 times. Certain of What happens to the somatic cells of the among the simplest organisms to exhibit a these divisions are asymmetrical and pro- “parent” Volvox now that its young have division of labor between two completely duce the 16 large cells that will become a “left home”? Having produced offspring different cell types. As a consequence, it is new set of gonidia in the next generation. and being incapable of further reproduc- among the simplest organisms to include At the end of cleavage, all the cells that tion, these somatic cells die. Actually, these death as a regular, genetically regulated will be present in an adult have been pro- cells commit suicide, synthesizing a set of part of its life history. duced from the gonidium. But the result- proteins that cause the death and dis- ing embryo is “inside out”: it is now a hol- solution of the cells that make them Death and Differentiation low sphere with its gonidia on the outside (Pommerville and Kochert 1982). Moreover, Unicellular organisms that reproduce by and the flagella of its somatic cells pointing in death, the cells release for the use of oth- simple cell division, such as amoebae, are toward the interior. This predicament is ers, including their own offspring, all the potentially immortal. The amoeba you see corrected by a process called inversion, in nutrients that they had stored during life. today under the microscope has no dead which the embryo turns itself right side out “Thus emerges,” notes David Kirk, “one of ancestors. When an amoeba divides, nei- by a set of cell movements that resemble the great themes of life on planet Earth: ther of the two resulting cells can be con- the gastrulation movements of animal em- ‘Some die that others may live.’” sidered either ancestor or offspring; they bryos (Figure 2.13A–H). Clusters of bottle- In V. carteri, a specific gene, somatic are siblings. Death comes to an amoeba shaped cells open a hole at one end of the regulator A, or regA, plays a central role in only if it is eaten or meets with a fatal acci- embryo by producing tension on the inter- regulating cell death (Kirk 1988, 2001a). dent, and when it does, the dead cell connected cell sheet (Figure 2.13I). The em- This gene is expressed only in somatic cells, leaves no offspring. Death becomes an essential part of life, however, for any multicellular organism Figure 2.12 that establishes a division of labor between Asexual reproduction in V. carteri. (A) When reproductive cells (gonidia) are mature, they enter a somatic (body) cells and germ (reproduc- cleavage-like stage of embryonic development to produce juveniles within the adult. Through a se- tive) cells. Consider the life history of ries of cell movements resembling gastrulation, the embryonic Volvox invert and are eventually re- Volvox carteri when it is reproducing asex- leased from the parent. The somatic cells of the parent, lacking the gonidia, undergo senescence and ually (Figure 2.12). Each asexual adult is a die, while the juvenile Volvox mature. The entire asexual cycle takes 2 days. (B) Young adult spheres spheroid containing some 2000 small, bifla- of Volvox carteri being released from parent to become free-swimming individuals. Once the progeny leave, the parent undergoes programmed cell death. (A after Kirk 1988; B from Kirk 2001b.)

(A) (B) Expansion of both adult and juveniles Embryogenesis Adult with juveniles Adult with mature gonidia Continued fpo expansion of Maturation extracellular of gonidia matrix

Continued expansion of Release juveniles of juveniles Death of parental somatic cells 2ND PASS PAGE PROOFS Life cycles and the evolution of developmental patterns 37

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

(E) (F) (G) (H) (I)

Figure 2.13 Inversion of embryos of V. carteri.A–D are scanning electron micrographs of whole embryos. those times, V. carteri swims about, repro- E–H are sagittal sections through the center of the embryo, visualized by differential interference ducing asexually. These asexual volvoxes microscopy. Before inversion, the embryo is a hollow sphere of connected cells with the new goni- will die in minutes once the pond dries up. dia on the outside. When the “bottle cells” change their shape, a hole (the phialopore) opens at the V. carteri is able to survive by turning sexu- apex of the embryo (A, B, E, F). Cells then curl around and rejoin at the bottom (C, D, G, H). The al shortly before the pond disappears, pro- new gonidia are now inside. (I)“Bottle cells” near the opening of the phialopore in a V. carteri em- ducing dormant zygotes that survive the bryo. These cells remain tightly interconnected through cytoplasmic bridges near their elongated heat and drought of late summer and the apices, thereby creating the tension that causes the curvature of the interconnected cell sheet. cold of winter. When rain fills the pond in (From Kirk et al. 1982; photograph courtesy of D. Kirk.) spring, the zygotes break their dormancy and hatch out a new generation of individ- uals that reproduce asexually until the pond is about to dry up once more. and it prevents their expressing gonidial the animal kingdom and all for the sake of How do these simple organisms predict genes. In laboratory strains possessing reg- sex.’ And he asked: ‘Is it worth it?’ the coming of adverse conditions so accu- ulatory mutations of this gene, somatic rately that they can produce a sexual gen- cells begin expressing regA, abandon their For Volvox carteri, it most assuredly is eration in the nick of time, year after year? suicidal ways, gain the ability to reproduce worth it. V. carteri lives in shallow tempo- The stimulus for switching from the asexu- asexually, and become potentially immor- rary ponds that fill with spring rains but dry al to the sexual mode of reproduction in V. tal (Figure 2.14). The fact that such mutants out in the heat of late summer. Between have never been found in nature indicates that cell death most likely plays an impor- (A) (B) tant role in the survival of V. carteri under natural conditions.

Enter Sex Although V. carteri reproduces asexually much of the time, in nature it reproduces sexually once each year. When it does, one generation of individuals passes away and a new and genetically different genera- tion is produced. The naturalist Joseph Wood Krutch (1956, pp. 28–29) put it more poetically: The amoeba and the paramecium are po- tentially immortal. … But for Volvox, death seems to be as inevitable as it is in a mouse or in a man. Volvox must die as Figure 2.14 Leeuwenhoek saw it die because it had chil- Mutation of a single gene (somatic regenerator A) abolishes programmed cell death in V. carteri. dren and is no longer needed. When its (A) A newly hatched Volvox carrying this mutation is indistinguishable from the wild-type spher- time comes it drops quietly to the bottom oid. (B) Shortly before the time when the somatic cells of wild-type spheroids begin to die, the so- and joins its ancestors. As Hegner, the Johns matic cells of this mutant redifferentiate as gonidia (B). Eventually, every cell of the mutant will Hopkins zoologist, once wrote, ‘This is the divide to regenerate a new spheroid that will repeat this potentially immortal developmental cycle. first advent of inevitable natural death in (Photographs courtesy of D. Kirk.) 2ND PASS PAGE PROOFS 38 Chapter 2 carteri is known to be a 30-kDa sexual in- that might be expected in a shallow pond production in pools of rainwater of scarce- ducer protein. This protein is so powerful in late summer. When this was done, the ly a fortnight’s duration” (Powers 1908). that concentrations as low as 6 × 10–17 M somatic cells of the asexual volvoxes pro- Thus, in temporary ponds formed by spring cause gonidia to undergo a modified pat- duced the sexual inducer protein. Since the rains and dried up by summer’s heat, tern of embryonic development that re- amount of sexual inducer protein secreted Volvox has found a means of survival: it sults in the production of eggs or sperm, by one individual is sufficient to initiate uses that heat to induce the formation of depending on the genetic sex of the indi- sexual development in over 500 million sexual individuals whose mating produces vidual (Sumper et al. 1993). The sperm are asexual volvoxes, a single inducing volvox zygotes capable of surviving conditions released and swim to a female, where they can convert an entire pond to sexuality. that kill the adult organism. We see, too, fertilize eggs to produce dormant zygotes This discovery explained an observation that development is critically linked to the (Figure 2.15). The sexual inducer protein is made over 90 years ago that “in the full ecosystem in which the organism has able to work at such remarkably low con- blaze of Nebraska sunlight, Volvox is able adapted to survive. centrations by causing slight modifications to appear, multiply, and riot in sexual re- of the extracellular matrix. These modifica- tions appear to signal the transcription of a whole battery of genes that form the ga- Figure 2.15 metes (Sumper et al. 1993; Hallmann et al. Sexual reproduction in V. carteri. Males and females are indistinguishable in their asexual phase. 2001). When the sexual inducer protein is present, the gonidia of both mating types undergo a modified What is the source of this sexual induc- embryogenesis that leads to the formation of eggs in the females and sperm in the males. When er protein? Kirk and Kirk (1986) discovered the gametes are mature, sperm packets (containing 64 or 128 sperm each) are released and swim that the sexual cycle could be initiated by to the females. Upon reaching a female, the sperm packet breaks up into individual sperm, which heating dishes of V. carteri to temperatures can fertilize the eggs. The resulting dormant zygote has tough cell walls that can resist drying, heat, and cold. When spring rains cause the zygote to germinate, it undergoes meiosis to produce haploid males and females that reproduce asexually until heat induces the sexual cycle again.

Sperm packets Sexual development of gonidia Sexual inducer Sperm Modified embryonic Asexual male Gonidium development of Sexual male gonidia resulting in gamete production Ova

Sexual Zygotes inducer

Egg

Asexual female Sexual female Meiosis and germination

Although all the volvocaceans, like their unicellular rela- which involves the production of large, relatively immotile tive Chlamydomonas, reproduce predominantly by asexual eggs by one mating type and small, motile sperm by the other means, they are also capable of sexual reproduction, which in- (see Sidelights & Speculations). Here we see one type of ga- volves the production and fusion of haploid gametes. In many mete specialized for the retention of nutritional and develop- species of Chlamydomonas, including the one illustrated in mental resources and the other type of gamete specialized for Figure 2.10, sexual reproduction is isogamous (“the same ga- the transport of nuclei. Thus, the volvocaceans include the metes”), since the haploid gametes that meet are similar in simplest organisms that have distinguishable male and female size, structure, and motility. However, in other species of members of the species and that have distinct developmental Chlamydomonas—as well as many species of colonial volvo- pathways for the production of eggs or sperm. caceans—swimming gametes of very different sizes are pro- In all volvocaceans, the fertilization reaction resembles duced by the different mating types. This pattern is called het- that of Chlamydomonas in that it results in the production of erogamy (“different gametes”). But the larger volvocaceans a dormant diploid zygote that is capable of surviving harsh have evolved a specialized form of heterogamy called oogamy, environmental conditions. When conditions allow the zygote 2ND PASS PAGE PROOFS Life cycles and the evolution of developmental patterns 39 to germinate, it first undergoes meiosis to produce haploid over to produce the migrating slug (with the tip at the front). offspring of the two different mating types in equal numbers. The slug (often given the more dignified title of pseudoplas- modium or grex) is usually 2–4 mm long and is encased in a slimy sheath. The grex begins to migrate (if the environment Differentiation and morphogenesis in is dark and moist) with its anterior tip slightly raised. When it Dictyostelium: Cell adhesion reaches an illuminated area, migration ceases, and the culmi- THE LIFE CYCLE OF DICTYOSTELIUM. Another type of multi- nation stages of the life cycle take place as the grex differenti- cellular organization derived from unicellular organisms is ates into a fruiting body composed of spore cells and a stalk. found in Dictyostelium discoideum.* The life cycle of this fas- The anterior cells, representing 15–20% of the entire cellular cinating organism is illustrated in Figure 2.16. In its asexual population, form the tubed stalk. This process begins as some cycle, solitary haploid amoebae (called myxamoebae or “social of the central anterior cells, the prestalk cells, begin secreting amoebae” to distinguish them from amoeba species that al- an extracellular cellulose coat and extending a tube through ways remain solitary) live on decaying logs, eating bacteria the grex. As the prestalk cells differentiate, they form vacuoles and reproducing by binary fission. When they have exhausted and enlarge, lifting up the mass of prespore cells that made their food supply, tens of thousands of these myxamoebae up the posterior four-fifths of the grex (Jermyn and Williams join together to form moving streams of cells that converge at 1991). The stalk cells die, but the prespore cells, elevated a central point. Here they pile atop one another to produce a above the stalk, become spore cells. These spore cells disperse, conical mound called a tight aggregate. Subsequently, a tip each one becoming a new myxamoeba. arises at the top of this mound, and the tight aggregate bends WEBSITE 2.5 Slime mold life cycle. Check out this web- *Though colloquially called a “cellular slime mold,” Dictyostelium is not a site to see the digitized videos from which the photographs mold, nor is it consistently slimy. It is perhaps best to think of Dictyostelium in Figure 2.16 were made. as a social amoeba.

Figure 2.16 Low RES Life cycle of Dictyostelium dis- coideum. Haploid spores give rise fpo to myxamoebae, which can repro- duce asexually to form more hap- loid myxamoebae. As the food supply diminishes, aggregation oc- Low RES curs at central points, and a mi- grating pseudoplasmodium is Slug (pseudo- 15 hours 16 hours fpo plasmodium; formed. Eventually it stops moving grex) and culminates in a fruiting body 17 hours that releases more spores. The 14 hours times refer to hours since nutrient starvation began the developmen- 20 hours tal sequence. The prestalk cells MIGRATION have been indicated in yellow. Low RES fpo 12 hours Low RES CULMINATION Spore case fpo

Low RES 23 hours 10 hours Stalk AGGREGATION fpo Tight Spores aggregate 9 hours

24 hours Low RES 6 hours Mature Low RES fruiting fpo Loose body fpo aggregate

Cell streams Myxamoebae 2ND PASS PAGE PROOFS 40 Chapter 2

In addition to this asexual cycle, there is a possibility of The differentiation of individual myxamoebae into either sex for Dictyostelium. Two myxamoebae can fuse to create a stalk (somatic) or spore (reproductive) cells is a complex mat- giant cell, which digests all the other cells of the aggregate. ter. Raper (1940) and Bonner (1957) demonstrated that the When it has eaten all its neighbors, it encysts itself in a thick anterior cells normally become stalk, while the remaining, wall and undergoes meiotic and mitotic divisions; eventually, posterior cells are usually destined to form spores. However, new myxamoebae are liberated. surgically removing the anterior part of a slug does not abol- Dictyostelium has been a wonderful experimental organ- ish its ability to form a stalk. Rather, the cells that now find ism for developmental biologists because initially identical themselves at the anterior end (and which originally had been cells differentiate into two alternative cell types—spore and destined to produce spores) now form the stalk (Raper 1940). stalk. It is also an organism wherein individual cells come to- Somehow a decision is made so that whichever cells are ante- gether to form a cohesive structure composed of differenti- rior become stalk cells and whichever are posterior become ated cell types, a process akin to tissue formation in more spores. This ability of cells to change their developmental fates complex organisms. The aggregation of thousands of myxam- according to their location within the whole organism and oebae into a single organism is an incredible feat of organiza- thereby compensate for missing parts is called regulation.We tion that invites experimentation to answer questions about will see this phenomenon in many embryos, including those the mechanisms involved. of mammals.

VADE MECUM Slime mold life cycle. The life cycle of CELL ADHESION MOLECULES IN DICTYOSTELIUM. How do indi- Dictyostelium—the remarkable aggregation of myxamoe- vidual cells stick together to form a cohesive organism? This bae, the migration of the slug, and the truly awesome cul- problem is the same one that embryonic cells face, and the so- mination of the stalk and fruiting body—can best be viewed through movies. The Slime Mold segment in Vade lution that evolved in the protists is the same one used by em- Mecum contains a remarkable series of videos. bryos: developmentally regulated cell adhesion molecules. [Click on Slime Mold] While growing mitotically on bacteria, Dictyostelium cells do not adhere to one another. However, once cell division AGGREGATION OF DICTYOSTELIUM CELLS. The first of these stops, the cells become increasingly adhesive, reaching a questions is, What causes the myxamoebae to aggregate? plateau of maximum adhesiveness about 8 hours after starva- Time-lapse videomicroscopy has shown that no directed tion. The initial cell-cell adhesion is mediated by a 24-kilodal- movement occurs during the first 4–5 hours following nutri- ton glycoprotein (gp24) that is absent in myxamoebae but ap- ent starvation. During the next 5 hours, however, the cells can pears shortly after mitotic division ceases (Figure 2.18A; be seen moving at about 20 mm/min for 100 seconds. This Knecht et al. 1987; Wong et al. 1996). This protein is synthe- movement ceases for about 4 minutes, then resumes. Al- sized from newly transcribed mRNA and becomes localized in though the movement is directed toward a central point, it is the cell membranes of the myxamoebae. If myxamoebae are not a simple radial movement. Rather, cells join with one an- treated with antibodies that bind to and mask this protein, the other to form streams; the streams converge into larger cells will not stick to one another, and all subsequent develop- streams, and eventually all streams merge at the center. Bon- ment ceases. ner (1947) and Shaffer (1953) showed that this movement is a Once this initial aggregation has occurred, it is stabilized result of chemotaxis: the cells are guided to aggregation cen- by a second cell adhesion molecule. This 80-kDa glycoprotein ters by a soluble substance. This substance was later identified (gp80) is also synthesized during the aggregation phase. If it is as cyclic adenosine 3′5′-monophosphate (cAMP) (Konijn et defective or absent in the cells, small slugs will form, and their al. 1967; Bonner et al. 1969), the chemical structure of which fruiting bodies will be only about one-third the normal size. is shown in Figure 2.17A. Thus, the second cell adhesion system seems to be needed for Aggregation is initiated as each of the myxamoebae be- gins to synthesize cAMP. There are no dominant cells that *The biochemistry of this reaction involves a receptor that binds cAMP.When begin the secretion or control the others. Rather, the sites of this binding occurs, specific gene transcription takes place, motility toward the aggregation are determined by the distribution of the myxam- source of the cAMP is initiated, and adenyl cyclase enzymes (which synthesize oebae (Keller and Segal 1970; Tyson and Murray 1989). cAMP from ATP) are activated. The newly formed cAMP activates the cell’s own receptors as well as those of its neighbors. The cells in the area remain in- Neighboring cells respond to cAMP in two ways: they initiate sensitive to new waves of cAMP until the bound cAMP is removed from the a movement toward the cAMP pulse, and they release cAMP receptors by another cell surface enzyme, phosphodiesterase (Johnson et al. of their own (Robertson et al. 1972; Shaffer 1975). After this 1989). The mathematics of such oscillation reactions predict that the diffusion of cAMP should initially be circular. However, as cAMP interacts with the cells happens, the cell is unresponsive to further cAMP pulses for that receive and propagate the signal, the cells that receive the front part of the several minutes. The result is a rotating spiral wave of cAMP wave begin to migrate at a different rate than the cells behind them (see that is propagated throughout the population of cells (Figure Nanjundiah 1997 1998). The result is the rotating spiral of cAMP and migra- tion seen in Figure 2.17. Interestingly, the same mathematical formulas predict 2.17B–D). As each wave arrives, the cells take another step to- the behavior of certain chemical reactions and the formation of new stars in ward the center.* rotating spiral galaxies (Tyson and Murray 1989). 2ND PASS PAGE PROOFS (B) (A) Adenine

O CH 5' 2 O H H H H 3' OOP OH OH cAMP

(C)

(D)

Figure 2.17 Chemotaxis of Dictyostelium myxamoebae is a result of spiral waves of cAMP. (A) Chemical structure of cAMP. (B) Visualization of several cAMP “waves.” Central cells secrete cAMP at regular intervals, and each pulse diffuses outward as a concentric wave. The waves were charted by saturating filter paper with radioactive cAMP and placing it on an aggre- gating colony. The cAMP from the secreting cells dilutes the radioactive cAMP. When the radioactivity on the paper is recorded (by placing it over X-ray film), the regions of high cAMP concentration in the cul- ture appear lighter than those of low cAMP concentration. (C) Spiral waves of myxamoebae moving toward the initial source of cAMP. Because moving and nonmoving cells scatter light differently, the photograph reflects cell movement. The bright bands are composed of elongated migrating cells; the dark bands are cells that have stopped moving and have rounded up. As cells form streams, the spiral of move- ment can still be seen moving toward the center. (D) Computer simulation of cAMP wave spreading across migrating Dictyostelium cells. The model takes into account the reception and release of cAMP, and changes in cell density due to the movement of the cells. The cAMP wave is plotted in dark blue. The population of amoebae goes from green (low) to red (high). Compare with the actual culture shown in (C). (B from Tomchick and Devreotes 1981; C from Siegert and Weijer 1989; D from Dallon and Othmer 1997.)

(A) (B) (C)

Figure 2.18 as shown here—10 hours after cell division has ceased—individual The three cell adhesion molecules of Dictyostelium. (A) Dictyo- myxamoebae have this protein in their cell membranes and are ca- stelium cells synthesize an adhesive 24-kDa glycoprotein (gp24) pable of adhering to one another. (B) The gp80 protein, stained by shortly after nutrient starvation. These Dictyostelium cells were specific antibodies (green), is present at the cell membranes of stained with a fluorescently labeled (green) antibody that binds to streaming amoebae. (C) The gp150 protein (green) is present in the gp24 and were then observed under ultraviolet light. This protein is cells of the migrating grex (cross-sectioned). Photographs are not at not seen on myxamoebae that have just stopped dividing. However, the same magnification. (Photographs courtesy of W. Loomis.) 2ND PASS PAGE PROOFS 42 Chapter 2

Sidelights Speculations Rules of Evidence I

iology, like any other science, does Dictyostelium, the next step was to use the stance, da Silva and Klein (1990) and Faix not deal with Facts, but with evi- antibodies that bound to gp24 to block the and co-workers (1990) obtained such evi- Bdence. Several types of evidence will adhesion of myxamoebae. Using a tech- dence to show that the 80-kDa glycopro- be presented in this book, and they are not nique pioneered by Gerisch’s laboratory tein (gp80) is an adhesive molecule in equivalent in strength. As an example, we (Beug et al. 1970), Knecht and co-workers Dictyostelium. They isolated the gp80 gene will use the analysis of cell adhesion in (1987) isolated the antibodies’ antigen- and modified it in a way that would cause Dictyostelium. binding sites (the portions of the antibody it to be expressed all the time. They then The first, and weakest, type of evidence molecule that actually recognize the anti- placed it back into well-fed, dividing myx- is correlative evidence. Here, correlations gen). This step was necessary because the amoebae, which do not usually express this are observed between two or more events, whole antibody molecule contains two protein and are not usually able to adhere and there is an inference that one event antigen-binding sites and would therefore to one another. The presence of this pro- causes the other. Correlative evidence pro- artificially crosslink and agglutinate the tein on the membranes of these dividing vides a starting point for investigations, myxamoebae. When these antigen-binding cells was confirmed by antibody labeling. but one cannot say with certainty that one fragments (called Fab fragments) were Moreover, the treated cells now adhered to event causes the other based solely on cor- added to aggregation-competent cells, the one another even in the proliferative stage relations. As we have seen, fluorescently cells could not aggregate. The fragments (when they normally do not). Thus, they labeled antibodies to a certain 24-kDa gly- inhibited the cells’ adhesion, presumably had gained adhesive function solely by ex- coprotein (gp24) do not bind to dividing by binding to gp24 and blocking its func- pressing this particular glycoprotein on myxamoebae, but they do find this protein tion. This type of evidence is called loss-of- their cell surfaces. Similar experiments in myxamoeba cell membranes soon after function evidence. have recently been performed on mam- the cells stop dividing and become compe- While stronger than correlative evi- malian cells to demonstrate the presence tent to aggregate (see Figure 2.18A). Thus, dence, loss-of-function evidence still does of particular cell adhesion molecules in the there is a correlation between the presence not make other inferences impossible. For developing embryo. Such gain-of-function of this cell membrane glycoprotein and the instance, perhaps the antibody fragments evidence is more convincing than other ability to aggregate. kill the cells outright (as might have been types of evidence. Although one might infer that the syn- the case if gp24 were a critical transport This “find it; lose it; move it” progres- thesis of gp24 causes the adhesion of the channel); this would also stop the cells sion of evidence is at the core of nearly all cells, it is also possible that cell adhesion from adhering. Or perhaps gp24 has noth- studies of developmental mechanisms causes the cells to synthesize this glycopro- ing to do with adhesion itself, but is neces- (Adams 2000). Sometimes we find the en- tein, or that cell adhesion and the synthesis sary for the real adhesive molecule to func- tire progression in a single paper, but more of the glycoprotein are separate events ini- tion (perhaps, for example, it stabilizes often, as the case above illustrates, the ev- tiated by the same underlying cause. The membrane proteins in general). In this idence comes from many laboratories. Such simultaneous occurrence of the two events case, blocking the glycoprotein would sim- evidence must be taken together. “Every could even be coincidental, the events hav- ilarly cause the inhibition of cell aggrega- scientist,” writes Fleck (1979), “knows just ing no relationship to each other.* tion. Thus, loss-of-function evidence must how little a single experiment can prove or How, then, does one get beyond mere be bolstered by many controls demonstrat- convince. To establish proof, an entire sys- correlation? In the study of cell adhesion in ing that the agents causing the loss of tem of experiments and controls is need- function specifically knock out the particu- ed.” Science is a communal endeavor, and *In a tongue-in-cheek letter spoofing such correla- lar function and nothing else. it is doubtful that any great discovery is the tive inferences, Sies (1988) demonstrated a re- The strongest type of evidence is gain- achievement of a single experiment, or of markably good correlation between the number of of-function evidence. Here, the initiation any individual. Correlative, loss-of-func- storks seen in West Germany from 1965 to 1980 of the first event causes the second event tion, and gain-of-function evidence must and the number of babies born during those same to happen even under circumstances consistently support each other to establish years. where neither event usually occurs. For in- and solidify a conclusion.

retaining a large enough number of cells to form large fruiting has evolved three developmentally regulated systems of cell- bodies (Müller and Gerisch 1978; Loomis 1988). During late cell adhesion that are necessary for the morphogenesis of in- aggregation, the levels of gp80 decrease, and its role is taken dividual cells into a coherent organism. As we will see in sub- over by a third cell adhesion protein, a 150-kDa protein (gp150) whose synthesis becomes apparent just prior to ag- *The gp150 cell adhesion protein of Dictyostelium may be critical for the sort- gregation and which stays on the cell surface during grex mi- ing out of the prespore and prestalk cells in the grex. This protein is first ex- gration (Wang et al 2000; Figure 2.18). If Dictyostelium cells pressed in prestalk cells and leads to their sorting out from prespore cells. A lack functional genes for gp150, development is arrested at the few hours later it is also expressed in prespore cells but at a lower level. If the protein is absent, no sorting out occurs. Thus, it appears that the temporal dif- loose aggregate stage, and the prespore and prestalk cells fail ference in expression and the levels of expression of this protein in the cell to sort out into their respective regions.* Thus, Dictyostelium types allows them to sort out (W. Loomis, personal communication). 2ND PASS PAGE PROOFS Life cycles and the evolution of developmental patterns 43 sequent chapters, metazoan cells also use cell adhesion mole- one of the two alternative pathways (Figure 2.19). At first cules to form the tissues and organs of the embryo. there is a bias toward one path or another. Then, there is a la- Dictyostelium is a “part-time multicellular organism” that bile specification, a time when the cell will normally become does not form many cell types (Kay et al. 1989), and the more either a spore cell or a stalk cell, but when it can still change its complex multicellular organisms do not form by the aggrega- fate if placed in a different position in the organism. The third tion of formerly independent cells. Nevertheless, many of the and fourth stages are a firm commitment to a specific fate, fol- principles of development demonstrated by this “simple” or- lowed by the cell’s differentiation into a particular cell type, ei- ganism also appear in the embryos of more complex phyla ther a stalk cell or a spore cell. (see Loomis and Insall 1999). The ability of individual cells to sense a chemical gradient (as in the myxamoeba’s response to BIAS. Although the details are not fully known, a cell’s fate ap- cAMP) is crucial for cell migration and morphogenesis dur- pears to be regulated by both internal and external agents. Pre- ing animal development. Moreover, the role of cell surface aggregation myxamoebae are not all the same; they can differ proteins in cell cohesion is seen throughout the animal king- in several ways. The internal factors distinguishing individual dom, and differentiation-inducing molecules are now being myxamoebae include nutritional status, cell size, cell cycle isolated in metazoan organisms. phase at starvation, and intracellular calcium levels (Nanjun- diah 1997; Azhar et al. 2001). Each of these factors can act to Differentiation in Dictyostelium bias the cell toward a prespore or a prestalk pathway. For in- Differentiation into stalk cell or spore cell reflects another stance, cells starved in the S and early G2 phases of the cell major phenomenon of embryogenesis: the cell’s selection of a cycle have relatively high levels of calcium and display a ten- developmental pathway. Cells often select a particular devel- dency to become stalk cells, while those starved in mid- or late opmental fate when alternatives are available. A particular cell G2 have lower calcium levels and tend to become spore cells. in a vertebrate embryo, for instance, can become either an epidermal skin cell or a neuron. In Dictyostelium, we see a LABILE SPECIFICATION. Several external factors are also impor- simple dichotomous decision, because only two cell types are tant in specifying cells as stalk or spore. Ammonia, a product possible. How is it that a given cell becomes a stalk cell or a of protein degradation, stimulates prespore gene expression spore cell? There appears to be a progressive commitment to and suppresses the expression of those genes that would lead

(A) Bias (C) Stable commitment (D) (E) and differentation Amoebae

Upper cup Pre-stalk and stalk Spore head or cell strain fpo sorus (pre-spore cells) (F) (G) (B) Labile Lower cup commitment Grex Stalk Pre-spore and spore cell strain

Pre-stalk Pre-spore Basal disk cells cells

Figure 2.19 Alternative cell fates in Dictyostelium discoideum.(A–C) Progressive stalk-forming anterior is cut off, the anteriormost cells remaining commitment of cells to become either spore or stalk cells. (A) will convert from stem to stalk. (C) At culmination, the spore- Myxamoebae may have biases toward stalk or spore formation due forming cells are massed together in the spore sac. The stalk cells to the stage of the cell cycle they were in when starved. (B) As the form the cups of the spore sac, as well as the stalk and basal disc. grex migrates, most prestalk cells are in the anterior third of the (D, E) Grex and culminant stained with dye that recognizes the ex- grex, while most of the posterior two-thirds are prespore cells. tracellular matrix of the prestalk and stalk cells. (F, G) Grex and Some prestalk cells are also seen in the posterior, and these cells will culminant stained with a dye that recognizes the extracellular ma- contribute to the cups of the spore sac and to the basal disc at the trix of prespore and spore cells. (After Escalante and Vicente 2000. bottom of the stalk. The grex can still regulate, however, and if the Photographs courtesy of XXXX XXXXX.) 2ND PASS PAGE PROOFS 44 Chapter 2

the cell to become a stalk (Oyama and Blumberg 1986). Pre- creased by manipulating the slug to have higher calcium levels stalk cells are able to form only when ammonia is depleted, (Cubitt et al. 1995; Jaffe 1997). A secreted chlorinated lipid, and this appears to occur by the diffusion of ammonia at the DIF-1, also plays some role in making prestalk cells, and it raised anterior tip of the grex. Cyclic AMP may also function may induce those genes that make the stalk-specific extracel- to induce prespore cell formation (Ginsburg and Kimmel lular matrix. These two factors may act synergistically to push 1997). High concentrations of cAMP initiate the expression of cells with high calcium levels into the prestalk pathway. spore-specific mRNAs in aggregated myxamoebae. Moreover, when slugs are placed in a medium containing an enzyme that COMMITMENT AND DIFFERENTIATION. Two secreted proteins, destroys extracellular cAMP, the prespore cells lose their dif- spore differentiation factors SDF1 and SDF2, appear to be im- ferentiated characteristics (Figure 2.20; Schaap and van Driel portant in the final differentiation of the prespore cells into 1985; Wang et al. 1988a,b). Thus, cAMP works both as an ex- encapsulated spores (Anjard et al. 1998a,b). SDF1 is impor- tracellular signal (for chemotaxis) and an intracellular signal tant in initiating culmination, while SDF2 seems to cause the (to activate those genes responsible for spore formation). prespore cells (but not prestalk cells) to become spores. The In the pathway to stalk cells, calcium appears to play a prespore cells appear to have a receptor that enables them to critical role. High calcium levels appear to push cells into the respond to SDF2, while the prestalk cells lack this receptor prestalk pathway, and the percentage of stalk cells can be in- (Wang et al. 1999). The formation of stalk cells from prestalk cells is similarly complicated and may involve several factors working synergistically (Early 1999). The differentiation of stalk cells appears to need a signal from the intracellular en- (A) zyme PKA, and at least one type of stalk cell is induced by the DIF-1 lipid (Thompson and Kay 2000; Fukuzawa et al. 2001).

Developmental Patterns among the Metazoa Since most of the remainder of this book concerns the devel- opment of metazoans—multicellular animals* that pass through embryonic stages of development—we will present (B) an overview of their developmental patterns here. Figure 2.21 illustrates the major evolutionary trends of metazoan devel- opment. The most striking pattern is that life has not evolved in a straight line; rather, there are several branching evolution- ary paths. We can see that metazoans belong to one of three major branches: diploblasts, protostomes, and deuterostomes. The sponges (Porifera) develop in a manner so different from that of any other animal group that some taxonomists (C) do not consider them metazoans at all, and call them “para- zoans.” A sponge has three major types of somatic cells, but one of these, the archeocyte, can differentiate into all the other cell types in the body. Individual cells of a sponge passed through a sieve can reaggregate to form new sponges. Moreover, in some instances, such reaggregation is species- specific: if individual sponge cells from two different species are mixed together, each of the sponges that re-forms contains cells from only one species (Wilson 1907). In these cases, it is thought that the motile archeocytes collect cells from their Figure 2.20 own species and not from others (Turner 1978). Sponges con- Chemicals controlling differentiation in Dictyostelium. (A, B) Effects of placing a Dictyostelium grex into a medium containing enzymes tain no mesoderm, so the Porifera have no true organ sys- that destroy extracellular cAMP. (A) Control grex stained for the tems, nor do they have a digestive tube, circulatory system, presence of a prespore-specific protein (white regions). (B) Similar grex stained after treatment with cAMP-degrading enzymes. No *Plants undergo equally complex and fascinating patterns of embryonic and prespore-specific product is seen. (C) Higher magnification of a postembryonic development. However, plant development differs significant- slug treated with DIF (in the absence of ammonia). The stain used ly from that of animals, and the decision was made to focus this text on the de- here binds to the cellulose wall of the stalk cells. (A, B from Wang velopment of animals. Readers who wish to discover some of the differences etal., 1988a; C from Wang and Schaap, 1989; courtesy of the au- are referred to Chapter 20, which provides an overview of plant life cycles and thors.) the patterns of angiosperm (seed plant) development. 2ND PASS PAGE PROOFS Life cycles and the evolution of developmental patterns 45

Vertebrata

Cephalochordata (Amphioxus) Anus from blastopore Urochordata DEUTEROSTOMES (ascidians)

Hemichordata

Echinodermata

Bilateral symmetry, three germ layers (Bilateria) Arthropoda

Nematoda Ecdysozoa

Priapulida

Molting

Annelida

Mouth from Spiral Brachiopoda PROTOSTOMES blastopore cleavage

Radial symmetry, Sipunculida two germ layers (Radiata) Mollusca Lopho- trochozoa (Spiralia) Colonial protists

True Rotifera multicellularity

Platyhelminthes (flatworms)

Ctenophora (comb jellies) DIPLOBLASTIC Cnidaria ANIMALS (jellyfish)

Porifera (sponges) Figure 2.21 Major evolutionary divergences in extant animals. Other models of evolutionary relationships among the phyla are possible. This grouping of the Metazoa is based on embryonic, morphological, and molecular criteria. (Based on J. R. Garey, per- sonal communication.) 2ND PASS PAGE PROOFS 46 Chapter 2

nerves, or muscles. Thus, even though they pass through an Phyla in the deuterostome lineage include the chordates embryonic and a larval stage, sponges are very unlike most and echinoderms. Although it may seem strange to classify metazoans (Fell 1997). However, sponges do share many fea- humans, fish, and frogs in the same group as starfish and sea tures of development (including gene regulatory proteins and urchins, certain embryological features stress this kinship. signaling cascades) with all the other animal phyla, suggesting First, in deuterostomes (“mouth second”), the oral opening is that they share a common origin (Coutinho et al. 1998). formed after the anal opening. Also, whereas protostomes generally form their body cavities by hollowing out a solid The diploblasts block of mesoderm (schizocoelous formation of the body Diploblastic animals are those that have ectoderm and endo- cavity), most deuterostomes form their body cavities from derm, but no true mesoderm. The diploblasts include the mesodermal pouches extending from the gut (enterocoelous cnidarians (jellyfish and hydras) and the ctenophores (comb formation of the body cavity). It should be mentioned that jellies). there are many exceptions to these generalizations. Cnidarians and ctenophores constitute the Radiata, so The evolution of organisms depends on inherited called because they have radial symmetry, like that of a tube changes in their development. One of the greatest evolution- or a wheel. In these animals, the mesoderm is rudimentary, ary advances—the amniote egg—occurred among the consisting of sparsely scattered cells in a gelatinous matrix. deuterostomes. This type of egg, exemplified by that of a chicken (Figure 2.22), is thought to have originated in the am- Protostomes and deuterostomes phibian ancestors of reptiles about 255 million years ago. The Most metazoans have bilateral symmetry and three germ lay- amniote egg allowed vertebrates to roam on land, far from ex- ers. The evolution of the mesoderm enabled greater mobility isting ponds. Whereas most amphibians must return to water and larger bodies because it became the animal’s musculature to lay their eggs, the amniote egg carries its own water and and circulatory system. The animals of these phyla are known food supplies. It is fertilized internally and contains yolk to collectively as the Bilateria. All Bilateria are thought to have de- nourish the developing embryo. Moreover, the amniote egg scended from a primitive type of flatworm. These flatworms contains four sacs: the yolk sac, which stores nutritive pro- were the first to have a true mesoderm (although it was not hollowed out to form a body cavity), and they may have resembled the larvae of certain Embryo contemporary coelenterates. Amnion O2 Animals of the Bilataria are further classified CO2 as either protostomes or deuterostomes. Proto- Heart stomes (Greek, “mouth first”), which include the Allantois mollusc, arthropod, and worm phyla, are so called because the mouth is formed first, at or near the opening to the gut, which is produced during gas- Amniotic cavity trulation. The anus forms later at another location. The coelom, or body cavity, of these animals forms from the hollowing out of a previously solid cord of mesodermal cells. There are two major branches Vitelline vein of the protostomes. The Ecdysozoa includes the Vitelline artery animals that molt their exterior skeletons.* The major constituent of this group is Arthropoda, a phylum containing the insects, arachnids, mites, Shell Chorion crustaceans, and millipedes. The second major group of protostomes is the Lophotrochozoa. Shell Yolk These animals are characterized by a common type membrane sac of cleavage (spiral), a common larval form (the Yolk Figure 2.22 trochophore), and a distinctive feeding apparatus Diagram of the amniote egg of the chick, showing the membranes enfolding the (the lophophore) found in some species. Lopho- 7-day chick embryo. The yolk is eventually surrounded by the yolk sac, which al- trochozoan phyla include the flatworms, bry- lows the entry of nutrients into the blood vessels. The chorion is derived in part ozoans, annelids, and molluscs. from the ectoderm and extends from the embryo to the shell (where it will fuse with the blood vessel-rich allantois. This chorioallantoic membrane will ex- *The name Ecdysozoa is derived from the Greek ecdysis, “to change oxygen and carbon dioxide and absorb calcium from the shell). The am- shed” or “to get clear of.”Asked to provide a more dignified job nion provides the fluid medium in which the embryo grows, and the allantois description for a “stripper,” editor H. C. Mencken suggested collects nitrogenous wastes that would be dangerous to the embryo. Eventually the term “ecdysiast.” the endoderm becomes the gut tube and encircles the yolk. 2ND PASS PAGE PROOFS Life cycles and the evolution of developmental patterns 47 teins; the amnion, which contains the fluid bathing the em- Embryology provides an endless assortment of fascinat- bryo; the allantois, in which waste materials from embryonic ing animals and problems to study. In this text, we will use but metabolism collect; and the chorion, which interacts with the a small sample of them to illustrate the major principles of outside environment, selectively allowing materials to reach animal development. This sample is an incredibly small col- the embryo.* The entire structure is encased in a shell that al- lection. We are merely observing a small tide pool within our lows the diffusion of oxygen but is hard enough to protect the reach, while the whole ocean of developmental phenomena embryo from environmental assaults and dehydration. A sim- lies before us. ilar development of egg casings enabled arthropods to be the After a brief outline of the experimental and genetic ap- first terrestrial invertebrates. Thus, the final crossing of the proaches to developmental biology, we will investigate the boundary between water and land occurred with the modifi- early stages of animal embryogenesis: fertilization, cleavage, cation of the earliest stage in development: the egg. gastrulation, and the establishment of the body axes. Later chapters will concentrate on the genetic and cellular mecha- VADE MECUM The amniote egg. The egg of the chick, as nisms by which animal bodies are constructed. Although an detailed in this sequence, is a beautiful and readily accessi- attempt has been made to survey the important variations ble example of the amniote egg—a remarkable adaptation throughout the animal kingdom, a certain deuterostome to terrestrial life. [Click on Chick-early] chauvinism may be apparent. (For a more comprehensive sur- *In mammals, the chorion is modified to form the embryonic portion of the vey of the diversity of animal development across the phyla, placenta—another example of the modification of development to produce see Gilbert and Raunio 1997.) evolutionary change.

Principles of Development: Life Cycles and Developmental Patterns

1. The life cycle can be considered a central unit in biology. 8. There are three main ways to provide nutrition to the de- The adult form need not be paramount. In a sense, the life veloping embryo: (1) supply the embryo with yolk; (2) form cycle is the organism. a larval feeding stage between the embryo and the adult; or (3) create a placenta between the mother and the embryo. 2. The basic life cycle consists of fertilization, cleavage, gas- trulation, germ layer formation, organogenesis, metamor- 9. Life cycles must be adapted to the nonliving environment phosis, adulthood, and senescence. and interwoven with other life cycles. 3. Reproduction and sex are two separate processes that may 10.Don’t regress your tail until you’ve formed your hindlimbs. but do not necessarily occur together. Some organisms, 11. There are several types of evidence. Correlation between such as Volvox and Dictyostelium, exhibit both asexual re- phenomenon A and phenomenon B does not imply that A production and sexual reproduction. causes B or that B causes A. Loss-of-function data (if A is 4. Cleavage divides the zygote into numerous cells called experimentally removed, B does not occur) suggests that A blastomeres. causes B, but other explanations are possible. Gain-of- function data (if A happens where or when it does not 5. In animal development, gastrulation rearranges the blas- usually occur, then B also happens in this new time or tomeres and forms the three germ layers. place) is most convincing. 6. Organogenesis often involves interactions between germ 12. Protostomes and deuterostomes represent two different layers to produce distinct organs. sets of variations on development. Protostomes form the 7. Germ cells are the precursors of the gametes. Gameto- mouth first, while deuterostomes form their mouths later, genesis forms the sperm and the eggs. usually forming the anus first.

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