22.6 FINALE: MOLECULAR BIOLOGY, DEVELOPMENTAL BIOLOGY, AND EVOLUTION

Molecular biology is a story of biological information—the metabolism, maintenance, and transfer of that information from one generation to the next. As we go back almost unfathomable lengths of time, those generations link us to every living thing on our planet. Our genomic DNA makes our life possible. With its seemingly ragtag mix of piggy-backing transposons, integrated viruses, and genes, both borrowed and linearly evolved, our genome tells us about our past while at the same time linking us to a future rich with potential. Evolution continues.

Each topic in molecular biology has evolutionary significance. Errors or random events in DNA replication, recombination, and repair fuel genomic changes—some useful, many deleterious. Genomic changes are expressed, through transcription and translation, in the organismal phenotypes on which natural selection acts. However, there are few areas where molecular biology meets evolution more dramatically than in the regulation of organismal development. Developmental biology thus provides a fitting final topic for our exploration of molecular biology.

The Interface of Evolutionary and Developmental Biology Defines a New Field

South America has several species of seed-eating finches, commonly known as grassquits. About 3 million years ago, a small group of a single species of grassquits took flight from the continent’s Pacific coast. Perhaps driven by a storm, they lost sight of land and traveled nearly 1,000 km. Small birds such as these might easily have perished on such a journey, but the smallest of chances brought this group to a newly formed volcanic island in an archipelago later to be known as the Galápagos. It was a virgin landscape with untapped plant and insect food sources, and the newly arrived finches survived. Over many millennia, new islands formed and were colonized by new plants and insects—and by the finches. The birds exploited the new resources on the islands, and groups of birds gradually specialized and diverged into new species. By the time Charles Darwin stepped onto the islands in 1835, there were many different finch species in the archipelago, feeding on seeds, fruits, insects, pollen, and even blood. Some islands now have as many as 10 finch species, each adapted to a somewhat different lifestyle and food source.

The diversity of living creatures on our planet was a source of wonder for humans long before scientists sought to understand its origins. The extraordinary insight handed down to us by Darwin, inspired in part by his encounter with the Galápagos finches, provided a broad explanation for the existence of organisms with a vast array of appearances and characteristics. It also gave rise to many questions about the mechanisms underlying evolution. Answers to those questions have started to appear, first through the study of genomes and nucleic acid metabolism in the last half of the twentieth century, and more recently through an emerging field nicknamed evo-devo—a blend of evolutionary and developmental biology.

In its modern synthesis, the theory of evolution has two main elements: mutations in a population generate genetic diversity, and natural selection then acts on this diversity to favor individuals with more useful genomic tools and to disfavor others. Mutations occur at significant rates in every individual’s genome, in every cell (see Chapters 3 and 12). Advantageous mutations in single-celled organisms or in the germ line of multicellular organisms can be inherited, and they are more likely to be inherited (i.e., are passed on to greater numbers of offspring) if they confer an advantage. It is a straightforward scheme. But many have wondered whether that scheme is enough to explain, say, the many different beak shapes in the Galápagos finches, or the diversity of size and shape among mammals. Until recent decades, there were several widely held assumptions about the evolutionary process: that many mutations and new genes would be needed to bring about a new physical structure, that more-complex organisms would have larger genomes, and that very different species would have few genes in common and perhaps use very different patterns of gene regulation. All of these assumptions were wrong.

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Modern genomics has revealed that the human genome contains fewer genes than expected—not many more than the fruit fly genome, and fewer than some amphibian genomes. The genomes of every mammal, from mouse to human, are surprisingly similar in the number, types, and chromosomal arrangement of genes. Meanwhile, evo-devo is telling us how complex and very different creatures can evolve within these genomic realities.

Small Genetic Differences Can Produce Dramatic Phenotypic Changes

The kinds of mutant organisms shown in Figure 22-30 were studied by the English biologist William Bateson in the late nineteenth century. Bateson used his observations to challenge the Darwinian notion that evolutionary change would have to be gradual. Recent studies of the genes that control organismal development have strongly supported Bateson’s ideas. Subtle changes in regulatory patterns during development, reflecting just one or a few mutations, can result in startling physical changes and fuel surprisingly rapid evolution.

The Galápagos finches provide a wonderful example of the link between evolution and development. There are at least 14 species (some specialists list 15), and they are distinguished in large measure by their beak structure. The ground finches, for example, have broad, heavy beaks adapted to crushing hard, large seeds. The cactus finches have longer, slender beaks, ideal for probing cactus flowers and fruit (Figure 22-33).

Figure 22-33: The evolution of new beak structures to exploit new food sources. Galápagos finches that feed on different, specialized food sources have different beak structures, as shown for the cactus finch and the large ground finch. (a) The beak structures can be varied along three dimensions. (b) The differences observed in the two finch species were produced largely through natural selection acting on a few mutations that altered the timing and level of expression of just two genes: those encoding Bmp4 and calmodulin (CaM).

Clifford Tabin and his colleagues carefully surveyed a set of genes expressed during avian craniofacial development. They identified a single gene, Bmp4, whose expression level correlated with the formation of the more robust beaks of the ground finches. More robust beaks were also formed in chicken embryos when high levels of the Bmp4 protein were artificially expressed in the appropriate tissues, confirming the importance of Bmp4. In a similar study, the formation of long, slender beaks was linked to the expression of the protein calmodulin in particular tissues at appropriate developmental stages. Thus, major changes in the shape and function of the beak can be brought about by subtle changes in the expression of just two genes involved in developmental regulation. Very few mutations are required, and the needed mutations affect regulation. New genes are not required. Note that Bmp4 is a member of a family of signaling proteins, with roles in development similar to those of the Wnt and Hedgehog proteins. Like Wnt and Hedgehog, Bmp homologs are widely conserved in eukaryotes. And as in the other signaling pathways, alterations in Bmp signaling pathways can have large effects on development.

The system of regulatory genes that guides development is remarkably conserved among all vertebrates. Elevated expression of Bmp4 in the right tissue at the right time leads to more robust jaw parts in zebra fish. The same gene plays a key role in tooth development in mammals. The development of eyes is triggered by the expression of a single gene, Pax6, in fruit flies and in mammals. The mouse Pax6 gene will trigger the development of fruit fly eyes in the fruit fly, and the fruit fly Pax6 gene will trigger the development of mouse eyes in the mouse. In each organism, these genes are part of the much larger regulatory cascade that ultimately creates the correct structures in the correct locations in each organism. The cascade is ancient; the Hox genes have been part of the developmental program of multicellular eukaryotes for more than 500 million years. Subtle changes in the cascade can have large effects on development, and thus on the ultimate appearance of the organism. These same subtle changes can promote rapid evolution. For example, the 400 to 500 described species of cichlids (spiny-finned fish) in Lake Malawi and Lake Victoria on the African continent are all derived from one or a few populations that colonized each lake over the past 100,000 to 200,000 years. The Galápagos finches simply followed a path of evolution and change that living creatures have been traveling for billions of years.

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Our discussion of developmental regulation brings us full circle, back to a biochemical beginning—both figuratively and literally. Evolution appropriately provides a key backdrop for the first and last chapters in this book. If evolution is to generate the kind of changes in an organism that we associate with a different species, it is the developmental program that must be affected. Developmental and evolutionary processes are closely allied, each informing the other. Molecular biology ties the fields together, informs us about molecular mechanisms that underpin the changes, and provides the technology needed for new discovery. The continuing study of molecular biology has everything to do with enriching the future of humanity and understanding our origins.

SECTION 22.6 SUMMARY

  • Developmental biology and evolutionary biology are closely related. The two fields inform each other, and molecular biology is intimately intertwined with both.

  • Major changes in the appearance and/or function of multicellular eukaryotes can be effected by subtle changes in an organism’s developmental program, involving mutations in the regulatory genes that guide the process.

UNANSWERED QUESTIONS

Our understanding of gene regulation remains incomplete in many areas. Indeed, the recent discovery of RNA interference, which has proved to be a major mode of regulation, underscores the likelihood that more fundamentals remain to be elucidated.

  1. How is alternative splicing regulated? Mounting evidence suggests that alternative splicing accounts for a much greater degree of protein complexity in higher eukaryotes than would be predicted by simply counting the number of open reading frames in a genome. How such alternative splicing is regulated is not well understood, nor have mechanisms of tissue-specific splicing been worked out.

  2. How and when do miRNAs control gene expression in human cells? The human genome encodes several hundred miRNAs, yet the targets and functions of most of these are currently unknown. How do we harness these newly discovered regulatory mechanisms to provide new therapies for cancer and other diseases?

  3. What other regulatory mechanisms have we just not uncovered yet? RNA interference is a fairly recent discovery. What are the functions of all the new RNAs being discovered in eukaryotic genomes?

  4. Are transcriptional and posttranscriptional steps in gene expression coordinately regulated? What kinds of mechanisms might enable communication between the cytoplasm and the nucleus to adjust transcription, splicing, and mRNA transport rates in response to increased or decreased translation of a particular mRNA?

  5. What are all of the signals that guide the action of regulatory proteins and the development of specific tissues? A much more detailed understanding is needed to completely unleash the potential of stem cell technologies, particularly with respect to the control of differentiation of induced pluripotent stem cells. That potential includes new cancer treatments, the regeneration of lost limbs, and the replacement of diseased tissues (e.g., heart, lung, kidney) without the danger of tissue rejection.

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A Natural Collaboration Reveals a Binding Protein for a 3′UTR

Zhang, B., M. Gallegos, A. Puoti, E. Durkin, S. Fields, J. Kimble, and M.P. Wickens. 1997. A conserved RNA-binding protein that regulates sexual fates in the C. elegans hermaphrodite germ line. Nature 390:477–484.

Marvin Wickens

Scientific collaborations come about in many ways, as the discovery of one gene-regulatory protein illustrates. The importance of the untranslated parts of an mRNA, particularly the 3′UTR, gradually became apparent over the course of the 1990s. In 1991, Judith Kimble’s lab reported the discovery of a 3′UTR regulatory element in the fem-3 gene of the nematode C. elegans, a sequence called PME (point mutation element) (see this chapter’s Moment of Discovery). Single base-pair changes in this element had a dramatic effect on germ cell fate, specifically in the switch from sperm to eggs during germ-line development in the hermaphrodite. The PME sequence had to be interacting with something, but what? An RNA-binding protein seemed a likely candidate, but what protein? How could it be identified? For Kimble, the obvious approach was unusually close at hand.

In 1996, Marvin Wickens and his coworkers reported the invention of the three-hybrid method to identify proteins that bind to particular RNA sequences (see Figure 7-29). The problem posed by the Kimble group was a perfect test of this new technology. Happily, not only was the Wickens lab quite near the Kimble lab at the University of Wisconsin, but Wickens and Kimble were husband and wife. A new kind of collaboration (for them) was soon hatched.

The investigators initiated a screen of a cDNA library in which C. elegans genes were fused to the gene encoding the Gal4p activation domain. The new three-hybrid method worked as advertised; one clone was found that met all criteria for the study and activated expression of the reporter gene, a his3-lacZ fusion. Testing a wide array of RNA-binding substrates, they demonstrated that the protein encoded by the cloned nematode gene bound only to the target sequence in the fem-3mRNA (Figure 1). The protein was named FBF-1 (fem-3 binding factor). The fbf-1 gene sequence was then used to search DNA databases for homologs. A second gene, fbf-2—91% identical to fbf-1 and encoding another protein that bound to the fem-3 3′UTR—was identified in the C. elegans genome. Perhaps more significant was that both gene products, FBF-1 and FBF-2, were identified as members of the newly described PUF family of RNA-binding proteins, a group that includes the protein Pumilio.

RNAi experiments confirmed the role of FBF proteins in germ cell fate. Studies of the FBF proteins quickly became part of a still expanding effort to characterize the function of PUF family proteins.

FIGURE 1 (a) The protein FBF-1 was identified in a three-hybrid screen. The hybrid RNA engineered to be the link between the binding protein MS2 and the (unknown) protein X–Gal4p fusion contained two PME sequences. (b) Activation of the his3-lacZ reporter gene led to production of β-galactosidase, which catalyzes the conversion of X-gal to a colored product. The color is seen only in cells expressing RNA containing PMEs of the proper sequence. The RNAs in other lanes of the gel provide controls that demonstrate specificity of binding. IRE is iron response element; A30, a sequence of 30 A residues; and HIV-E, a 573-nucleotide RNA sequence derived from HIV.

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Little RNAs Play a Big Role in Controlling Gene Expression

Lagos-Quintana, M., R. Rauhut, W. Lendeckel, and T. Tuschl. 2001. Identification of novel genes coding for small expressed RNAs. Science 294:853–858.

Lau, N.C., L.P. Lim, E.G. Weinstein, and D.P. Bartel. 2001. An abundant class of tiny RNAs with probable regulatory roles in Caenorhabditis elegans. Science 294:858–862.

Lee, R.C., and V. Ambros. 2001. An extensive class of small RNAs in Caenorhabditis elegans. Science 294:862–864.

Scientific discovery has a way of occurring in bursts of insight, often with input from multiple research teams whose ideas and experiments converge on a new line of thinking. In the field of RNA interference, such a conceptual breakthrough occurred in 2001 with the finding by three different labs that small regulatory RNA molecules are abundant in eukaryotic cells. Scientists had come to suspect that small RNAs might normally be produced in cells as a means of controlling gene expression. This suspicion was based on the discovery by Craig Mello and Andrew Fire that double-stranded RNA, when fed to C. elegans, could silence gene expression. Research teams led by Victor Ambros, David Bartel, and Thomas Tuschl set out to find evidence of small regulatory RNAs that might be produced naturally in cells.

Each team took a similar experimental approach, in which C. elegans or mammalian cells were grown in the laboratory and total cellular RNA was isolated. The total RNA was fractionated by size to enable purification of RNA molecules about 20 to 30 nucleotides long, the size of the molecules used in the Mello and Fire experiments. To identify these molecules, the RNAs were covalently linked at their 3′ ends to oligonucleotide sequences that provided binding sites for a complementary oligonucleotide, which could be used to prime the reverse transcription of the RNA into DNA. The complementary strand of this DNA sequence could be produced in a similar fashion, by covalently attaching it to a second oligonucleotide of defined sequence at its 3′ end. Once the small RNAs had been copied into double-stranded DNA, they were cloned into plasmids using standard techniques (see Chapter 7). The plasmids could then be propagated in bacterial cell culture, purified, and sequenced. The sizes of the small RNAs identified in C. elegans all fell within a narrow range (19 to 24 nucleotides), as opposed to the sizes of RNAs originating from E. coli, generated in a separate control experiment (Figure 2). These results suggested that the small RNAs cloned from C. elegans were indeed the product of transcription of the C. elegans genome, and not simply short degradation products.

The sequences of these small RNAs proved very exciting, because in many cases they were complementary to sequences found in the host genome. This finding suggested that the small RNAs were produced as part of a large regulatory pathway in which small RNA molecules, dubbed microRNAs (miRNAs), could base-pair with target sequences in mRNAs. Subsequent experiments verified that this mechanism occurs widely in eukaryotes.

Why were miRNAs overlooked by molecular biologists for so long? One reason is simply size: because they are so small, they tended to be ignored or were thought to be irrelevant degradation products rather than functional RNAs produced by the cells.

FIGURE 2 One class of short RNAs derived from C. elegans show a characteristically narrow length distribution (darker bars) compared with clones of E. coli RNA fragments (lighter bars) produced using the same protocol in an organism that lacks miRNAs. The short but uniform lengths of the C. elegans RNAs provided some of the evidence that the RNAs were functional, and not degradation products.

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Everything Old Is New Again: Beauty at the Turn of a Developmental Switch

Carroll, S.B., J. Gates, D.N. Keys, S.W. Paddock, G.E. Panganiban, J.E. Selegue, and J.A. Williams.1994. Pattern formation and eyespot determination in butterfly wings. Science 265:109–114.

Sean Carroll

We might not notice the diminutive fruit fly, but butterflies rarely fail to inspire fascination. The bold colors and patterns in a butterfly wing that catch our eye—surely these are the product of an elaborate developmental program that is distinct from the program operative in fruit flies?

Sean Carroll’s boyhood fascination with butterflies was eventually translated into research in a lab at the University of Wisconsin, where he studies insect development. In the early 1990s, it was already clear that many genes that control development are highly conserved, not just in insects, but in all higher eukaryotes. In setting out to decipher the development of butterfly wing patterns, Carroll decided that the genes known to affect the development of Drosophila wings were a good place to start.

Carroll’s subject was the butterfly Precis coenia, also called the Buckeye, found each summer over much of the United States. His laboratory succeeded in cloning a series of P. coenia genes homologous to Drosophila genes known to control wing development, including the genes for signaling proteins called Wingless and Decapentaplegic, and transcription factors called Apterous, Invected, Scalloped, and Distal-less. From the cloned genes, Carroll and his colleagues made labeled DNA probes and developed in situ hybridization methods to reveal the location of mRNAs in the butterfly wing at different stages of development. For months, the expression patterns looked identical to those already defined for the same genes in Drosophila. That changed when they got to distal-less, a gene that helps control appendage development in animals from insects to humans (Figure 3). Carroll describes it as one of his most thrilling moments in science.

One day, Carroll’s student, Julie Gates, called him over because she saw a pattern of genes turned on in so-called eyespots, the concentric rings of pigment in butterfly wings that look like eyes and are used in both mate recognition and predator avoidance. Carroll was stunned to be staring at the developing spotted pattern of gene expression, realizing that the gene involved, distal-less, had been around for at least 500 million years and was used in other organisms for appendage patterning. In fruit flies, there is no counterpart to eyespot development, and it was suddenly clear that the ancient gene had been recruited to an entirely new function in butterflies. This turned out to be the first example of what became a major theme in understanding the evolution of animal form: old genes occasionally evolve to do something completely new.

FIGURE 3 Expression of the distal-less gene (dark coloring) is revealed by in situ hybridization at three different stages of upper wing development in P. coenia. (a) A wing bud. (b) Partially developed wing. Expression of distal-less is evident at the point where an eyespot will develop (arrow). (c) Fully developed wing with eyespot.

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