Nobel Prize winner (Medicine, 1995) Christiane Nüsslein-Volhard has been director of the Max Planck Institute for Developmental Biology since 1985, and also leads their Genetics Institute. Her 2006 book, Coming To Life, describes the genetics behind the process by which organisms develop from a single fertilized cell into highly differentiated forms with over 200 types of cells.
The author’s highly readable prose paints in broad strokes, providing only the detail essential for understanding. In the sense that perfection is defined by ‘nothing more can be removed’, her book is perfection. For backup detail, the author recommends ‘Fat Alberts’, the standard text on the subject (Molecular Biology of the Cell, Bruce Alberts et al, 1500 pages of details that the author skillfully omits). Fat Alberts is available online in a searchable, but not browsable edition.
The first third of the book provides the biological and genetic information necessary to understand the rest. Throughout this part, the history of pivotal discoveries is folded in to create an accurate sense of timing of the development of our knowledge. An understanding of how various bits of the puzzle were discovered enhances our own sense of discovery, and also adds further illustration of the concepts.
Origin and Heredity
Popular historical thinking on organism development could only imagine a finished creature in great miniature, a homunculus, residing in either the sperm or egg. The logical fallacy of this preformation concept was that all future generations would also have to be present, a set of Russian dolls without end. The notion of spontaneous generation, creating something out of nothing, was also widespread. Yet the fact that the offspring always resembled the parents suggested inheritance of individual characteristics.
Not all people of antiquity were so easy to confound. Aristotle, the father of biology, considered preformation, but favored a concept of new patterns arising in each generation. Studying the development of a chick in an egg, he concluded that the complex forms of life must originate from simpler ones. But just as scientists of the present time get confused and classify things by their appearance, Aristotle could be excused from positing a developmental link between worms and eels. In the end, his characterizing living organisms by appearance rather than manner of living was a great step forward.
Over time, the understanding and experience of the ever increasing variety of life made the management of the relationships increasingly unwieldy until 2000 years after Aristotle, Carl Linnaeus founded the basis for classification still in use for all living organisms, based on their observable similarities. Organisms are ordered into groups called taxa (sing. taxon), based on similar attributes whose sum define the taxon’s phenotype. Taxa are ordered hierarchically, proceeding to ever increasing levels of abstraction. The entire ordered structure of life is called its taxonomy.
The fundamental (lowest hierarchical layer) taxon of organisms is the species, roughly defined as a group of organisms similar enough that they can reproduce among themselves. Similar species are grouped in genera, which are grouped into families, and so on up to the two original great kingdoms, Plantae and Animalia, recognized from the time of Aristotle. As the level of abstraction grows greater climbing the tree of life, the divisions become more speculative. Over time, the classifications at the top of the tree of life have changed, with 2, 3, 4, 5, or even 6 Kingdoms at the top. As of 2010 there is still no general agreement about the apical topology.
[WS Aside: Taxonomy has given way in the last two decades to cladistics, a similar but more rigorous classification based on DNA similarity rather than morphological similarity. In cladistics, a taxon is called a clade.]
Cells and Chromosomes
Nearly a century after Linnaeus, von Baer recognized that there are more similarities between embryos than between adult organisms. But he didn’t know where to go with this except to attribute lack of creativity to the Creator. Goethe observed that all animals are related and suggested that humans be considered a species of Mammalia. It was Darwin who recognized embryonic similarities as true biological links, pointing backward to a common origin of life. This became one of the pillars of his monumental opus, Origin of Species by Means of Natural Selection.
Darwin recognized the changeability of all things, from living organisms to the Earth itself. He learned from fossils that organisms that once lived now live no longer. Species could go extinct. They could also change, as he observed with finches in the Galapagos, with similar but different species on each islet.
The Darwinian hypothesis has three components: surplus, variation, and selection. A surplus of offspring are produced, enough to guarantee species continuity. The amount of reproductive wastefulness varies by species. He observed offspring could vary slightly from the parents, more so with sexual than with asexual reproduction, and speculated such variation, combined with natural selection of the fittest variants, was the engine that powered the variety of life. And since variations that favored reproduction, both survivability and sexual attractiveness traits, were heritable, good traits could be passed to the next generation. He hypothesized that a natural selection was happening over long periods of time that would favor survivability, reproduction, and adaptability traits when they occurred and would extinguish other traits, gradually causing species to morph. He observed that population isolation could cause the morphing to accelerate.
Following Darwin’s evolutionary arrow backward in time, one reaches the primordial single living cell, progenitor of all life on Earth, that lived over 3 billion years ago. For a span of perhaps 2.5 billion years, only microscopic single cell life forms existed. Then 600 million years ago, multicell life forms began appearing, one of which is ancestor to all animals that have ever lived. Of these animal species, fewer than 1% still survive. Since living animals are leaves on the great tree of life, most have not evolved from other concurrent living animals, but rather from extinct ancestors that lived millions of years ago.
The idea of evolution through selection accommodated the much lesser variation of embryos. Relatively stable embryonic forms are buffered from selection, removing the force that drives variation. Thus human embryos can still exhibit gill slits early in development, because there is no reproductive penalty that entails. Also, the initial steps of embryonic development are quite rigidly determined, so any variation would not result in a functional organism.
Simultaneously with Darwin’s work, the German Remak discovered that every cell originates from a precursor cell. Improved microscopes and staining techniques were enabling plant and animal cells to be studied. The research on continuity of cells, published just 4 years before Origin of Species, may have given Darwin further insight and confirmation into the mechanism of evolution.
Darwin himself never understood how variation and inheritance actually worked, which makes his achievement even greater. Mendel, 13 years junior to Darwin, studied inheritance by cross-breeding peas with distinct traits and observed how the traits were distributed in offspring in certain numerical ratios. He speculated there were factors in cells that determined the traits. (We now call these factors genes.) From his observations, he deduced that organisms have two copies of each factor; inheritance involves passing discrete heritable factors equally from father and mother.
Like Darwin, Mendel did not know the details behind his theory. Chromosomes and the mechanism of inheritance had not yet been discovered. But he was able to deduce the abstract rules for heritability from his detailed observations. His brilliant work was overlooked for 35 years, partly because he was obscure and partly because he knew his rules must apply to all living things, but it was difficult at the time to confirm the theory for a sufficient variety of organisms. It was only in 1900 that his work was instated into the mainstream of science.
Cells and Chromosomes
With the increasing capabilities of microscopy in the late 19th century, it became possible to observe the development of an embryo from a simple to complex cellular form, and even to study the nucleus of a cell, observing that as cells divide, the division of the nucleus happens first. Then at the beginning of the 20th century, observations of the chromosomes within the nucleus led to the deduction that the chromosomes must carry the heritable factors.
Animals differ in cellular complexity and usually animals of the same type will have differing numbers of cells. However, some simple animals always have the same number, such as the worm Caenorhabditis elegans (C. elegans) with its 959 body cells. Human cells come in over 200 different sizes and functions. The cells in larger animals are both more numerous and larger in size. A mouse has 2×10^9 cells, a human 3×10^13. Cells have a minimum possible size. Mammalian cells are on average around 10 micrometers in diameter.
In the fertilization process of sexual reproduction, the zygote is the cell formed by the merging of two gametes, the tiny, mobile sperm and the large, immobile egg. It is called totipotent, because it is capable of becoming the entire organism. The cytoplasm in the zygote comes exclusively from the egg, but the nucleus contains equal parts of egg and sperm chromatin.
The fertilizing sperm then contributes the centrosome, an organelle that pulls single chromosomes from the cell nucleus to each of the organizer points prior to cell mitosis. But in some organisms (e.g. bees), the egg provides its own centrosome, which allows cell division to commence without contribution from a sperm cell. This ‘virgin’ conception process is called parthenogenesis; all male bees are conceived this way.
Once fertilized, the zygote cleaves into two cells, then each of these again and again, with the daughter cells initially successively smaller, creating a collection of identical cells called the blastula. Subsequently, embryo building begins, with the cells forming different groupings and differentiating by groups. At this phase, there is growth in the cytoplasm after nuclear cleavage, so that daughter cells can be as big as the mother cell.
A cell grouping folds inward creating the basic early embryonic form during the early gastrulation process. Differentiation and growth proceeds to create the primordia for the various tissue types and later the organs themselves.
The German zoologist Boveri answered the question of where the heritable factors reside in the cell. He observed that by using a sperm from a different species to fertilize an egg lacking a nucleus (by heavily shaking it to remove the nucleus), the resulting embryos resembled the father’s species, proving the nucleus rather than the cytoplasm codes the heritable traits.
The American biologist Sutton studied grasshopper cells and observed 11 distinct chromosomes in the nucleus. In body (somatic) cells, the chromosomes appeared in duplicate (diploid 2N), while in gametes, only one chromosome of each type is present (haploid 1N). Boveri showed the chromosomes each carried independent heritable factors (genes) by fertilizing an egg with two sperm, causing unusual combinations of chromosomes in resulting zygotes. The abnormal cells failed to develop and showed characteristic problems, implying that genes on different chromosomes could not substitute for one another.
When a germ stem cell undergoes meiosis (divides), only one of the chromosomes in each pair is passed on to the gamete. Only the germ cell line has the capability to pass on the genes to subsequent generations. Similarly, gene mutations can only be passed on if the mutation happens in a germ cell. Germ stem cells migrate early in embryo development to the gonads and likely contain special programs to prevent chromosomal damage.
[WS Aside: Gametes are created by division of diploid germ stem cells. The germ cell begins division as in mitosis with cell growth and replication of the chromosomes. But the process diverges after this into two separate complex division steps, a reduction division that creates two haploid 2N cells with sister chromatids still attached to each other in pairs, followed by a final equational division that separates and crosses the sister chromatids, producing four gametes (haploid 1N). In males, all four resulting gametes become sperm. In females, one gamete becomes the egg and the other three are pinched off and die. In female mammals, meiosis of germ cells begins in the embryonic womb, but is arrested at the reduction step until the cells (oogonia) are released in an adult menstrual cycle, at which point they complete reduction division and arrest at the final division stage, completing meiosis only if fertilized.]
Since all cells contain all genes, one wonders what causes cells to differentiate from one another. It became known that substances within the cytoplasm turn individual genes in the cell on and off. Up to the completed blastoma, cleavage cells are totipotent. But after that, the cytoplasm begins to differentiate. Boveri observed polarity in egg cytoplasm; splitting along the polar axis enabled the two halves to develop, but splitting along a perpendicular axis did not produce viable eggs.
The Sutton and Boveri results were published in 1903, by which time it was known that most animals have several chromosomes in each cell nucleus. These chromosomes exist in homologous pairs, one of each from father and mother. In 1903, recombination of chromosomes was not yet understood, but it was known that chromosomes are the sole carriers of genes, the source of our heritable traits. We have two copies (alleles) of each gene in each cell. The cytoplasm influences embryonic development by scheduling gene activity.
This introduces the subject of the rest of the book: the control of gene activity in time and space to produce a new organism from the simple group of identical cells in the blastula. The genes and the cytoplasm provide the molecular machinery to implement the process. Signals from neighboring cells and from the cell environment drive the process. Tantalizing evidence of how the process worked was available early, as in German zoologist Spemann’s 1923 experiment involving a special region of the egg called the organizer. By transplanting organizer cells into another embryo, a new body axis was formed in the recipient embryo as a result of reaction to the foreign cells. The foreign cells did not directly contribute to the new body, but rather directed the host to make another axis. The organizer cells then became a focus of study for embryology, but it took another 70 years of development in molecular genetics to coax the mechanism details.
Genes and Proteins
Further sophistication in the genetic model of the cell was deduced from experiments with Drosophila (fruit fly), whose lowly four chromosomes, giant polyploid larval cells, 12 day life cycle, and easily observed phenotypic traits made them ideal genetics subjects. Using flies, Mendelian laws were quickly confirmed for animal life as well as for plants. But exceptions to the expected Mendelian results were noted that required explanation.
American biologist Morgan was an early researcher who noted that the dominant red eye trait in the flies did not produce Mendelian results. Mating red-eyed males with white-eyed females should have produced red-eyed offspring, but all the males were still white-eyed. This was evidence for sex-specific chromosomes, and is explained because the eye color gene is on the X chromosome. In most male organisms, this chromosome is always present in a single copy inherited from the mother. Males pair this chromosome with a Y chromosome inherited from the father. Females have two paired X chromosomes, one from mother and one from father.
Linkage between genes was also deduced from fly experiments whose results differed from Mendelian expectations. Two genes on the same chromosome are said to be linked because their traits are inherited together. Then further experiments brought to light that even factoring in gene linkage, some results could still not be explained when it was observed that two mutations on the same chromosome are not always linked. This led to a hypothesis of genetic recombination, the great source of variation in life.
In meiosis, homologous chromosomes are randomly mixed and matched in sections prior to division, a process called crossing over. Thus the resulting gamete will not receive an entire chromosome from father or mother, but rather pieces from one and pieces from the other. Initial genetic maps of the chromosomes resulted from the observation that crossing over more often unlinks genes that are some distance from one another. Put another way, the closer two genes are on the chromosome, the more likely they will remain linked, so frequency of linkage correlates with gene proximity.
Fly larvae do not undergo cell division; their cells just grow larger with multiple chromosomal duplications, leading to massive polyploidy (multiplicity of chromosomes) within the cells. The resulting thick bundles of chromosomes show visible banding patterns, most apparent in the salivary glands. While the bands do not correspond to individual genes, this was the first visible evidence of gene existence. American geneticist Bridges mapped the X chromosome using these bands, discovering the location of some important genes.
Yet the Drosophila was still too complex an organism to study at the molecular and biochemical level. The biochemical connection between genotype and phenotype was later deduced from study of much simpler bacteria and fungi, which are haploid and hence directly show, within the cell, the phenotype associated with each gene allele.
It was learned, while growing genetic variants in media, that cultures containing certain alleles would only grow with addition of nutrients such as glucose or its precursors. From this, it was deduced that such alleles prevented the gene from generating enzymes needed for some metabolism steps. Thus a gene-enzyme correlation was deduced. This was soon generalized to a notion that a gene’s function is to produce protein.
It was not until 1944 that bacteriologist Avery demonstrated that genes are made of nucleic acids rather than protein (even though DNA had been known since 1869, discovered by Swiss scientist Miescher). Avery exposed cell-free gene extracts to enzymes that attack protein, but the genes remained active; exposure to DNase enzyme immediately destroyed the gene extract. In 1951, American Biologists Hershey and Chase showed that viral cell infections only transfer DNA into the cell.
The relation between nucleic acids in a gene and the protein synthesized by the gene is direct. The order of the bases comprising the gene determines the order of the amino acids in the corresponding protein. A two step process is involved, transcription followed by translation. During transcription, the DNA is unwound and the RNA polymerase enzyme reads the bases in the sense strand and generates a single strand of compatible bases in a molecule called messenger RNA (mRNA). mRNA differs from DNA; the sugar desoxyribose is replaced with ribose, and the thymine base is replaced by uracil.
Transcription typically proceeds automatically to translation. Transcription control can both determine when the gene is turned on (i.e. when a protein is required) and when it is turned off. Every gene has a control region called the promoter, which binds to an RNA polymerase enzyme to begin transcription. Protein transcription factors located near the promoter can either block (repressor) or stimulate binding of the RNA polymerase. In more complex organisms, a variety of control factors are needed to regulate transcription.
During translation, the protein is synthesized, one amino acid at a time, alongside the mRNA strand in the cytoplasm. The mRNA is interpreted as a sequence of codons, where a codon is a series of 3 consecutive bases. Translation involves reading a codon and producing the amino acid coded for by that codon. Codons are read by ribosomes that employ tRNA (transfer RNA) molecules, having an anticodon on one end to match the current codon, and having a corresponding amino acid attached to the other end. The anticodon match is not necessarily 1-1 with the codon, because the anticodon can have more general molecules that match more than one base. In a strictly 1-1 scheme, it would take 61 types of tRNA molecule to code for all amino acids, but a minimum of 31 will still do the job.
A ribosome ‘marches’ along the mRNA strand one codon at a time, acquiring the appropriate tRNA strand to match the current codon. The matching tRNA molecule gives up its amino acid, which is linked by enzymes within the ribosome to the peptide chain being synthesized. The empty tRNA molecule then exits the ribosome to pick up another amino acid matching its anticodon. The synthesized three-dimensional protein folds in a characteristic manner determined by the sequence of the amino acids. Just as empty tRNA molecules can be reused, the ribosome parts (subunits) themselves are reused after each translation.
Proteins are difficult to analyze and synthesize. They are complex, some unstable, and some available only in miniscule quantities. The ability to analyze a DNA sequence and predict the structure of the corresponding protein was a big advance. Once a gene is isolated, large quantities of its protein can be synthesized.
Proteins can have common domains (sequences of amino acids) related to their function. Several transcription factors contain just such a domain of 60 amino acids, called the homeodomain, that binds to DNA. Other domains are repeating motifs within a protein, such as the immunoglobulin domain of antibodies, consisting of 110 amino acids. Proteins to be secreted from the cell have a typical domain at the start region. Other proteins have domains that serve to anchor them to cell membranes. Computerized searches can recognize recurrent domains and hypothesize protein roles based on them.
In eukaryotes, DNA is not freely distributed in the cytoplasm, but is wrapped up within the nuclear membrane where it is associated with proteins called histones. Histones condense and shorten the long DNA strands into chromatin. We don’t yet know how this packaging is undone prior to (or redone after) transcription.
Most eukaryotic DNA is not transcribed and hence is non-coding. Coding regions can be recognized by the absence of stop codons. Non-coding regions can occur between genes or within genes, where thay are called introns. Coding sections are called exons. During transcription, an RNA transcript is produced that contains introns and exons. The RNA is then processed by enzymes that remove the introns and produce mRNA.
Mammals have long stretches of non-coding DNA. In these stretches are found control regions that bind to transcription factors to control gene activity. In addition to the promoter region, there are also enhancer regions that can affect gene transcription over long distances. Enhancer regions can occur in introns. The more specialized the gene’s role, the more complex its control regions; they will bind with many different transcription factors, both activating and inhibiting.
Animals can have foreign genes added to all their cells, making them transgenic. By injecting copies of the gene, synthesized in bacteria, into cytoplasm at the posterior of an egg, the gene becomes encased within the pole cells from which the germ line develops, causing some gametes to contain the foreign gene. From these gametes, transgenic offspring develop. Transgenic animals aid understanding of gene function, malfunction, and relation to other genes.
Development and Genetics
Until the 1970s, there was not enough interest in, or tools for, understanding the underlying mechanisms of the embryo development process outlined at the start of the century by Spemann. In the interim, only theoretical discussions were advanced that discussed how embryo development might work. One such argument by Wolpert proposed the position of cells in the embryo determines their fate, based on observing the regenerating polyp Hydra.
A gradient mechanism was theorized, whereby a Spemann organizer secretes a substance called a morphogen that lessens in concentration as it spreads away from the organizer. Above some gradient threshold, cells in the organizer vicinity have their state controlled by the organizer’s secretion. Different cells may have different morphogenetic thresholds and achieve different states, all under control of one organizer. This theory did not gain traction until the morphogens could be identified and observed.
It was presumed that the morphogens were proteins, so that genetic approaches to identification would be useful when the technology became available. Thus spawned the field of developmental genetics in the 1970s. Care was needed to choose a model organism for study, because such studies require crossbreeding of mutants that is only possible within a species. Breeding space and time both needed to be minimized, and efficacy of breeding and visibility of genetic traits maximized. Both the fly Drosophila and the worm C. elegans proved good choices.
The Drosophila development from conception to hatched larva is only 24 hours. The egg is created from germ cells and helper nurse cells, accompanied by somatic follicle cells that develop into the shell. The front, rear, top, and bottom of the egg are easily discriminated by shape of the shell, but the only detectable internal structure is the clear region at the posterior pole, called pole plasm. This substance is incorporated into early embryonic pole cells that become the germ cell line.
The egg nucleus is found at the top frontal part. It divides many times within the cytoplasm, each nucleus migrating to the surface. At 1.5 hours, this single large cell with all its nuclei and its posterior pole cells is called the syncytial blastoderm. Nutrients and other substances flow freely, unhindered by cell membranes. Then at 3 hours, all 6,000 nuclei develop enclosing cellular membranes simultaneously, creating the cellular blastoderm.
At 3.5 hours, gastrulation has caused some cells to migrate inward, forming three layers of stem cells: ectoderm, mesoderm, and endoderm. The ectoderm develops into skin and nerve tissue; the mesoderm develops into musculature, inner organs, and blood; the endoderm develops into intestines and stomach. A furrow develops along the ventral side of the egg, and endoderm cells fold into it from each end. On the dorsal side, the embryo stretches forward at 4.5 hours, the cells there further dividing to create the nervous system, beginning with neuroblast formation. Subsequently this stretching is reversed while organ tissues and body segmentation begin to appear. By 24 hours, further migration and cell differentiation create the larval form. The nervous system develops from neuroblasts into a brain and a ventral cord, a ladder-structure of knots and strings.
The larva grows by increasing cell size, rather than by cell division. While growing, it sheds its skin twice before the three day pupation stage where the larva metamorphizes into an adult fly. The hormone ecdysone triggers metamorphosis in which cuticular structures differentiate into bristles, hairs, eyes, sexual organs, and wings. These features originate in undifferentiated cells called imaginal discs, which come in right/left pairs, three pairs for the legs, two for the wings and thorax, two for the head with eyes and antennae, and a few smaller for abdomen and head. Each growing from 3-20 precursor cells, the fully developed imaginal discs consist of up to 40,000 cells. Imaginal discs develop as an inner fold and an enveloped part. At the last moment, the enveloped part migrates outside; examples are thorax enveloping wing, and head enveloping antennae.
Cell differentiation rules can be visualized as a fate map. The map begins in two dimensions with two orthogonal axes, anterior-posterior (A-P) and dorsal-ventral (D-V). For example, the major segments of the abdomen and thorax each begin from a strip 3 cells wide in the center of the embryo. A small group of genes in the embryonic cells is responsible for implementing the fate map. But prior to this, there is a skeletal initial fate map provided by the mother’s germ cell genes. The embryo’s genes take over beginning with the blastoderm phase. Mutants of either the maternal or zygotic development genes produce bizarre structural defects. By stimulating mutations and then studying their effects, 40 maternal and 120 zygotic genes have been found that influence Drosophila development, a very small fraction of the genome.
The gene functions are largely time and spatially oriented, rather than targeted to a specific feature of the phenotype. Thus a small contingent of genes can effect the entire complex development. By studying the effects of mutations, these genes can be grouped, where a group effects similar changes in the phenotype and hence may operate in a common way. The earlier a gene operates in development, the more profound the results of mutations. Thus mutant maternal alleles will have the greatest impact on the resulting larvae.
Only a simple set of such maternal gene defects is observed, affecting growth either along one axis or the other. Thus, it is realized that development along the two axes occurs independently. One set of maternal genes controls D-V axis development, and three groups affect the A-P axis (Bicoid, the front; Oskar, the rear; Torso, the two non-segmented ends at the egg poles). The four groups thus generate four developmental cues, positioned at the ends of the two axes. These cues then spread to take control of the entire embryonic development.
In zygotic genes, one group again affects the D-V growth, but expressing much smaller differences in phenotype. Again thee zygotic groups are observed in A-P axis development of segmentation. The first group are called gap genes, because mutations cause missing body regions, but smaller than those resulting from the parallel maternal mutant alleles. The second and third are the pair-rule and segment-polarity gene groups. A hierarchy of gene group operation is observed, with genes guiding development by regulating activity of other genes. Early on, large regions are defined coarsely by interplay between maternal and gap genes; these interplays occur in the context of one large cell prior to creation of the blastoderm. Then a periodic pattern of double (paired) segments appears, followed by a splitting into single segments, under control of the remaining two groups of zygotic genes.
Effects of gene expression prior to the blastoderm generation are not visible without special techniques; they are referred to as prepatterns (zones of molecular concentration). Genes produce proteins that diffuse away from the cell nuclei and bind to enhancer regions of other genes. Through the interplay of mutual activation and repression, complex molecular patterns emerge throughout the single-cell zygote. The final prepattern triggers the first changes in cell shape with the formation of the blastoderm.
The morphogenic factors overlap and affect other genes in various ways in response to concentration gradients. The two principles that create the required complexity of signaling are morphogen overlaps and gene concentration sensitivity thresholds; innumerable different signals can arise from the confluence of these principles. Prior to blastoderm, these principles operate somewhat amorphously via prepatterns. After cellular membrane creation, these principles operate between cells using signal transduction pathways to diffuse morphogens from one cell to neighboring cells. A gradient is only established if the morphogen is unstable; without continuous decay, it would just continuously accumulate in the cell.
For example, consider the bicoid maternal gene at the anterior pole of the zygotic cell. Its transcribed and anchored mRNA emits the bicoid protein that diffuses out toward the opposite pole. This protein contains a homeodomain that binds to the promoters of various segmentation genes, such as the hunchback gap gene. Where concentrations are high enough in the anterior third of the zygotic cell, the hunchback gene is transcribed. If another gene with higher threshold were transcribed at the anterior pole, three transcription zones would be defined: both genes active, one gene active, and no genes active.
The zygote cell contains four gradients emanating from axis poles, corresponding to four maternal gene groups. One is the bicoid gradient (pole A), one is Oskar gradient (pole P). Two gradients from the torso gene group act at shorter range, one from each A-P pole. Finally, a dorsal gradient is present (D), activating genes that control dorsal-ventral differentiation. These maternal gradients activate zygotic segmentation gap genes (e.g. hunchback, knirps) within their ranges. The overall A-P axis differentiation is effected by a series of transient prepatterns that eventually produce segmentation.
The D-V gradient is triggered by the Toll protein, anchored in the membrane throughout the zygote. It causes the dorsal protein, a transcription factor present throughout the cytoplasm, to enter the nuclei exclusively on the ventral side, by signal transduction through the ventral membrane from outside the cell; its gradient diminishes toward the dorsal pole. At high concentrations, dorsal protein activates the twist gene for formation of musculature, and in lesser concentrations activates genes on right and left sides of the embryo. It also represses certain genes, so those genes are only active near the dorsal pole. Toll thus divides the zygote into four longitudinal zones defined by the protein transcribed: twist, sog (L, R), and Dpp at the dorsal side.
There are six gap genes that are transcribed, producing broad zones determined by maternal gradients. These zones are further differentiated by interactions between the gap genes, because they code for a transcription factor that affects other genes, diffusing in the cytoplasm and forming short-range concentration gradients. At its highest concentration near the transcribed gene, the coded protein acts as a repressor to other gap genes. At lower gradients, the repression is inactive and genes may be activated. Thus adjacent peaks of activity occur across the A-P axis.
As all gap genes are transcribed, the confluence of their protein interactions create a simple morphogen pattern of several broad stripes. Further interactions with the pair-rule genes create seven stripes per gene, one stripe for every future segment pair. The periodicity of these stripes may appear to indicate some general harmonic process, but rather, each is created independently by gene transcription logic. Combinations of gap genes can turn on different pair-rule genes. These pair-rule genes, such as fushi-tarazu and even-skipped, then interact with mutual repression so the result is 14 interlaced pair-rule gene gradients.
Finally, under influence of the pair-ruled gene gradients, the segment-polarity genes (engrailed) are activated in zygotic cells, delineating 14 evenly distributed regions of cells that will produce the 14 segments of the adult fly. The engrailed genes are activated in rows of cells that will become the posterior part of each segment.
Selector genes respond to overall embryo spatial subdivisions (stripes) to set cells to special states related to their developmental function. Selector genes encode transcription factors that will activate groups of genes later in development for detailed differentiation. The twist gene is a selector gene, as is the tinman gene which drives formation of heart muscle.
American biologist Lewis discovered a group of genes, the homeotic genes, that control body development from front to back. Drosophila have eight homeotic genes, organized into two gene complexes, Antennapedia and Bithorax. The order of the genes on their chromosome corresponds to the order of the structures they influence, from head to tail. Their products work in combination: one is active at the very front, then two, and then more and more working backward. They determine the nature of the segments along the body axis.
Homeotic genes code for transcription factors containing a homeodomain, which is transcribed from the homeobox segment of the gene (thus the name hox gene). Hox genes are activated or repressed early on in response to gap gene gradient stripes. Hox genes can maintain a state through many cell divisions, unlike gap genes that assume temporary states. Hox genes occur in every animal yet studied; humans have 13 arranged in a single cluster, occurring four times on different chromosomes. The discovery of Hox genes and their ubiquity tells us that animals are all built on a similar basic body plan via homologous genes arising during evolution from a common ancestor.
Inter-cell signaling is by induction or signal transduction. In induction, layers of cells in a tissue respond to a morphogen gradient in the extracellular space, influencing cell activity. In transduction, one cell secretes a molecule and another cell receives it. Small messenger molecules such as hormones can directly penetrate the cell membrane and bind with a transcription factor to regulate gene activity in the target cell. But protein signaling molecules are too big to pass through membranes. Rather, transmembrane receptor proteins bind to the the signal protein as a ligand; the receptor then transmits the signal from the ligand into the cell nucleus.
Binding a ligand activates a receptor protein. Often, a phosphate group attaches to the active receptor inside the cell. When another protein binds to a phosphorylated receptor, the receptor is again activated and transfers its phosphate group. Proteins that transfer phosphate groups are called kinases. Signal transduction can initiate a signal cascade within the target cell. The final step in the cascade is activation of a transcription factor that will activate one or several genes in the nucleus. A limited set of signal cascades is used repeatedly within the organism and across the animal kingdom to induce cell differentiation, form patterns, and initiate growth.
The maternal dorsal gradient, with Toll as receptor, involves 10 genes in producing the signal or passing it on to the dorsal protein transcription factor, which enters the nucleus when activated. Four common cascade pathways are used during gastrulation, and again in pattern formation for tissues. They are named for the gene coding for the signaling molecule.
Decapentaplegic (Dpp): The name means 15 mishaps in Greek, explaining the extent of mischief that can accrue from a mutation in this gene. The Dpp protein is one of a class of growth factors, TGF-β (transforming growth factor) or bone morphogenic protein (BMP). It is an important pathway in the dorsal-ventral larval development.
Delta-Notch: This pathway operates between adjacent cells, since both signaling protein Delta and receptor Notch are anchored in cell membrane. Initially, the adjacent cells have both Delta and Notch protein at the surface. But once Delta activates Notch, the activated cell represses Delta, so fewer Delta signals are returned. This causes the signaling cell to make more Delta. Eventually, only the initiating cell makes Delta, and the others repress it. This is a standard mechanism for making initially similar cells different. In the nervous system, this pathway is used to differentiate sensory cells (Delta producers) from epidermal cells (Notch producers).
Hedgehog and wingless (Sonic Hedgehog and wnt): Examples of these pathway types are the segment polarity genes in Drosophila. The engrailed transcription factor causes hedgehog to be synthesized in strips of cells, after which it is secreted and diffuses into the extracellular matrix near an adjacent row of cells, where it binds to receptors. An activated receptor cell’s gene for transcription factor Ci is then activated, triggering synthesis of wingless protein. Wingless protein then signals back to activate engrailed protein, a positive feedback mechanism ensuring production of Ci and engrailed proteins in neighboring rows of cells.
Imaginal discs emerge from such rows. As their cells continue division, the front part produces Ci and wingless while the back part produces engrailed and hedgehog proteins. The neighboring epidermal cells do not divide, but simply continue to grow in size.
Morphogenic gradients, combining factors, and signal transduction cascades lead to ever finer details in embryo tissues. Selector genes define cell groups and fixes their fate.
Cellular Form and Form Change; Tissue Building
Cells are organized into tissues embedded in a gel-like or fibrous material called the extracellular matrix. Tissues can be highly organized, such as the epithelium comprising skin and gut lining. Alternatively, they can be loosely scattered through the matrix, called mesemchyme tissue.
The shape of a cell is determined by its inner cytoskeleton, by its connection to other cells, and by the extracellular matrix, the gel-like or fibrous substance in the extracellular space. Many form-giving proteins establish and maintain cell forms, often polymers arranged in long chains and bundles.
The cytoskeleton comprises a group of several types of protein fibers extending through the cell. Microfiliments comprised of long chains of actin molecules organize in bundles and tightly-knit three-dimensional networks of fine threads. They are particularly dense just beneath the cell membrane, stiffening and strengthening it. Actin chains can adjust their length, and associate with the myosin motor proteins, which can move actin chains. Myosins also are used in highly organized fibers to generate force in muscle cells, operating the same way within the cytoskeleton to divide a cell or change its shape. When a cell moves, it forms extensions called filopodia in which microfiliments change continuously.
Microtubules are stiff, helical chains of molecules forming a hollow tube. They usually emanate from an organizer, the centrosome, and lengthen or shorten by adding or removing molecules at the free ends. Special proteins can attach the free ends to the actin cortex, further shaping and stabilizing the cell. The versitility of cytoskeletons and their component tubules and filaments supports formation of an extraordinary variety of cell forms.
Beyond cell structure, major functions of microtubules are pulling apart duplicated chromosomes during mitosis/meiosis. Microtubule spindles from the centrosome attach to chromosomes at special points, and then pull the chromosomes toward the connected organizer. Microtubules also provide transfer tracks for transport of proteins and organelles within the cell, powered by motor proteins either to or from the cell center. For example, the Bicoid RNA is transported to the anterior pole of the zygote along microtubule tracks.
Cell adhesion occurs when proteins (e.g. Cadherins), extending from the surfaces of adjacent cells, bind to one another. The inner end of the binding proteins binds to the internal cytoskeleton of the adhered cells. There are different types of binding proteins, some being cell specific, and usually only binding to like proteins. Selective cell adhesion serves both to stabilize a tissue type, and to segregate it from a different tissue type.
Collegen is a typical extracellular matrix component, found in bone and connective tissues. Glycoproteins are typical components of a gel-like matrix. The basal membrane of the epithelia is composed of complex molecules such as fibronectin and laminin, which facilitate movement of cells through the matrix. Basal membrane proteins called integrins bind to fibronectin, anchoring epithelial cells to the membrane.
During gastrulation, a band of cells folds inward in a process called invagination, to form an inner cell layer called the mesoderm. These cells develop microfilament rings at the outward side that constrict that side; in concert, they bend their row of cells inward. Once in the interior, the cell complex loosens and spreads along the inner ectoderm. Neural tubes, glands, and sensory organs are formed by invagination.
Cells can ingress into the embryo from the surface epithelium, individually or in groups. Once inside, they loosely associate with other cells. The Drosophila nervous system develops via this process. Neuroblasts, defined by Delta-Notch signaling, ingress and then divide several times, forming nerve cells, ventral cord, and surrounding cells. Blastoderm cells ingress to develop the precursor of the central gut, later organizing as the epithelial lining of the gut.
Gastrulation causes extensive rearrangement, changing relative positions of cells. Stretching of the embryo in this process is due to cell shape shange caused by microfilaments. During the extended state, cell segment borders form and the cells of the central nervous system move inside.
The cells of the central nervous system follow directed pathways defined by attractive and rejecting signals that are sensed by the membrane of a migrating cell. The signals are sensed directly by the cytoskeleton, not requiring transcription. Such cell shape changes allow nerve cells to move along complex tracks, eventually connecting from the central nervous systems to the organ or muscle tissue to be controlled. Working in reverse, cells migrate from sensory organs to attach to the brain. Nerves grow by cell stretching. The cell remains in situ, but sends out long extensions called axons that are stiffened by microtubules. The tip of a growing axon, the growth cone, extends filopodia that move and explore their environment, searching for guidance signals.
Cells do not grow until connected to a supply of nutrients. Thus, initial cell divisions in a zygote are via simple cleavage, producing smaller and smaller replicas. However, in zygotes accompanied by large yolks, such as in birds and reptiles, cell growth can commence at the beginning. For insects, the larval stage marks the beginning of cell growth, and in mammals, it is marked by implantation in the uterus. After connection to nutrients, cells grow before dividing.
Cell division almost always occurs in undifferentiated tissues. Prior to differentiation, cell divison stops and the switch is irreversible. Up until this point, divisions proceed in a controlled manner, signaled by external growth factors.
Cell division begins with replication of DNA. Newly synthesized proteins named cyclins bind to kinase proteins to activate them and initiate the replication, after which the cyclins disappear and the kinases deactivate. A similar cyclin-kinase initiation sequence controls the start of mitosis after the DNA replication is completed. If cell division is coupled with cell growth, the initiator does not trigger until growth is complete.
Cells can be programmed for death, a process called apoptosis. Cell division stop signals and cell growth factors control cell early growth and division. When both stop and growth factors are missing, the cell disappears without a trace. Excess nerve cell culling involves apoptosis. Also it can be triggered by signals from unhealthy cells.
Tissue growth involves repetitions of cell growth followed by cell division. In growth, molecules are added to structures such as membranes and organelles. In Drosophila, the larval shape increases by 20-fold, entirely through cell growth (excepting the imaginal discs that experience cell divisions). Only after this growth phase is complete does morphogenesis occur from the imaginal discs.
In an adult animal, growth ceases, but some tissues are constantly renewed from stem cells: skin and intestinal lining, blood, and to a lesser degree, muscle and nerve tissue. Stem cells experience unequal division, resulting in a replica stem cell and a specialized cell. This results from an uneven distribution of internal RNA, or from signals from neighbor cells, preventing differentiation of one of the daughter cells. The cell in contact with the neighbor cells (the niche) will remain a stem cell, while the daughter cell facing away from the niche will differentiate after several divisions. Unequal division in germ stem cells gives rise to sperm and egg precursor cells. The germ plasm plays a large role in maintaining the germ line.
Frogs and chickens are typical model organisms used to study vertebrata, although the zebrafish is better in that individual genes can be more readily turned off and the eggs are plentiful and transparent. Lack of these efficacies makes the mouse a much less effective model. In favor of the mouse, embryonic mouse cells can be isolated, cultured, genetically modified, and transplanted back into the embryo, after which these embryonic stem cells can become any body part. Genes can be ‘knocked out’ to directly support genetic analysis. Such mouse knock out analyses have direct support for modern medical research.
For all their differences, all vertebrates are similar in general body organization, due to their evolutionary homologies. The commonality begins with a notochord, a rod-shaped structure defining and strengthening the A-P axis of the embryo. It supports the neural tube, the forerunner of the spinal cord that broadens at the anterior to form the brain. It is ultimately protected by the skull and bones of the spine. The muscles of the trunk develop in segmented blocks called somites, to left and right of the notochord. The digestive tract and heart develop on the ventral side of the notochord, the heart pumping blood through a closed circulatory system. The embryo forms of all vertebrates have these similar features right after the formation of the major organ precursors, although the embryos are quite different in size and other features. Other homologies appear later, including jaws and four paired limbs.
Beyond the phenotypic homologies, the genetic homologies are even more striking. Homologous genes serve as convenient markers to identify related features and assist in comparing embryos of different vertebrate classes.
In the frog, fish, and chicken, the embryo develops outside the mother in an egg. The yolk contains the necessary nutrients to develop the embryo within the egg, requiring only an environment with adequate temperature, humidity, and oxygen to mature. Mammal embryos grow inside the mother in an egg zygote with no yolk; the mother fullfils the function of the yolk, via the umbilical cord. Egg layers provide their future embryos with nutrition before fertilization. Mammals provide embryonic nutrition only after implantation of the egg in the uterine wall.
The first zygote cleavage creates the left/right halves, the second the dorsal/ventral pair as a second layer, the third orthogonally bisects the first four, and so on until the blastula results, several layers of cells around a hollow, fluid-filled center. All cells have yolks, but those at the bottom have more and will become the intestinal tract.
During gastrulation, cells migrate into the embryo forming the mesoderm and leaving a ring-shaped opening called the blastopore just below the equator. Cells on the dorsal side of the opening become the notocord and those on the ventral become blood. A multi-layer horizontal axis develops on the dorsal side, beginning at the dorsal lip, the area identified as the organizer by Spemann. Mesoderm cells converge on the dorsal side and migrate inward and upward. The first cells to invaginate position themselves in front of the organizer where they pair with the overlying ectoderm to become the head. Cells with later ingress squeeze between the cells already in place, elongating the horizontal axis. The ectoderm stretches downward to envelop the lower half. The endoderm forms from the cells with the most yolk. The blastopore cells become the anus and the mouth and tail bud form at either end of the now obvious A-P axis.
Development next proceeds front to rear, with notochord and somites. The somites will become the cartilage and bones of the vertebrae, the musculature, and the inner layer of the skin. The outer skin forms from the ectdoderm. Also from the ectoderm will form the nervous system, via neurulation. A groove forms in the ectdoderm on the dorsal side above the notochord. It widens at the front into three larger vessicles that will house forebrain, midbrain, and hindbrain. The rest of the neural tube becomes the spinal cord that will become encased by the vertebrae, built from somites and the notochord. Eyes form as bulges on the forebrain; lenses, nose, and ears form as ectoderm thickenings called placodes.
Special cells over the dorsal neural tube form the neural crest. They migrate large distances, contributing to head bones, skin pigmentation, innervation of sensory organs, and a variety of surface features such as antlers/horns, head shapes, and color patterns.
Zebrafish and Chicken
A major developmental difference from that of frogs is that cell cleavage does not involve the yolk. In the fish, the yolk remains and the divided cells, containing only clear cytoplasm, form a surface layer over it, like a cap. Once these cells reach the equator, they invaginate to form the mesoderm and ectdoderm.The A-P axis then forms, beginning with the thickening called the shield, homologous to the frog organizer. the intestinal tract will form enclosing the yolk, which will provide the growing embryo nourishment.
In chickens, the separation of yolk and egg cells is even more dramatic; the yolk is the egg. The blastoderm forms from a small island of fertilized cytoplasm, becoming the blastodisc on top of the yolk. Embryo development is almost two dimensional, making it much easier to visualize than that of the frog.
The primitive streak forms as an indentation from the center to posterior of the blastodisc, homologous to the frog blastopore. Hensen’s node develops at the anterior of the primitive streak, homologous to the frog organizer. Beginning at Hensen’s node, cells ingress along the primitive streak to form mesoderm and endoderm, migrating to the anterior to form the head, while the embryonic A-P axis elongates and the node moves back toward the posterior. Unlike frogs and fish, chicken cells at this stage already grow before dividing.
A layer of cells spread above the yolk, forming the extraembryonic membranes and tissues, and serving to temporarily nourish and protect the embryo. One such tissue is the yolk sac, emerging from the endoderm forming in the blastodisc, and surrounding the yolk. The sac is covered with mesoderm cells that will become the blood and blood vessels and will connect to embryonic vessels to transport nutrients from yolk to embryo. Another extraembryonic tissue, the amnion, emerges from ectodermal folds to enclose the embryo in a protective, fluid-filled cavity.
The heart develops after two days from paired mesoderm structures on the ventral side; it moves blood through the vessels around the yolk to provide nutrients for growing embryonic cells, long before the embryo’s circulatory system will emerge. The allantois is a sac also filled with blood vessels for transporting oxygen to the embryo and removing waste products. It functions as lungs and kidneys for the growing embryo.
Aristotle hypothesized that yolk circulation in the chicken embryo was homologous to the workings of the placenta in mammals.
Mammal eggs, some of the smallest known, are wrapped in a clear shell, the zona pellucida. The egg is fertilized in the fallopian tube while swimming in a fluid. Cleavage divisions happen initially slowly, every 12 to 24 hours. The cells are loosely associated until the third division, when the compact ball of 8 pluripotent cells is formed via expression of cadherin cell adhesion molecules. After this stage, the embryo is called the morula. After three more divisions, a central cavity develops in the 64-cell blastocyst containing inner and outer cell layers that behave as separate entities. The outer cells form an extraembryonic epithelium, the trophectoderm, contributing chorion that will combine with maternal tissue to become the placenta.
The first four cells are totipotent, but after more divisions the cells become too small for viability. One or two cells from an 8-cell embryo can be removed and the result will still form a complete embryo. Cells of two 8-cell embryos can be mixed, forming an embryo with characteristics of both, a chimera. Without the trophectoderm cells, the embryo cannot implant in the uterus and grow.
Parthenogenesis cannot be induced in mammalian reproduction because both sperm and oocyte genes must contribute to a balancing of the cells in the trophectoderm and the inner cell mass. Different genes are involved based on sex, and both male and female sets are required, a process called imprinting.
The blastocyst ‘hatches’ from the zona pellucida and attaches to the extracellular matrix of the uterus mucous membrane. The trophectoderm cells imbed to a point where the blastocyst is covered with uterine tissue, then divide and penetrate surrounding tissue to eventually form the placenta. Later the umbilical forms to circulate maternal blood to the embryo, allowing the maternal organs to substitute until those of the embryo have formed.
After implantation, the embryo and its extraembryonic layers (amnion, yolk sac, allantois) emerge from the inner cell mass. The embryo develops from an epithelial structure, the egg cylinder, comparable to the chicken’s early embryo, but with shape of an indented cup. The main developmental A-P axis aligns with a primitive streak, as in the chicken, controlled by a node that is homologous to the chicken’s organizer; the streak elongates and the node moves toward the bottom. Final arrangement of embryonic structures is reached later by a twisting about this axis until the embryo is turned inside-out, becoming wrapped up in the amnion.
The cells within the blastocyst are embryonic stem (ES) cells; in compatible culture media, they can be multiplied without changing their undifferentiated character. In other media, they can randomly create different tissue types, but cannot be used to create an entire embryo, since the required imprinting would be missing. ES cells treated with appropriate growth factors can be differentiated in vitro and then transplanted into a host to combine with host tissue, a hope for future therapeutic genetic interventions. When stem cell genes are manipulated in a cell culture, and mutated cells are re-inserted into the blastocyst, a transgenic embryo can be created that will have transgenic offspring if its germ line cells also express the mutation. A transgenic mouse in which an entire gene has been changed or deactivated is called a knockout mouse, useful in studies of gene function.
Gradients, Prepatterns, and Induction in Vertebrates
These general mechanisms are operative in vertebrates, as in Drosophila, to create initial spatial changes in embryonic form. However, in vertebrates, the individual steps are not as well delineated, and since cellular membranes exist from the beginning of development, all signaling must be intercellular. Also, the development of the vertebrate embryo is ‘more messy’, in that there is significant cell mobility, and different phased stages of development in different parts, so that the relationship of the prepatterns to the developing forms is less well observed than in Drosophila.
During gastrulation, maternal factors, asymmetrically localized mRNAs, determine top-bottom axis of frog and fish eggs. Then diffusable factors determine the endoderm and induce the mesoderm in a neighboring belt-shaped region. A signal in a future dorsal region forms the organizer within which, wingless signaling molecules activate several genes, encoding transcription factors such as goosecoid that activate even more genes. The organizer chordin molecules (inhibitors) create a gradient determining the D-V axis, by repressing action of the BMP morphogen (closely related to the Drosophila Dpp). High BMP induces formation of dorsal tissues (blood, kidneys) while repressing nerve tissue. Low BMP induces mesodermal structures (somites), while absence of BMP permits nerve tissue to develop. The organizer develops into the notochord.
The ordered ingression of mesoderm cells begins after the dorsal side has been established, depending on simultaneous activation of several transcription factors, such as T-gene (for short-Tail phenotype, called brachyury in frogs and no tail in fish). T-gene is transcribed in all invaginating cells of the blastopore, then becomes limited only to notochord cells. In the mouse, T-gene activates first in the primitive streak, then the notochord.
Neurulation begins with establishment of the A-P axis in the three germ layers, with notochord at the center. A-P is laterally confined by somite precursors, and covered by ectoderm cells, where signals from the organizer induce the central nervous system to form. The organizer suppresses BMP where the ectoderm should be favored over nervous tissue development.
Operation of the organizer explains things that were historic mysteries. If frog blastomeres are separated after first cleavage, both right and left halves inherit part of the organizer, so two smaller frogs will develop. Transplantation of an organizer produced chordin on the ventral side, suppressing BMP, and inducing an additional axis in neighboring tissue.
Segmentation in vertebrates is not as clear as in arthropods, and is mainly expressed internally in musculature and vertebrae organization. The mesoderm is divided into regular blocks, the somites, arising sequentially from front to rear. Segmentation results from timed pulses of gene transcription, such as in the her-gene of the tail bud. The rhythmic gene expression propagates a wave toward the anterior of the germ band. When a wave reaches the edge of the last formed somite, a new somite boundary is formed. The rhythm results from a complex interaction between Delta-Notch proteins. The transcription of the prepattern myoD-gene signals the front margins of the somites.
Somite differentiation on either side of the notochord is signaled by the protein coded by sonic hedgehog-gene in the notochord. This signaling induces organization of the nervous system above the notochord, by formation of motor neurons in the spinal chord.
Hox-genes are activated along the A-P axis. The position of the hox-genes within their complex determines where along the A-P axis they are transcribed, influencing shape of vertebrae and position of ribs and limbs.
Limbs develop from buds, aggregations of mesoderm cells. Growth factor FGF initiates emergence of limbs from the bud tips. Sonic hedgehog signaling proteins determine the limb A-P axis pattern. Mesenchyme cells in the emerging limbs condense, forming cartilage that will become bone. Fingers and toes develop from plates of cartilage, by controlled death of cells between emerging digits.
Due to human ethics, human gene functions are not studied directly, but rather via the understood homologies with the model organisms previously discussed. This situation is in contrast to the vast available detail regarding general human nutrition, physiology, biochemistry, cell biology, immune system, and diseases.
Mammalian sex is determined by the presence or absense of the sry-gene, found only on the Y-chromosome. The sry-gene causes the gonads to produce testosterone, inducing testes rather than ovaries. Without this gene, gonads produce different hormones to induce ovaries. The corresponding secondary sexual characteristics are due to yet other hormones. Other than sry and a few other Y-chromosome genes that cause sperm gametes to be produced, female and male genes are identical. To ensure the male and female bodies receive the same amount of X-chromosome gene products, one (randomly selected) of the female X-chromosomes is shut down in all somatic cells early in development.
The germ cells from a group of 50 cells in the yolk sac. During gastrulation, these stem cells migrate into the embryo and associate with the gonads. The female prospective egg cells are all formed in the embryo. Male sperm are produced from sperm stem cells continuously from puberty. Only 400 or so female eggs mature in a lifetime. They are 100 times the size of the typical cell, but contain no yolk. Their size provides sufficient cytoplasm to permit a 100 cell blastocyst to form by successive cleavage divisions.
Meiosis is a complex and fragile process prone to chromosomal recombination errors: defective distribution and breaks. Repair enzymes can fix some and other defectives undergo apoptosis. The error rate increases with age, and a few eggs with chromosomal irregularities (aneuploidies) may survive, producing miscarriages and sterility. Defects involving three copies of a chromosome (trisomies) almost always result in miscarriage (the lone exception is the chromosome 21 trisomy, causing Down syndrome). Monosomies are also lethal; a few may survive until shortly after birth.
Human eggs mature in the ovaries, one per month after puberty, surrounded by supportive cells forming a follicle. The maturing egg grows from 10 to 100 micrometers in diameter. The first (reduction) meiotic division, suspended since embryonic time, completes, and meiosis again suspends until fertilization. One of the complete daughter nuclei, with a complete set of chromosomes, is extruded from the egg as a ‘polar body’ that subsequently disappears. When ready for fertilization, the ovulation process pushes the egg from its ovary follicle into the connected fallopian tube.
At fertilization, a second polar body is extruded and meiosis is completed. The sperm and egg pronuclei migrate toward each other. Upon meeting, their chromosomes double and the first cell division commences, but in a manner unlike other organisms because the pronuclei do not fuse, but divide separately. At the two cell stage, the male and female chromosomes combine and cleavage divisions commence, followed by compacting into a blastocyst. During cleavage, the egg moves down the fallopian tube to the uterus, where it hatches out of the zona pellucida. By the fifth day after fertilization, the blastocyst is attached to the uterine wall.
Embryonic enzymes dissolve components of the extracellular matrix of the uterine mucous membrane, allowing the trophectoderm cells to ingress into the lining and form the syncytiotrophoblast by cell fusion. The syncytiotrophoblast later joins cells in the extraembryonic mesoderm to form the chorion, which covers the embryo and penetrates deeply into uterine tissue by many branched extensions. The chorion, together with cells of the uterine mucous membrane, form the placenta. Uterine cavities develop around the chorion intrusions and fill with maternal blood. In the chorion, embryonic capillaries develop and spread into the cavities, where they bathe in maternal blood, facilitating molecular interchange of nutrients and waste products. Additionally, signals are being exchanged; embryonic factors initiate the provision of blood supply in the uterine wall, and maternal factors stimulate embryo growth.
After implantation, the inner cell mass resembles the two-layered disk of the chicken embryo. Cells of the lower layer spread to cover the inside wall of the trophectoderm, a homolog of the chicken yolk sac. From the upper layer, the epiblast, the amnion membrane develops to enclose the fluid-filled cavity above the embryo. The epiblast develops into the embryo, developing a primitive streak and initiating gastrulation as in the chicken embryo. After 2 weeks, the germ layers have formed, the somites, central nervous system, and head are apparent, and the heart begins to beat. The amnion encloses the embryo completely and the yolk sac and blood vessels bundle together to form the umbilical cord. At 5 weeks after fertilization, most embryonic structures have formed in miniature. The remaining gestation entails growth and the final differentiation of structures.
Because of the connection to the maternal blood stream, during pregnancy and particularly at the beginning, the mother needs to avoid intake of any substance toxic to the fetus. Morning sickness is thought to be a defense mechanism for the potential for poisoning. Although large molecules cannot bridge the maternal-fetal blood barrier, chemicals including drugs and alcohol can. On the positive side, the mother’s antibodies can pass to the fetus.
Monozygotic twins can form at the early cleavage stages, in which case each fetus gets its individual chorion and placenta. Twinning can also occur after blastocyst formation, so that the chorion is shared. Up to 14 days after fertilization, until gastrulation commences, twinning can still occur with shared amnion, although at increasing risk for connected body parts. The implication is that up to two weeks after conception, there is as yet no individual identity; the embryonic form still has potential to become one or two separate fetuses.
Because the relatively large size of the fetal head presents a maternal birthing risk, birth in humans occurs at the earliest possible time when separation from the mother is survivable. Thus human infants are less mature at birth than are other animals. The development of sensory organs and their nerve connections is continued after human birth. Healthy development then depends on external sensory stimuli, because the body generates a surplus of nerve connections, but only those that are used are retained; once connections go missing, they can never be recreated. Connections within the brain need similar activity; the human capacity for language and music requires earliest possible stimulation to realize maximum retainable capabilities.
Evolution, Body Plans, Genomes
The bacteria represents the primitive origins of life on Earth, a single cell organism with no nucleus, and with rigid cell walls that determine cell shape. Already, bacteria possess the basic mechanisms for cell replication: DNA, RNA, protein synthesis, ribosomes. The DNA molecule is ring-shaped and loosely arranged in the cytoplasm. There is no true sex, but a form of genetic exchange does occur between cells, even cells of different species in a process called horizontal gene transfer. Some bacteria can form aggregates of cells. Bacteria possess remarkable biochemical ability to convert and build materials of all kinds, and are adaptable to any extreme environment condition found on Earth.
Next in complexity are the eukaryotes, created perhaps 2 billion years ago as a single-celled organism, but with a nuclear membrane enclosing nuclear chromatids. Particles can enter the cell by folding in of the cell membrane. The cytoplasm has internal structure in the form of membrane-enclosed organelles, providing chemically-segregated environments within the cell. Eukaryote cytoplasm also contains energy producing and regulating mitochondria, probably from ancient enslaved bacterial invaders that left their DNA behind. Eukaryotes have an internal cytoskeleton to replace the rigid cell wall of the more primitive bacteria; it stabilizes the cell, yet enables shape change and motility. The nuclear chromatins are partitioned into chromosomes prior to cell division, ensuring correct distribution to daughter cells. Yeast is an example of a single-celled eukaryote. Animals and plants are multi-celled eukaryotes.
Bacteria can form collections, but their rigid cell walls prohibit adhesion and communication. Eukaryotes solved that by losing the rigid cell wall in favor of the internal cytoskeleton that facilitated cell adhesion and communication. In plants and fungi, multicellular forms probably originated in multiple independent episodes. But it only happened once in animals. Cells of the eukaryote organism are free to achieve distinct type characteristics, such as membrane, nerve, and sensory cells. The first simple body plan appeared with radial symmetry and a blind gut, perhaps looking similar to today’s Cnidarians, corals and jellyfish.
Around 600 million years ago, life began to greatly diversify, a period called the Cambrian explosion. Earth’s climate and atmosphere resembled today’s, the result of oxygen provided by photosynthetic bacteria and algae. Over a period of 50 million years, all the basic animal body plans developed; all the phyla present today were represented in that period, along with others whose fossil forms have been discovered.
The first step beyond simple radial symmetry was bilateral symmetry with a distinct top, bottom, front, back. Roundish flatworm organisms were probably the first examples of the elongated bilaterian form. The embryo had become flatter and added a mesoderm to create a third germ cell layer. The endoderm still produced the gut, but now it was a through gut. Ectodermal sensory organs and mouth formed at the front to facilitate feeding. The new mesoderm added circulatory system and muscle to the plan. The organism’s essential developmental mechanisms (genes and signaling cascades) must have been those of today’s animal life, such as Delta-Notch and wingless, D-V axis gradients via Dpp and sog, the Hox genes eyeless and tinman for positioning of sense organs and heart on the A-P axis.
Most animal phyla today fall in one of the two basic bilaterian body plans, depending on whether gastrulation invagination occurs through the mouth (protostomes) or through the anus (deuterostomes). Vertebrates and echinoderms are deuterostomes; arthropods and mollusks are protostomes. Thus arthropods and vertebrates have opposite left and right organization, a result of the inversion of the D-V body axis. Deuterostome embryos have heart on ventral side, central nervous system on dorsal side; protostomes are the opposite.
In deuterostomes, the D-V axis gradients are determined by BMP and chordin, homologs for the protostomes’ Dpp and sog, but with opposite orientation. The many molecular feedback mechanisms shared by deuterostomes and protostomes clearly suggest that they were operative in ancestors of the bilaterians. There seem two possibilities for the existence of bilaterians of opposing D-V polarity; either these developed independently, or one derives from the other by a twisting during gastrulation.
A subsequent advance in body plan variety comes via a basic iteration process called segmentation. Most phyla plans utilize some form of segmentation, but the mechanisms can be very different. Sometimes the segmentation is not obvious in the adult form, because the repetitive units formed via an embryonic prepattern are then individually modified in the maturing embryo. In annelids and many arthropods, stem cells in repetitive growth zones are precursors to segments. In Drosophila, segmentation forms via simultaneous subdivisions of the blastoderm. Arthropods including Drosophila use segment polarity genes to link pairs of segments. The bicoid, hunchback, and knirps genes that operate early in Drosophila segmentation are not preserved in other arthropods, so may be a later invention of the insects. A totally different segmentation mechanism is active in vertebrates, a time-delayed pulsing of spatial waves utilizing Delta-Notch signaling. In both arthropods and vertebrates, head and frontal segmentation are via different mechanisms than the body segmentation.
Just as bacterial cells with rigid outer membranes advanced to eukaryotes with internal support structures, body plans also advanced to internal stabilizing structure with chordata and its successor, vertebrata. Here, the selective advantage was greatly enhanced potential for continuous growth.
Greater function and control were added to the basic body plans by the addition of appendages: arms and legs, claws and jaws, wings and antennae. In arthropods, the large variety of appendages are all formed by a common construction principle. Heads are adorned with sensory and defense organs and chewing tools. Vertebrates have two pairs of limbs, arms and legs, or their wing and fin homologs. Limbs arise from buds on the body wall using pattern formation molecules homologous to those in arthropods, perhaps implying a common ancestor with appendages.
Vertebrates exhibit greater complexity than arthropods; one characteristic stands out in this respect, ever increasing brain size and complexity. Large brains need skulls for protection, and they support complex sensory organs. Vertebrate cranial developments derive from the embryonic neural crest, arising from the dorsal ectoderm, whose cells migrate throughout the body, differentiating to furnish the body’s periphery with specialized functions, among them bones of the skull and jaw, teeth, claws, horns, beaks, and body pigmentation.
Genomes drive body formation, and the complexity of a body plan is mirrored in the richness of its genome. Bacterial genomes are smallest and human the largest:
Smallest 600 genes
E. coli 4,300 genes, 4 million base pairs (bps)
Yeast (S. cerevisiae) 6,000 genes
Drosophila 13,000 genes
C. Elegans 19,000 genes
Mammals 32,000 genes; 3 billion bps
The large genome growth in higher order organisms is due mainly to DNA dark matter that does not code for protein, and whose real purpose, if any, is not yet understood. Around half of mammalian DNA is such ‘junk’, mainly repeated sequences of bps. Such repetitive DNA may offer insight into genome history, but does not seem to have much utility to the current organism. Because they are not harmful, selection has no say in the matter, so the junk persists. The lack of function can be inferred by comparing genomes of related organisms. For example, the puffer fish Fugu lives with a genome a quarter the size of the zebrafish, although both have around the same number of genes.
Genes exist in families, with new ones evolving from existing ones in accordance with three principles: mutation of individual bases; gene duplication; combining parts of different genes. Bacteria add a fourth principle, horizontal gene transfer.
Most proteins are modularly constructed from domains of short amino acid sequences that confer specific biochemical properties, such as DNA binding or enzymatic activity. Around 1,200 protein domains are known, of which 200 are common to all animal life. Domains occur in many combinations, defining many gene families arising from gene duplication. In homologous genes from different species, often only the sequences coding for these domains are preserved. Although the surrounding sequences can be significantly changed, the original function of protein coding is preserved.
Compared to a simple organism such as yeast, multicellular eukaryote genes usually code for several domains. The proteins seem to have evolved by combining parts of different genes, creating protein useful for previously unavailable function. In mammals, the number of different proteins is greater than number of distinct genes. About one third of mammalian genes can be spliced in multiple ways, each producing a different protein.
The genome of vertebrates seems to have been duplicated twice in a relatively short time early on. Duplication occurred before the appearance of fish, because chordates such as the lancet, without a skull, have only one Hox gene complex while most vertebrates have four. There is a range of genes that appear only once in Drosophila, but four times in the mouse. Such duplicates can be expected to be preserved only if they take on different functions via a sequence of mutations. Additional genome duplication is evident in fish, but only 20% is preserved.
Entire genome sequencing is invaluable for identifying genes and finding homologs across species. Two mammalian genomes have been sequenced, the mouse and human. The last common ancestor lived about 100 million years ago. 99% of mouse genes have human gene homologs; 78% of amino acids in the respective proteins are identical. The differences are somewhat larger than indicated, however, because control regions of the genome are not accounted for, degree of gene similarity varies, and functional variation depends in complex ways on the primary sequence of the protein. A better feel for the differences is obtained by the number of genes influencing certain protein families; mice, for instance, have many more genes than humans that influence sensing smell.
Going further back, 60% of Drosophila genes and 50% of worm and yeast genes have mammalian homologs. Rather than indicating common function, these numbers should be understood to mean that the genes have a common origin. Thus, it is more a metric on the conservative nature of evolution. But in spite of its conserving operation, the process has brought forth a dazzling variety of organism form, function, and lifestyle.